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A Los1p-independent pathway for nuclear export of intronless tRNAs in Saccharomyces cerevisiae
Edited by Christine Guthrie, University of California, San Francisco, CA, and approved February 22, 2002 (received for review December 19, 2001)
Los1p, the Saccharomyces cerevisiae exportin-t homologue, binds tRNA and functions in pre-tRNA splicing and export of mature tRNA from the nucleus to the cytosol. Because LOS1 is unessential in yeast, other pathways for tRNA nuclear export must exist. We report that Cca1p, which adds nucleotides C, C, and A to the 3′ end of tRNAs, is a multicopy suppressor of the defect in tRNA nuclear export caused by los1 null mutations. Mes1p, methionyl-tRNA synthetase, also suppresses the defect in nuclear export of tRNAMet in los1 cells. Thus, Cca1p and Mes1p seem to function in a Los1p-independent tRNA nuclear export pathway. Heterokaryon analysis indicates that Cca1p is a nucleus/cytosol-shuttling protein, providing the potential for Cca1p to function as an exporter or an adapter in this tRNA nuclear export pathway. In yeast, most mutations that affect tRNA nuclear export also cause defects in pre-tRNA splicing leading to tight coupling of the splicing and export processes. In contrast, we show that overexpressed Cca1p corrects the nuclear export, but not the pre-tRNA-splicing defects of los1∷Kanr cells, thereby uncoupling pre-tRNA splicing and tRNA nuclear export.
In eukaryotic cells most RNAs are transcribed in the nucleus, but function in the cytosol, whereas karyophilic proteins are translated in the cytosol, but function in the nucleus. Movement of macromolecules between the nucleus and the cytoplasm is a signal-mediated process with localization signals within cargoes. Transport of many macromolecules across the nuclear pore complex (NPC) requires Ran, a small GTPase, and its regulators, members of the RanGTPase-binding importin-β family, as well as functional NPCs (for review, see ref. 1). Importin-β family members, also known as importins, exportins, or karyopherins, in addition to binding to Ran, interact with NPC components and cargo and shuttle between the nucleus and cytosol. The Saccharomyces cerevisiae importin-β family has 14 members and the human genome has more than 20 (1). According to a simple model, a given importin-β family member would provide unidirectional nucleus/cytosol movement for a specific subset of cargoes (1). However, departures from this simple model occur. For example, Msn5p/Kap142p mediates nuclear import of some proteins and export of others (2); nuclear import of histones H2A, H2B, and some ribosomal proteins are also mediated by multiple importin-β family members (3, 4). Moreover, macromolecules, such as mRNAs, can be exchanged across the nuclear membrane by both Ran-independent (for review, see ref. 5) and Ran-dependent mechanisms (6, 7).
Exportin-t, a member of the importin-β family (8), binds tRNAs directly by a Ran-GTP-dependent mechanism and it functions to export tRNAs from the nucleus to the cytosol (9, 10). Los1p is the yeast exportin-t homologue (8, 11–13). Because LOS1 is unessential (14), alternative pathway(s) for tRNA nuclear export must exist, at least in budding yeast. Aminoacylation of tRNAs in nuclei facilitates tRNA nuclear export in Xenopus oocytes (15) and S. cerevisiae (16, 17), and a priori nuclear tRNA aminoacylation could provide a yeast Los1p-independent tRNA nuclear export pathway. However, our previous studies failed to resolve whether nuclear tRNA aminoacylation functions in series or in parallel with Los1p (18).
To address this problem we sought to identify gene products that participate in tRNA nuclear export. We used fluorescence in situ hybridization (FISH) and screened a collection of yeast thermosensitive (ts) mutants (19) for candidates with specific defects in tRNA nuclear export at the nonpermissive temperature. This screen uncovered ts352, which possesses a mutation of CCA1 (20). CCA1 is an essential gene encoding ATP(CTP):tRNA nucleotidyltransferase (Cca1p) that catalyzes addition of C, C, and A nucleotides to the tRNA 3′ termini in nuclei, cytosol, and mitochondria (21). Cca1p was previously implicated in tRNA nuclear export (15–17, 22, 23). Here, we report that Cca1p can function in a Los1p-independent tRNA nuclear export pathway. We also show that Cca1p shuttles between the nucleus and the cytosol, providing the potential for it to function as an exporter or an adapter for tRNA nuclear export.
Recently it was shown that mRNA nuclear export and mRNA splicing are coupled (24, 25). A similar situation exists in yeast for tRNA splicing and tRNA nuclear export. Most mutations that should affect only the export process, in fact, also affect pre-tRNA intron removal. Examples include mutations of LOS1, RNA1 encoding RanGAP, PRP20 encoding RanGEF, and several genes encoding components of the NPC (26–29). Here we show that unlike the Los1p-dependent pathway, the Los1p-independent nuclear export pathway is uncoupled from pre-tRNA processing.
Materials and Methods
Strains and Media.
The yeast strains used were: SS328 [MATα ade2–101 his3Δ200 lys2–801 ura3–52; 19); SS330 (MATa ade2–101 his3Δ200 tyr1 ura3–52 (19)]; ts352 [MATa cca1–1 ade2–101 his3Δ200 tyr1 ura3–52 (20)]; X2316–3C [MATα LOS1 SUP4 ade2–1 can1–100 lys1–1 his5–2 trp5–48 ura3–1 (26)]; 201–1-5 [MATα los1–1 SUP4 his5–2 lys1–1 can1–100 trp5–48 ade2–1 ura3–1 (26)]; SS700 [MATα los1∷Kanr SUP4 ade2–1 can1–100 lys1–1 his5–2 trp5–48 ura3–1; constructed by a Kanr disruption of LOS1 in strain X2316–3C by the methods described (18)]; SWY27 (MATα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 nup116Δ∷HIS3) from S. Wente, Washington University School of Medicine, St. Louis (30); YM6154 (MATa MSN5 ura3–52 ADE2 his3–200 lys2–801 met− gal80− GAL1-LacZ∷LEU2) and YM6141 (MATa msn5∷HIS3 ura3–52 ADE2 his3–200 lys2–801 met− gal80− GAL1-LacZ∷LEU2), from M. Johnston, Washington University School of Medicine; Y1706 (MATα ura3–52 ade2–101 his3–11 trp1-Δ901 CSE1) and Y1709 (MATa ura3–52 ade2–101 his3–11 trp1-Δ901 cse1–1), both from M. Fitzgerald-Hayes, University of Massachusetts Amherst (31); PSY1200 (MATa sxm1∷HIS3 leu2Δ1 his3Δ200 trp1Δ63 ura3–52), PSY1201 (MATa pse1–1 ura3–52 trp1Δ63 leu2Δ1), and Kap123Δ (MATa kap123∷HIS3 lys2–801 leu2–3, 2–112 his3Δ200 trp1–1), all from P. Silver, Dana Farber Cancer Institute, Boston (32); DF5α (MATα MTR10 lys2–801 leu2–3, 2–112 ura3–52 his3Δ200 trp1–1), mtr10Δ (MATα mtr10∷HIS3 lys2–801 leu2–3, 2–112 ura3–52 his3Δ200 trp1–1), kap123 (MATα kap123∷URA3 lys2–801 leu2–3, 2–112 ura3–52 his3Δ200 trp1–1), and kap104–16 [MATα kap104∷ura3∷HIS3 lys2–801 leu2–3, 2–112 ura3–52 his3Δ200 trp1–1 and containing pRS314kap104–16 (ts allele)], all from L. Pemberton, University of Virginia Medical School, Charlottesville (33, 34); W303 XPO1 (MATa xpo1∷LEU2 ade2–1 ura3–1 his3–1,115 trp1–1 leu2–3,2–112 can1–100 ura3–1 and containing plasmid pKW440 encoding XPO1) and W303 xpo1–1 (MATa xpo1∷LEU2 ade2–1 ura3–1 his3–1,115 trp1–1 leu2–3, 2–112 can1–100 ura3–1 ura3–1 and containing plasmid pKW457 encoding xpo1–1) from C. Guthrie, University of California Medical School, San Francisco (35); MS739 [MATα ura3–52 leu2–3, 112 ade2–101 kar1–1 (36)] from M. Rose, Princeton University, Princeton, NJ.
Yeast extract/peptone glucose or synthetic defined media lacking appropriate nutritional ingredients were used to grow yeast. For growth assays, cells were grown to saturation in liquid media, and then 10 μl of serial dilutions of cells was applied to solid media and incubated for 3–4 days at the indicated temperatures.
CCA1 Constructs.
Escherichia coli DH5α was used to propagate recombinant DNA. To generate nuclear localization sequence (NLS)-CCA1-III-GFP-pRS416GAL1/10F, a 0.7-kb DNA encoding the green fluorescent protein (GFP) ORF was excised from pSEY181GAL1–10/GFP (provided by R. Y. Tsien, University of California, San Diego) by BamHI digestion and fused into pRS426NLS-CCA1 (21) at the BclI site located at the 3′ end of CCA1. The 2.6-kb fragment of CCA1-GFP was excised from pRS426NLS-CCA1-GFP by EcoRV digestion and ligated into pRS416GAL1/10F (from G. Peng, Penn State University, Hershey) at the SmaI site. NLS-Cca1p-III-GFP in this plasmid is controlled by the GAL1 promoter.
FISH was performed according to published procedures (12) with the following modifications: prehybridized and hybridized temperatures were 39°C and washing with 2× SSC was performed at 50°C for tRNA probes.
Heterokaryon Analysis.
The heterokaryon assay was a modification of published procedures (37, 38). SS330 cells transformed with SV40NLS-CCA1-III-GFP-pRS416GAL1/10F or pGAL-H2B-GFP (37, 38) were grown to midlog phase in media containing 2% glucose as the carbon source. The cultures were diluted and grown overnight to ≈5 × 106 cells per ml in media containing 2% raffinose as the carbon source. NLS-Cca1p-GFP synthesis was induced by addition of 2% galactose for 2 h. Switching the carbon source from galactose to glucose terminated further NLS-Cca1p-GFP synthesis. Matings were initiated by mixing 3 × 106 cells with an equal number of MS739 cells (relevant genotype: kar1–1), concentrating the cells on a 25-mm-diameter, 0.45-μm-pore nitrocellulose filter, and placing the filter on a yeast extract/peptone glucose plate at 23°C. After ≈1.5 h about 30% of the cells were zygotes. At this time the subcellular location of GFP was determined in live cells by microscopy. For indirect immunofluorescence, ≈107 cells were mixed with an equal number of MS739 cells and concentrated on an 82-mm-diameter filter. After ≈1.5 h, the cells were washed off the filter and immunofluorescence was performed (39).
Fluorescence images were observed by using a Nikon Microphot-FX microscope and were captured with a SenSys charge-coupled device camera (Photometrics, Tucson, AZ) with QED software (QED Imaging, Pittsburgh, PA). Images were assembled with Adobe PHOTOSHOP 5.0.
Preparation of RNA and Northern Blot Analysis.
RNA was isolated from log-phase yeast cells by phenol extraction as described (26). Northern blot analysis was performed according to Sarkar and Hopper (12). Blots were probed with tRNAIleUAU and each band was quantitated by phosphoimaging. The percentages of end-matured intron-containing pre-tRNA relative to the total of precursors and mature tRNAs were calculated.
CCA1 Is a Multicopy Suppressor of the los1∷Kanr Defect of tRNA Nuclear Export.
We screened ≈200 ts mutants (19) by FISH with oligonucleotides specific to the intronless tRNAs, tRNAMet, and tRNAIleAAU, to identify variants that accumulate tRNA, but not mRNA, in the nucleus at the nonpermissive temperature of 37°C. Three mutants were identified and mutant ts352 was analyzed in detail. SS328 cells, one of two parents of this ts collection, do not accumulate either mRNA or tRNA (Fig. 1A) in the nucleus. Cells with a deletion of the nucleoporin, Nup116p, accumulate both tRNA and mRNA in the nucleus at 37°C (refs. 12 and 30; Fig. 1A). In contrast, ts352, accumulates only tRNA in the nucleus at 37°C (Fig. 1A). As determined by tetrad analyses ts352 possesses a single ts mutation that cosegregates with the defect in tRNA nuclear export (data not shown). Multicopy and single-copy yeast genomic libraries (40, 41) were transformed into ts352 to clone the wild-type counterpart of the mutated gene by complementation of the ts growth defect. Plasmid DNA was isolated from transformants showing concomitant loss of plasmid and temperature resistance and restoration of tRNA nuclear export. Sequence alignment showed that the identified plasmids had only one gene in common, CCA1. Thus, as reported, the gene mutated in ts352 is CCA1 (20).
CCA1 was previously implicated in tRNA nuclear export because Cca1p catalyzes addition of C, C, and A nucleotides to the tRNA 3′ end and exportin-t preferentially binds tRNAs with mature CCA 3′ termini (22, 23). Also, the 3′ CCA nucleotides are required for tRNA aminoacylation and tRNA nuclear aminoacylation facilitates tRNA nuclear export (15–17). Moreover, in Xenopus oocytes tRNAs lacking 3′ CCA nucleotides are not exported to the cytosol (15, 22). Finally, the cca1–1 mutant has been reported to accumulate tRNA in nuclei (16, 17). However, it was unknown whether CCA addition and/or tRNA aminoacylation functions in the yeast Los1p-dependent tRNA nuclear export pathway and/or in Los1-independent pathways (18). To learn whether CCA addition and/or tRNA aminoacylation function in series or in parallel with Los1p we tested whether overexpressed Cca1p would suppress los1 mutations. Overexpression of Cca1p should not suppress a los1 null mutation if they function in an obligatory series, but it could if they function in parallel.
We first tested whether multicopy plasmids encoding CCA1 affect growth of cells with LOS1 mutations. Although LOS1 is unessential, mutations of it cause detectable phenotypes (14, 26). At elevated temperatures (34–38°C), los1 cells grow slower than the parental LOS1 strain, X2316–3C. The growth characteristics of LOS1, los1–1, and los1∷Kanr with vector alone or with the multicopy vector carrying CCA1 (YEpCCA1) were determined. Los1–1 and los1∷Kanr cells with YEpCCA1 grew better than the same strains with vector alone (Fig. 2A). Therefore, Cca1p functions as a multicopy suppressor of the growth defect caused by los1 mutations.
To determine whether suppression of the los1 growth defect relates to tRNA nuclear export, we determined the consequences of Cca1p overexpression upon tRNA nuclear export by using FISH. Los1ΔV (12) and los1∷Kanr (Fig. 1B, 1 and 1′) cells have defects in export of the intronless tRNAMet from the nucleus to the cytosol. The defect is suppressed by Cca1p overexpression provided by YEpCCA1 (Fig. 1B, 2 and 2′). The defect is also corrected by a multiple-copy vector containing LOS1 [YEpLOS1-ET (13); Fig. 1B, 3 and 3′]. Thus, Cca1p is a multicopy suppressor of the defect in tRNA nuclear export caused by a los1 null mutation, consistent with its suppression of the growth defect described above. Together the data support the model that Cca1p functions in a Los1p-independent pathway for tRNA nuclear export.
To address the mechanism by which this Los1p-independent tRNA nuclear export pathway operates, we tested whether it uses a member of the importin-β family. We screened yeast with mutations of genes encoding eight members including all known exportins and those importins implicated in RNA metabolism (Transportin/Kap104p, Yrb4p/Kap123p, Pse1p/Kap121p, Sxm1p/Kap108p, Mtr10p, Crm1p/Xpo1p, Cse1p and Msn5p; see Materials and Methods for strains) for defects in tRNA nuclear export. Only the xpo1–1/crm1–1 mutant showed tRNA nuclear accumulation, and it was weak and detectable only after 2 h at the nonpermissive temperature (data not shown). Because under these conditions Rna1p accumulates in yeast nuclei, the effect of xpo1–1/crm1–1 on tRNA nuclear export may be a secondary consequence of the predicted Ran-GTP nucleus/cytosol gradient collapse (42).
MES1 Is a Multicopy Suppressor of the los1∷Kanr Defect of tRNA Nuclear Export.
Aminoacyl tRNA synthetases (aaRS) catalyze addition of amino acids to the 3′ CCA termini of tRNAs. Because Cca1p seems to function in a Los1p-independent pathway for tRNA nuclear export, it is possible that tRNA nuclear aminoacylation also functions in a Los1p-independent pathway. As cells have 20 different aaRS, it is not expected that overexpression of a single one would suppress the growth defect of a los1 null mutation. However, overexpression of a single aaRS might suppress a nuclear export defect of its cognate tRNA.
We tested whether overexpressed MES1, encoding methionyl-tRNA synthetase (43), suppresses the defect of intronless tRNAMet nuclear export in los1∷Kanr cells. As expected, los1∷Kanr cells with vector alone accumulate tRNAMet in the nucleus (Fig. 3 A and A′). In contrast, los1∷Kanr cells with YEpMES1 (43) show little tRNAMet nuclear accumulation (Fig. 3 B and B′) and seem similar to los1∷Kanr cells with YEpLOS1-ET (Fig. 3 C and C′). Overexpression of Mes1p suppresses the defect in tRNAMet nuclear export of a los1 null mutation. If suppression is caused by methionine addition to tRNA rather than to a nonspecific affect on cellular metabolism, then suppression may be specific to the cognate tRNAMet. To test this hypothesis, we assessed the location of another intronless tRNA, tRNAIleAAU. tRNAIleAAU accumulates in the nucleus of los1∷Kanr cells with YEp24 (Fig. 3 D and D′) and in nuclei of los1∷Kanr with YEpMES1 (Fig. 3 E and E′). However, little nuclear accumulation of tRNAIleAAU occurs in los1∷Kanr cells with YEpLOS1-ET (Fig. 3 F and F′). Therefore, MES1 seems to function as a cognate-specific multicopy suppressor of the los1∷Kanr defect of tRNAMet nuclear export. The data indicate that Mes1p may operate in a Los1p-independent pathway for tRNA nuclear export. Cca1p and Mes1p could either function in series with each other in a single Los1p-independent pathway for tRNA nuclear export, or they could function in parallel in separate Los1p-independent pathways.
CCA1 Is Not a Multicopy Suppressor of Defective pre-tRNA Splicing Caused by los1 Mutations.
LOS1 was initially identified as a gene involved in pre-tRNA splicing (26). It is not clear why a tRNA exportin should cause defects in pre-tRNA splicing that precedes nuclear export. Nevertheless, the data are consistent with other reports showing that mutations of the RanGTPase pathway [rna1–1 (27) and prp20 (28)] and alterations of genes encoding NPC components (29) affect pre-tRNA splicing as well as tRNA nuclear export. Because Cca1p seems to function in a Los1p-independent pathway for tRNA nuclear export, we were interested in determining whether pre-tRNA splicing and tRNA nuclear export are coupled in this pathway as they are in the Los1p-dependent pathway. If so, multicopy Cca1p should correct the splicing defect of los1∷Kanr cells; if pre-tRNA splicing and tRNA nuclear export are not coupled, multicopy Cca1p should not correct the splicing defect of los1∷Kanr cells.
We performed Northern blot analyses to determine the ratios of end-matured, intervening sequence (intron) (IVS)-containing pre-tRNA (IVS-tRNAs) to total tRNAs by using probes specific to the intron-containing tRNAIleUAU family (12). LOS1 and los1∷Kanr cells bearing YEp24, YEpCCA1, or YEpLOS1-ET were shifted to elevated temperatures (34°C, 37°C, and 38°C) for 1–2 h before RNA isolation. LOS1 cells with YEp24, YEpCCA1, or YEpLOS1, or los1∷Kanr cells with YEpLOS1 have low levels of IVS-tRNAs (≈10%; Fig. 4, data shown only for 37°C). As predicted, los1∷Kanr cells with YEp24 accumulate greater amounts of IVS-tRNAs (30%). No significant difference existed in the relative levels of IVS-tRNAs between los1∷Kanr cells with YEp24 and los1∷Kanr cells with YEpCCA1 (34%; Fig. 4); so, Cca1p overexpression does not correct the los1∷Kanr pre-tRNA-splicing defect. The data indicate that pre-tRNA splicing and tRNA nuclear export are uncoupled in the Los1p-independent pathway.
Cca1p Seems to Be a Nucleus/Cytosol-Shuttling Protein.
We considered possible mechanisms by which Cca1p could participate in a Los1p-independent tRNA nuclear export pathway. It could act catalytically, preparing tRNA for interaction with an unknown exportin. Alternatively, Cca1p could function as an "exportin" to move tRNA to the cytosol or as an adapter for an unidentified "exportin." The latter possibilities predict that Cca1p would shuttle between the nucleus and the cytosol.
To test whether Cca1p is a nucleus/cytosol-shuttling protein we used a heterokaryon assay (37) by using the kar1–1 mutation that prevents nuclear fusion in zygotes. Movement of a reporter protein from a KAR1 nucleus to the kar1 nucleus, in the absence of new synthesis, is indicative of nucleus/cytosol shuttling. This assay requires that the reporter protein be primarily nucleoplasmic so that movement of reporter cytosolic pools from the joint cytoplasm to the second nucleus cannot occur. Assessment of Cca1p nucleus/cytosol shuttling by this assay is complicated by the fact that Cca1p is a sorting isozyme residing in the nucleus, cytoplasm, and mitochondria (21). CCA1 has three in-frame ATGs; Cca1p-I, translated from AUG1, is primarily located in mitochondria, whereas Cca1p-II and III, translated from AUG2 and AUG3, respectively, are primarily located in the cytosol with small functional nuclear pools (21). To perform the heterokaryon analysis for Cca1p, we first created a CCA1 allele encoding a modified Cca1p-III residing primarily in the nucleus. Addition of the simian virus 40 (SV40) large T antigen NLS to Cca1p-III (NLS-Cca1p) generated a nucleus located catalytically active Cca1p able to complement cca1–1 and suppress los1 when expressed from pRS426 (ref. 21; Fig. 2 B and C; data not shown). Addition of GFP at the NLS-Cca1p carboxyl terminus generated a tagged version of Cca1p efficiently located to nuclei with slightly reduced ability to complement cca1–1 (Fig. 2 B and C). GFP tagged NLS-Cca1p was inserted into pRS416GAL1/10F (James E. Hopper, personal communication) generating a protein tightly controlled by the GAL1 promoter (data not shown) allowing manipulation of conditions to prevent synthesis of Cca1p during mating.
NLS-Cca1p-GFP synthesis was induced for 2 h in SS330 cells; synthesis was terminated by change in the carbon source and then the cells were mated to the kar1–1 cells. As a control, the location of histone 2B (H2B)-GFP (37) was monitored. As H2B-GFP is not a shuttling protein, it was expected to remain in the KAR1 nucleus. For heterokaryons generated by mating SS330 cells containing H2B-GFP to kar1–1 cells, the GFP signal was in only one nucleus in all observed zygotes as viewed in live cells (Fig. 5 B1 and B2) or by immunofluorescence (data not shown). In contrast, for heterokaryons generated by mating SS330 cells containing NLS-Cca1p-GFP with kar1–1 cells, the GFP signal distributed equally between both nuclei (Fig. 5 A1–A4; data not shown). Thus, NLS-Cca1p can move between the KAR1 and the newly introduced kar1–1 nucleus, supporting the notion that Cca1p may be a nucleus/cytosol-shuttling protein, thereby meeting a requirement for a direct role in tRNA nuclear export.
Redundant Pathways for tRNA Nuclear Export.
We report that the cca1–1 mutation causes defects in tRNA nuclear export and that CCA1 and MES1 (in a cognate tRNA-specific fashion) function as multicopy suppressors of the los1∷Kanr defect of tRNA nuclear export. The data support the notion that Cca1p and tRNA nuclear aminoacylation can function in a Los1p-independent tRNA nuclear export pathway, leading to the model that yeast cells possess at least two tRNA nuclear export pathways for intronless tRNAs (Fig. 6). Do vertebrate cells also possess exportin-t-independent tRNA nuclear export pathways? It is unknown whether exportin-t is unessential for life in vertebrates, like it is for yeast. However, the existing data indicate that exportin-t provides the major export pathway for tRNAs in Xenopus oocytes because anti-exportin-t injection into nuclei reduced tRNA export by >80% (22, 23). Nevertheless, residual tRNA export, regardless of the amount of introduced antibody, could indicate that Xenopus, like yeast, possesses an alternative tRNA nuclear export pathway(s).
Cca1p and aaRS may function in both the Los1p-dependent and the Los1p-independent pathways. It is unknown whether Los1p, like exportin-t, preferentially binds to tRNAs with mature CCA 3′ termini (22, 23). Also, although exportin-t binds uncharged tRNAs in vitro (22, 23), no data address the relative affinities of exportin-t or Los1p to aminoacylated versus nonaminoacylated tRNAs. Our genetic studies provide no evidence for synthetic interactions between los1 null and cca1–1 ts mutations (data not shown). Lack of synthetic interactions is compatible with either the model that Cca1p functions in both Los1p-dependent and Los1p-independent pathways or with the model that there are more than two export pathways. Nevertheless, we showed that most yeast nuclear tRNAs seem to be aminoacylated when nuclear export is inhibited (17), indicating that under these conditions there are sufficient nuclear aaRS pools to charge most nuclear tRNAs. Therefore, most yeast tRNAs encountered by all export pathways likely possess 3′ CCA nucleotides and are aminoacylated.
The most straightforward mechanism for the Los1p-independent pathway would be for Cca1p and aaRS to act catalytically, modifying tRNA transcripts for efficient binding to an unknown importin-β family member as it does for exportin-t in vertebrate cells (Fig. 6 2a). However, we were unable to identify a member of this family other than Los1p that affects tRNA nuclear export. Vertebrate cells may possess a minor tRNA nuclear export pathway that is Ran-independent, because tRNA nuclear export is less sensitive to perturbations of Ran cycle than are other RNAs, such as snRNAs in Xenopus oocytes (44, 45). If the yeast Los1p-independent pathway is Ran-independent, CCA addition to nuclear tRNAs may prepare them for interactions with a RNA-binding protein that mediates tRNA export. In this case, this tRNA export pathway would resemble the currently favored model for Ran-independent mRNA nuclear export (5).
On the other hand, we show that Cca1p is capable of nucleus/cytosol shuttling. If Cca1p remained bound to tRNAs long enough, it might export tRNA in tow upon exiting the nucleus (Fig. 6 2b). However, because genome wide two-hybrid analyses provide no evidence for Cca1p interactions with nucleoporins (46), Cca1p could function as an adapter for an unknown protein that interacts with the NPC. These possibilities require the unusual behavior for an enzyme (Cca1p) to remain associated with its product after catalysis. The Sulfolobus shibatae CCA-adding enzyme forms stable complexes with mature tRNA-CCA (47) providing precedence for this behavior. Alternatively, Cca1p shuttling may function solely for its appropriate nuclear and cytosolic subcellular distribution. These alternatives could be distinguished by using Cca1p mutants that fail to shuttle. Unfortunately, such mutants are not yet available because the Cca1p NLS seems not to be one of the characterized motifs (21) and the nuclear export sequence is not sensitive to Crm1p (data not shown).
We showed that small nuclear pools of tyrosyl-tRNA synthetase, Tys1p, exist. The Tys1p NLS is sufficient to target a large passenger protein into nuclei even though most endogenous Tys1p is cytosolic (18). Thus, either Tys1p is tethered in the cytosol as has been recently reported for methionyl- and glutamyl-tRNA synthetases (48) or it can shuttle between the nucleus and the cytosol. In the latter case, Tys1p and possibly other aaRS theoretically could bind and export tRNA in tow, thereby generating numerous parallel export pathways (Fig. 6 2c).
Coupling of tRNA Splicing and tRNA Nuclear Export.
tRNA nuclear export in yeast is thought to be tightly coupled to pre-tRNA splicing because mutations affecting Los1p-dependent tRNA nuclear export cause IVS-tRNA accumulation. Nevertheless, this coupling cannot be absolute because spliced cytosolic pools of functional tRNA are essential and Los1p is unessential. The situation for yeast is in contrast to Xenopus oocytes for which depletion of exportin-t or the Ran-GTP nucleus/cytosol gradient has no affect upon pre-tRNA splicing (15, 22). The differences in processing pathways between these systems might account for the differences in the presence/absence of the coupling of splicing and nuclear export. In Xenopus, tRNA splicing usually precedes end-processing (15), whereas in yeast end-processing usually precedes splicing, although spliced tRNAs with unprocessed termini can be detected (26, 27, 49). Lund and Dahlberg (15) proposed that combinations of the tRNA kinetic processing pathway and proofreading of tRNA end-processing by aaRS assures that most tRNAs exported to the cytosol are correctly matured. We report that although overexpressed Cca1p does not suppress the pre-tRNA splicing defect of los1∷Kanr cells, it mediates export of intronless tRNAs to the cytosol. Moreover, we reported that cca1–1 mutants do not accumulate unspliced pre-tRNAs (17). Thus, the Los1p-independent pathway resembles the Xenopus exportin-t pathway in that it appears to be uncoupled from pre-tRNA splicing. Perhaps, tRNA aminoacylation serves a proofreading role for this yeast pathway as it does for Xenopus oocytes.
Curiously, Cca1p can catalyze CCA 3′ addition to IVS-tRNAs (21), even though overexpressed Cca1p fails to suppress the los1 pre-tRNA-splicing defect. A possible mechanism for this would be for overexpressed Cca1p to cause nuclear export of IVS-tRNAs, separating them from the splicing endonuclease located in the inner nuclear membrane (50), similar to the consequences of overproduction of exportin-t or tRNAs in vertebrate cells (15, 22). However, because we were unable to detect decreased nuclear pools or increased cytosolic pools of IVS-tRNAs in los1∷Kanr cells containing YEpCCA1 (data not shown), we think this possibility is unlikely. Another possibility is that the Los1p-independent pathway is dedicated solely to export of tRNAs encoded by genes lacking IVSs, which, however, is inconsistent with the ability of overexpressed Cca1p to suppress the los1 null growth defect. Perhaps, Cca1p also functions to export tRNAs encoded by IVS-containing genes that are processed by the splicing-first pathway.
How are pre-tRNA splicing and tRNA nuclear export coupled in the Los1p-dependent pathway? Considering the recently proposed model for coupling pre-mRNA splicing and nonsense mediated decay (ref. 51 and references therein), we propose a similar model for the spliced last kinetic pathway whereby tRNA introns are marked by a tRNA nuclear retention protein(s) that is removed by components of the Los1p-dependent tRNA pathway when IVS-tRNAs reach the inner nuclear membrane. Accordingly, the Los1p-dependent tRNA nuclear export pathway would also provide a proofreading role for tRNAs whose termini are processed before intron removal, assuring that only mature tRNAs are exported to the cytosol.
We thank J. Abelson for the ts yeast collection, N. C. Martin for SV40NLS-Cca1p-IIIpRS426, G. Peng for pRS416Gal1/10F, R. Y. Tsien for pSEY181GAL1-10/GFP, and G. R. Fink for YEpMES1. We acknowledge M. Fitzgerald-Hayes, C. Guthrie, M. Johnston, L. Pemberton, M. Rose, S. Sarkar, P. Silver, and S. Wente for providing yeast strains. We thank A. Weiner for insightful discussions, A. Azad and S. Sarkar for comments on the manuscript, and D. Stanford and other members of the A.K.H. laboratory for numerous scientific interactions and help in manuscript preparation. This work was supported by Public Health Service Grant GM27930 from the National Institutes of Health to A.K.H.
- nuclear pore complex;
- fluorescence in situ hybridization;
- ts,
- thermosensitive;
- nuclear localization sequence;
- green fluorescent protein;
- aaRS,
- aminoacyl tRNA synthetase;
- intervening sequence or intron
- Received December 19, 2001.
- Copyright © 2002, The National Academy of Sciences
- ↵
- ↵
- Yoshida K,
- Blobel G
- ↵
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- Mosammaparast N,
- Jackson K R,
- Guo Y,
- Brame C J,
- Shabanowitz J,
- Hunt D F,
- Pemberton L F
- ↵
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- Steitz J A
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- Görlich D,
- Dabrowski M,
- Bischoff F R,
- Kutay U,
- Bork P,
- Hartmann E,
- Prehn S,
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- Bischoff F R,
- Kunzler M,
- Hurt E,
- Simos G
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- Sarkar S,
- Hopper A K
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- Selvakumar D,
- Stanford D R,
- Hopper A K
- ↵
- Hurt D J,
- Wang S S,
- Lin Y H,
- Hopper A K
- ↵
- Lund E,
- Dahlberg J E
- ↵
- Grosshans H,
- Hurt E,
- Simos G
- ↵
- Sarkar S,
- Azad A K,
- Hopper A K
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- Azad A K,
- Stanford D R,
- Sarkar S,
- Hopper A K
- ↵
- Vijayraghavan U,
- Company M,
- Abelson J
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- Aebi M,
- Kirchner G,
- Chen J Y,
- Vijayraghavan U,
- Jacobson A,
- Martin N C,
- Abelson J
- ↵
- Wolfe C L,
- Hopper A K,
- Martin N C
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- Sharma K,
- Fabre E,
- Tekotte H,
- Hurt E C,
- Tollervey D
- ↵
- Wente S R,
- Blobel G
- ↵
- Xiao Z,
- McGrew J T,
- Schroeder A J,
- Fitzgerald-Hayes M
- ↵
- Seedorf M,
- Silver P A
- ↵
- Aitchison J D,
- Blobel G,
- Rout M P
- ↵
- Pemberton L F,
- Rosenblum J S,
- Blobel G
- ↵
- ↵
- Vallen E A,
- Hiller M A,
- Scherson T Y,
- Rose M D
- ↵
- Flach J,
- Bossie M,
- Vogel J,
- Corbett A,
- Jinks T,
- Willins D A,
- Silver P A
- ↵
- Peng G,
- Hopper J E
- ↵
- Hopper A K,
- Traglia H M,
- Dunst R W
- ↵
- ↵
- Kuo C-L,
- Campbell J L
- ↵
- ↵
- Meussdoerffer F,
- Fink G R
- ↵
- Cheng Y,
- Dahlberg J E,
- Lund E
- ↵
- ↵
- ↵
- ↵
- ↵
- O'Connor J P,
- Peebles C L
- ↵
- ↵


text:
Managing app reviews
After a merchant installs your app, they can leave a review for it. Positive reviews can encourage merchants to use your app and increase the numbers of installs it gets. If your app is listed on the Shopify App Store, then positive reviews can make your app appear higher in search results or on category pages. Reviews are also an important part of getting featured on the Shopify App Store.
When a merchant leaves a review for your app, they rate it on a scale of 1 to 5 and leave a comment. To leave a review, the merchant must have installed your app to their store. After a merchant uninstalls an app, they have 45 days to leave a review before the privilege is revoked.
Asking for app reviews
You can ask merchants to leave reviews as long as you don't encourage them to leave a good review. You should use neutral language, and never attach a review with an offer. For example:
"We value feedback! It helps us make our product better and keeps us energized. Let us know how we're doing."
Here are some examples of inappropriate ways to request feedback:
- "Like our service? Leave us a positive review!": This only motivates users with a favourable perception of your services.
- "Get one month free by leaving us a review!": If you're giving something to someone for free, then you're influencing them to leave a positive review.
- "Positive feedback keeps us going! Help by leaving a review now!": Even though you're just asking for a review, you're suggesting that the review should be positive.
Replying to app reviews
Replying to app reviews can help you engage with merchants, identify issues, and encourage merchants to improve the rating they gave to your app. Replying to reviews can also show potential merchants how engaged you are as a developer, which can encourage them to download your app.
You can reply to app reviews if you have an owner account or if you have a staff account with the Manage public listings permission. You can change an account's permissions from the Partner Dashboard.
To reply to reviews:
- Log into your Partner Dashboard.
- Click Apps.
- Click the name of your app.
- In the App actions drop-down, click View Shopify App Store listing.
- In the Most recent reviews section, go to any review and click Reply. If you don't see the review you're looking for, then click View all reviews to see more reviews.
- Enter your reply, then click Submit reply.
When you reply to a review, the merchant who left the review is notified. They can then decide to edit the review itself, including the rating, which will then replace the original review and rating on your app listing page. You receive an email notification when a merchant edits their original review. You can edit your response at any time, even if the merchant hasn't updated their review. Only your latest response will be shown on the app listing page.
Although you can reply to every review, consider replying first to reviews that have the lowest star rating, or that mention an issue the merchant has experienced. Prompt replies can help engage the merchant in a meaningful conversation.
Ideally, your response should address the merchant's review in a clear and concise way, and the language you use should be consistent with your brand. If possible, personalize the response for the merchant instead of giving them a generic response. However, make sure to not share any personal or financial information. Anyone who visits your app listing can see your responses, so make sure every response is professional and respectful, and doesn't include any technical jargon, marketing language, or spam. For more best practices, see the blog post Replying to Reviews in the Shopify App Store.
Managing negative reviews
Negative reviews can be helpful at identifying issues with your app. If a merchant leaves a negative review because of a bug or other issue, then you should work with the merchant to fix the issue. After the issue is fixed, you can ask the merchant to consider updating their review.
Sometimes, negative reviews are not helpful. There are specific circumstances where we will intervene and potentially remove reviews from app listings. We will investigate reviews that include:
Inappropriate content: the review is clearly inflammatory or derogatory.
Personal information: reviews that contain phone numbers, email addresses, mailing addresses, or other personal information will be deleted to protect that person's privacy.
Conflict of interest: reviews written by merchants who were offered something in exchange for a review. We will also remove reviews left by an app developer or affiliated employee on a competing app or on their own app.
If you see a review that violates these terms, please contact us.

subset: commoncrawl


text:
dm+d VMPP - 1127411000001105
Furosemide, sold under the brand name Lasix among others, is a medication used to treat fluid build-up due to heart failure, liver scarring, or kidney disease. It may also be used for the treatment of high blood pressure. It can be taken by injection into a vein or by mouth. When taken by mouth, it typically begins working within an hour, while intravenously, it typically begins working within five minutes.Common side effects include feeling lightheaded with standing, ringing in the ears, and sensitivity to light. Potentially serious side effects include electrolyte abnormalities, low blood pressure, and hearing loss. Blood tests are recommended regularly for those on treatment. Furosemide is a type of loop diuretic that works by decreasing the reabsorption of sodium by the kidneys.Furosemide was patented in 1959 and approved for medical use in 1964. It is on the World Health Organization's List of Essential Medicines, which lists the most effective and safe medicines needed in a health system. The wholesale price in the developing world is between US$0.004 and US$0.02 per day. In the United States, it is available as a generic medication and costs about US$0.15 per day. In 2016, it was the 15th most prescribed medication in the United States, with more than 32 million prescriptions. It is on the World Anti-Doping Agency's banned drug list due to concerns that it may mask other drugs. It has also been used in race horses for the treatment and prevention of exercise-induced pulmonary hemorrhage.
This information is from Wikipedia and may not be 100% accurate, its here to give a helping hand but please refer to the BNF if unsure


text:
I knew a few members of my family, and my partner's family, were less than thrilled that I didn't want a lot of people in the delivery room when my son was born. It wasn't a secret that all the parents, and siblings, wanted in. I think even a bold aunt tried to muscle her way onto the birth squad. When I shut them down and said, without hesitation, that my kid's dad should be the only person in the delivery room, they were begrudgingly compliant. They didn't throw themselves on the ground, start weeping, and threaten to sue or anything — they were (mostly) adults about it — but they were deeply displeased.
What I didn't know at the time, but was later unhappily surprised to learn, is how few women feel like they can say, "I don't want you and/or anyone in my delivery room" to family and friends without creating intense (and sometimes, insurmountable) friction within their families. And hey, I get not wanting to deal with that, especially when you're pregnant. Taking the path of least bullsh*t is always understandable, never more so than when you're staring down the barrel of expelling a person from your body through an upsettingly small opening. Who wants to deal with a pissed off mother-in-law on top of that, right?
Evidently, I did. To me, being in control of what happened in my labor and birthing space — and what didn't — meant everything. So with that in mind, here are the reasons I didn't let anyone in the delivery room except my partner, why I would for sure do it again, and why I'm convinced one of the best decisions you can make before you bring a human into the world is to kick everyone else out of the damn delivery room:
Because You Won't Want Anyone Else There
We could truthfully just stop the list here. I wanted my partner there, and I didn't want anyone else there, and that never felt like a choice I needed to defend. Yes, other people absolutely thought I needed to defend that choice, but whatever. When they had a baby, they could decide who would watch it come out of their body. Call me crazy, but it just kinda felt like a "me" thing.
Because Chaotic Birthing Environments Are Confusing
I'm not about to tell anyone what they should or shouldn't do during their labor (because in the end and always it is your decision, even if I have some feels about the entire thing) but I cannot understand the logic behind having so many people in the delivery room. Who came up with that plan? I'm guessing it wasn't a laboring mom. I'm guessing it was her mom... and her partner's mom, and their sisters, and their moms, and maybe some sisters or cousins or whatever, all of whom want to feel essential and important. And nothing is more validating than being part of the inner circle around someone's Big Life Event. Hand-to-god, I'm certain the widely accepted notion of a mega-packed delivery room was originally built in service to relatives' egos.
Anyway, I wasn't having it.
Because You'll Feel More In Control
Fewer people in the room meant less distraction, and less distraction meant it would be easier for me to attain and maintain whatever mental state and environmental vibe was most conducive to me getting the job done. Basically, people are full of free will and variables, both of which felt like a liability that could f*ck up my flow.
Because You'll Have Fewer People's Feelings To Deal With
Yeah, man, labor and birth are times of intense feelings all over the place. Totally. I feel you. Excitement, fear, anticipation, just to name the broad ones and ignore the countless much more specific emotions that anyone and everyone in a birthing room is unavoidably condemned to feel at some point. It's a lot to handle. And honestly, It was more than enough just to deal with my feelings. Even my kid's dad was on alert that he might get thrown out if he didn't keep it together enough for me to do my work.
Because You'll Feel Less Pressure
I thought I knew exactly how my labor was going to go, largely because I was 25, basically competent at most things, insufferably stubborn, and had encountered very few challenges in my short life that I hadn't been able to ultimately bend to my will, so why would this be any different?
In addition to being obnoxious, I was obviously quite wrong. And something in me must've known that there was a strong possibility of birth being a stressful and unpredictable experience, since I very strongly dismissed the idea of having an audience in the room.
I know my irritating self, and trust me, if I'd really thought my labor experience was going to be the effortless, curated endeavor I would've told you beforehand that it would definitely be, I would've wanted as many people there to witness it. I knew it was going to be an unmitigated mess, and it was, and I'm glad no one was there watching me expectantly while I was contemplating any one of the dozens of little choices I had to made about how to proceed in the middle of an especially arduous labor.
Because Your Baby Will Be Born In A Quieter Room
I mean, being that I was the person who was going to be pushing a 9-pound beast out of my vagina, my needs and preferences took priority. If I could tend to other people's needs while also serving my own, I was delighted. If that wasn't possible on any point, I didn't sweat it. My baby was the only other person whose needs and comfort were arguably tied for top billing with my own. As it happened, we needed a lot of the same thing: space and quiet to get our bearings, adjust to the reality shift we were actively navigating, and figure out the complicated task of breastfeeding.
I was thrilled that so many people were so eager to get busy loving on my kid that they were inconvenienced by having to wait a few extra hours and days to do so — but I'm incredibly thankful that I made them do just that.


text:
At the end of March 2012, Isle of Man law firm Dougherty Quinn ("DQ") together with Döhle (IOM) Limited, Freeport Trust Company Limited and the Isle of Man Ship Registry facilitated the delivery, flagging and registration at the Isle of Man Ship Registry of the first of nine 13,100 TEU container vessels being built by Hyundai Heavy Industries in Korea, the largest shipbuilder and shipyard in the world. At over 360 metres long and 48.2 metres wide, these vessels are the largest to be registered in the Isle of Man and some of the largest vessels currently in operation in the world. To date three of the vessels (named Hanjin Sooho, Hanjin Asia and Hanjin Europe) have been registered at the Isle of Man Ship Registry.
The remaining vessels are scheduled to be delivered during 2012
and the first half of 2013. Each of these nine container ships is
owned by nine Isle of Man entities, all administered from the
Island by John Middleton of Freeport Trust Company Limited. Five
ships are directly chartered to Hanjin Shipping and the remaining
four are provided to the Döhle Group, who provide the ships to
Hanjin Shipping under charters which include the provision of
technical services. Hanjin Shipping is Korea's largest
container operating company and the ninth largest in the world,
managing over 3% of the world's container tonnage, in addition
to having a notable presence in the tanker and bulk carrier
The transaction was structured and arranged by Crédit Agricole CIB and the nine vessels were financed using a facility provided by a number of banks, led by HSH Nordbank. Legal advice outside of the Isle of Man was provided by Norton Rose and Ince & Co from London acting for the banks and the builder respectively. The flag registration for all nine vessels is being coordinated by Eckhard Garbers and Luke Tippett from the Döhle Group working together with the Isle of Man Ship Registry. Isle of Man legal representation is provided by Mark Dougherty at DQ.
Mark Dougherty, director in DQ's corporate & commercial department and head of the shipping team commented:
"DQ was delighted to work with such large names in the shipping industry as well as with the Ship Registry itself in facilitating the delivery and registration of these vessels. The transaction highlights the enhanced reputation of the Isle of Man as a leading location for ship financing and registration. I know that all parties involved were impressed by what the Island had to offer and further work will emanate as a result of this. We must continue this support between the public and private sectors in order to continue building the Island's name as a leading jurisdiction within the global shipping industry."
The Isle of Man Ship Registry received no negative performance indicators in the 2011 Shipping Industry Flag State Performance, an annual exercise undertaken by the leading ship-owner groups. This recognition enforces its continuing commitment to maintain the Isle of Man as a leading international business centre renowned for professionalism and a longstanding policy of positive engagement with international standards.
Dick Welsh Director the Isle of Man Ship Registry, commented on the recent registrations:-
"This is a very important project for the Ship Registry. Adding nine such prestigious ships will enhance our reputation and add significant tonnage to the register with corresponding benefits for the Isle of Man. It has been a pleasure to work with all of the partners involved in bringing these ships on to the register as smoothly and efficiently as they have been throughout the process."
Photo shows the vessel Hanjin Sooho.
The content of this article is intended to provide a general guide to the subject matter. Specialist advice should be sought about your specific circumstances.


text:
We,The World and The WE Campaign
We, The World (at WE.net) is a global coalition-building non-profit organization based in New York City.
Seven principles for visual climate change communication
This report summarises the research underpinning climatevisuals.org and presents the key findings so that practitioners can take an evidence-based approach to visual communication.
Blue State Digital
Hi. We're Blue State Digital. We build and galvanize communities on behalf of some of the world's leading nonprofits, advocacy groups, and brands.
Concern Worldwide
Geo-Environmental Resource Association
Geo-Environmental Resource Association (GERAS) is a Cameroonian Geo-Environmental and Ecological Association that is non-profit making, non denominational, charitable, development


text:
The Happy Camper Good Lunch® Snack Container Set features a cute canine with all his camping necessities.
Food stays fresh and tasty in these clear, durable, leak-proof and safe containers. Sized just right for lunches large or small, BPA-free, odor, stain, shatter and scratch-resistant. Light, practical, and stackable for compact storage, Good Lunch® Snack Container Sets come in two convenient sizes to make snack-packing a snap!
Perfect for microwaving and freezing food portions, and taking snacks on outings or in school lunches. Use them to prep and store meal ingredients, snacks and more.
Details and Dimensions
Enjoy meal time gathered 'round the campfire with the Happy Camper Divided Suction Plate. The...
The Happy Camper Good Lunch® Box features a cute canine with all his camping necessities.Good...
Gather 'round the campfire, kids, a hearty meal is on its way!Whatever the destination, the...
$9.95 $18.95
The Happy Camper Mini Bib Gift Set will have them demanding 'smores around the campfire....


text:
Hawaiian Breakfast Pizza
Tropical and flavorful, this pizza puts a fun spin on breakfast for dinner. Kids love layering on the toppings, and it's a great way to use leftover ham. —Holly Ciani, Taberg, New York
Total TimePrep/Total Time: 30 min.
Makes6 slices
- 1 prebaked 12-inch thin whole wheat pizza crust
- 6 large eggs
- 1/4 teaspoon pepper
- 1 cup cubed fully cooked ham
- 1 cup unsweetened pineapple tidbits, drained
- 3/4 cup shredded cheddar cheese
- 1 tablespoon minced fresh parsley
- Salsa and reduced-fat sour cream, optional
- Place crust on an ungreased 12-in. pizza pan; set aside.
- Whisk eggs and pepper; stir into a large nonstick skillet coated with cooking spray. Cook and stir over medium heat until almost set. Transfer to pizza crust. Top with ham and pineapple. Sprinkle with cheese.
- Bake at 400° for 9-12 minutes or until cheese is melted. Sprinkle with parsley. Serve with salsa and sour cream if desired.
Nutrition Facts1 slice (calculated without optional ingredients): 294 calories, 12g fat (6g saturated fat), 240mg cholesterol, 688mg sodium, 28g carbohydrate (6g sugars, 4g fiber), 20g protein.
Every product is independently selected by our editors. If you buy something through our links, we may earn an affiliate commission.
Recommended Video
Oct 2, 2012
No comment left
Aug 23, 2012
I made half the way the recipe called..it tasted a bit bland. The other half I added a BBQ sauce to the base and a little chicken to the top. It made the difference.


text:
Keeping this in mind, here are some things you should check before picking a good short term accommodation in Sydney CBD. Research is the key. One of the first things you should check is the type of accommodation you need. Now there are hotels, bed and breakfast inns, and motels. If you travel as a Continue Reading
Common house problems in Adelaide, Australia
A household is a place where we need to feel safe and comfortable. It is where we need to have that complete ease of mind and be free from all other problems that we may have from work or from anywhere else. But what if we will also have some problems back at our houses? Continue Reading


text:
Opened 4 years ago
Closed 4 years ago
#5397 closed defect (fixed)
Incorrect curves filter work
|Reported by:
|Owned by:
|Blocked By:
|Reproduced by developer:
|Analyzed by developer:
Summary of the bug: curves video filter provides much darker picture than it should be
How to reproduce:
% ffplay "curves.mkv" -vf curves=r='0.037516/0':g='0.040103/0':b='0.059508/0' Result - FFmpeg colours are much darker than in Gimp. ffmpeg version N-79087-gefa98cd built on x86_64 Arch Linux
Patches should be submitted to the ffmpeg-devel mailing list and not this bug tracker.
Attachments (11)
Change History (22)
Changed 4 years ago by Ilya87
ffmpeg version
Changed 4 years ago by Ilya87
GIMP version
Changed 4 years ago by Ilya87
Video file to test
comment:1 Changed 4 years ago by Ilya87
Hello, does anyone read this bug report?
comment:2 Changed 4 years ago by cehoyos
At least a command line together with complete, uncut console output is missing. Please only use FFplay to report issues if the issue is not reproducible with FFmpeg (the application).
comment:3 Changed 4 years ago by Ilya87
Ok. Result is the same:
ffmpeg -i "curves.mkv" -pix_fmt yuv420p -vf curves=r='0.037516/0':g='0.040103/0':b='0.059508/0' -c:v libx264 -preset ultrafast -qp 0 output.mkv
ffmpeg version N-79273-gd433623 Copyright (c) 2000-2016 the FFmpeg developers
built with gcc 5.3.0 (GCC)
configuration: --prefix=/usr --disable-debug --disable-static --enable-stripping --enable-avisynth --enable-avresample --enable-fontconfig --enable-gnutls --enable-gpl --enable-ladspa --enable-libass --enable-libbluray --enable-libfreetype --enable-libfribidi --enable-libgsm --enable-libmodplug --enable-libmp3lame --enable-libopencore_amrnb --enable-libopencore_amrwb --enable-libopenjpeg --enable-libopus --enable-libpulse --enable-libschroedinger --enable-libsoxr --enable-libspeex --enable-libssh --enable-libtheora --enable-libv4l2 --enable-libvidstab --enable-libvorbis --enable-libvpx --enable-libwebp --enable-libx264 --enable-libx265 --enable-libxvid --enable-shared --enable-version3 --enable-x11grab
libavutil 55. 20.100 / 55. 20.100
libavcodec 57. 34.100 / 57. 34.100
libavformat 57. 31.100 / 57. 31.100
libavdevice 57. 0.101 / 57. 0.101
libavfilter 6. 41.100 / 6. 41.100
libavresample 3. 0. 0 / 3. 0. 0
libswscale 4. 1.100 / 4. 1.100
libswresample 2. 0.101 / 2. 0.101
libpostproc 54. 0.100 / 54. 0.100
Input #0, matroska,webm, from 'curves.mkv':
ENCODER : Lavf57.28.102
Duration: 00:00:03.42, start: 0.000000, bitrate: 1049 kb/s
Chapter #0:0: start 0.000000, end 2.000000
title : Chapter 01
Stream #0:0(jpn): Video: h264 (High), yuv420p, 720x384 [SAR 1:1 DAR 15:8], 23.98 fps, 23.98 tbr, 1k tbn, 47.95 tbc (default)
title : Encoded by zdzdz
File 'output.mkv' already exists. Overwrite ? [y/N] y
[libx264 @ 0x5640bff4bb60] using SAR=1/1
[libx264 @ 0x5640bff4bb60] using cpu capabilities: MMX2 SSE2Fast SSSE3 SSE4.2 AVX
[libx264 @ 0x5640bff4bb60] profile High 4:4:4 Predictive, level 3.0, 4:2:0 8-bit
[libx264 @ 0x5640bff4bb60] 264 - core 148 r2643 5c65704 - H.264/MPEG-4 AVC codec - Copyleft 2003-2015 - http://www.videolan.org/x264.html - options: cabac=0 ref=1 deblock=0:0:0 analyse=0:0 me=dia subme=0 psy=0 mixed_ref=0 me_range=16 chroma_me=1 trellis=0 8x8dct=0 cqm=0 deadzone=21,11 fast_pskip=0 chroma_qp_offset=0 threads=6 lookahead_threads=1 sliced_threads=0 nr=0 decimate=1 interlaced=0 bluray_compat=0 constrained_intra=0 bframes=0 weightp=0 keyint=250 keyint_min=23 scenecut=0 intra_refresh=0 rc=cqp mbtree=0 qp=0
Output #0, matroska, to 'output.mkv':
encoder : Lavf57.31.100
Chapter #0:0: start 0.000000, end 2.000000
title : Chapter 01
Stream #0:0(jpn): Video: h264 (libx264) (H264 / 0x34363248), yuv420p, 720x384 [SAR 1:1 DAR 15:8], q=-1--1, 23.98 fps, 1k tbn, 23.98 tbc (default)
title : Encoded by zdzdz
encoder : Lavc57.34.100 libx264
Side data:
cpb: bitrate max/min/avg: 0/0/0 buffer size: 0 vbv_delay: -1
Stream mapping:
Press [q] to stop, ? for help
frame= 82 fps=0.0 q=-1.0 Lsize= 4257kB time=00:00:03.42 bitrate=10196.7kbits/s speed= 16x
video:4255kB audio:0kB subtitle:0kB other streams:0kB global headers:0kB muxing overhead: 0.035616%
[libx264 @ 0x5640bff4bb60] frame I:1 Avg QP: 0.00 size:154166
[libx264 @ 0x5640bff4bb60] frame P:81 Avg QP: 0.00 size: 51887
[libx264 @ 0x5640bff4bb60] mb I I16..4: 100.0% 0.0% 0.0%
[libx264 @ 0x5640bff4bb60] mb P I16..4: 5.3% 0.0% 0.0% P16..4: 82.9% 0.0% 0.0% 0.0% 0.0% skip:11.8%
[libx264 @ 0x5640bff4bb60] coded y,uvDC,uvAC intra: 79.5% 73.8% 73.6% inter: 71.3% 75.1% 73.9%
[libx264 @ 0x5640bff4bb60] i16 v,h,dc,p: 59% 38% 2% 1%
[libx264 @ 0x5640bff4bb60] i8c dc,h,v,p: 30% 34% 36% 0%
[libx264 @ 0x5640bff4bb60] kb/s:10191.59
comment:4 Changed 4 years ago by Ilya87
comment:5 Changed 4 years ago by Ilya87
comment:6 Changed 4 years ago by Ilya87
comment:7 Changed 4 years ago by richardpl
Consider adding bounty if you want to have better pong.
comment:8 Changed 4 years ago by ubitux
- Analyzed by developer set
- Reproduced by developer set
- Status changed from new to open
A patch can be found at http://ffmpeg.org/pipermail/ffmpeg-devel/2016-July/196740.html
Note that the curves in Gimp and Photoshop doesn't behave the same at all.
After the patch is applied, in order to get a close behavior to what you expect, you will have to use something like -vf curves=r='0.037516/0 1/1':g='0.040103/0 1/1':b='0.059508/0 1/1'. If you define points in (0;0) (-vf curves=r='0/0 0.037516/0 1/1':g='0/0 0.040103/0 1/1':b='0/0 0.059508/0 1/1'), the curves will be much curvy at the origin and you will get your unexpected darker output. This doesn't happen in Gimp but is closer to what you observe in Photoshop. I will attach screenshot of Gimp and PS to show this.
Edit: see in particular curves-ps-3pts-31.png vs curves-gimp-3pts-31.png. To mimic Gimp behaviour, you will have to use 2 points instead of 3, see curves-ps-2pts-31.png vs curves-gimp-2pts-31.png
Changed 4 years ago by ubitux
Curves Gimp with 2 points (31;0) and (255;255)
Changed 4 years ago by ubitux
Curves PS with 2 points (31;0) and (255;255)
Changed 4 years ago by ubitux
Curves Gimp with 3 points (0;0), (31;0) and (255;255)
Changed 4 years ago by ubitux
Curves PS with 3 points (0;0), (31;0) and (255;255)
comment:9 follow-up: ↓ 10 Changed 4 years ago by Ilya87
And what if I define not only 2 points (start point 0.037516/0 and 1/1) on channel, but more? For example,
-vf curves=r='0.037516/0 0.127516/0.4 1/1'
Will it work in Gimp-style?
comment:10 in reply to: ↑ 9 Changed 4 years ago by ubitux
Replying to Ilya87:
And what if I define not only 2 points (start point 0.037516/0 and 1/1) on channel, but more? For example,
-vf curves=r='0.037516/0 0.127516/0.4 1/1'
Will it work in Gimp-style?
Unfortunately no. It matches Photoshop behavior. I'm attaching 3 new images so you can compare the 3 tools. But basically, you can assume FFmpeg always matches Photoshop.
Also, you can try this branch: https://github.com/ubitux/FFmpeg/compare/curves (note to future reader: if this branch doesn't exist anymore, it's probably merged upstream).
Commit 2/2 of this branch adds a "plot" option you can use to get the curves used by FFmpeg. Quoting the example from the documentation:
ffmpeg -f lavfi -i color -vf curves=cross_process:plot=/tmp/curves.plt -frames:v 1 -f null - gnuplot -p /tmp/curves.plt
Changed 4 years ago by ubitux
3 points, not starting at zero, FFmpeg
Changed 4 years ago by ubitux
3 points, not starting at zero, Gimp
Changed 4 years ago by ubitux
3 points, not starting at zero, FPhotoshop
Changed 4 years ago by ubitux
3 points, not starting at zero, Photoshop
comment:11 Changed 4 years ago by ubitux
- Resolution set to fixed
- Status changed from open to closed
Fixed in 5c14018fc4f6ae9fcce7eaf8dd2d66cb6fbfc7bc
Plot option added in 4a8f5f1fd83dfa92e02901e543d79124c4e551dc
(Bonus 16-bit support in e30cdac14b55ef290e26e055a88055ba9cde2f46)
Thanks for the report.


text:
Antiquarian Books: A Companion for Booksellers, Librarians and Collectors
Aldershot: Scolar Press, 1994. First Edition. Signed by all three editors on full title to journalist and language writer (and book collector) William Safire with inscriptions "With best wishes to William Safire from Philippa Bernard," and in different ink below "and thanks from a regular reader of your column. Agnus..... More


text:
Daniel Murphy probably won't say it, but it has to feel good. Here he is, the Mets' spurned ex — the second baseman whom they never extended a real offer in the offseason — coming back to New York bigger and better than ever. First-place team? Check. A happy, comfortable life in D.C.? Check. Highest batting average in the majors. Check that one twice.
"We're playing well," Murphy said by way of understatement last weekend. "Bryce [Harper] is playing so well, really swinging the bat well, I know J-Dub [Jayson Werth] has had some good at-bats, Zim [Ryan Zimmerman] is coming around, hit two homers the other day, so, you know, you just try to get a good pitch to hit and hit it hard."
This is what the Mets have to deal with for the next three days, and for six of their next nine games: A murderers' row of big-swinging bats with something to prove, along with one of the most devastating pitching staffs in baseball. Just in the next three days, they'll face Max Scherzer (fanned 20 batters in his last start), Gio Gonzalez (1.93 ERA), and Stephen Strasburg (6-0, 2.95 ERA).
But perhaps the scariest thing about these Nationals, who split a four-game series with the Marlins last weekend and are 1.5 games in front of the Mets and Phillies, is that they don't think they've reached their full potential. Not even close.
"The big guys have got to pick it up," manager Dusty Baker said during the Marlins series, later adding that they'll be really dangerous when that happens.
After the Nationals won Game 1 of a doubleheader Saturday, centerfielder Michael A. Taylor also speculated what it would be like when the team finds its groove.
"If we can win these close games and tough games when the bats aren't going like we want," he said, "I think that puts us in a good position when we get hot."
And so, the lukewarm Nationals, at an apparently unimpressive eight games over .500, will have to make due with Murphy, who is hitting .400. They'll continue to labor on with their reigning NL MVP, Harper, who has teams so terrified, they've already walked him a league-high 41 times in 37 games. He's only actually had 113 at-bats, though he does tend to make the best of it: Harper has 11 home runs.
And then there's Scherzer, who threw two no-hitters last year (one against the Mets); On May 11, he became only the fourth pitcher to strike out 20 in a nine-inning game.
But no. They're not at their best yet.
"I think there's just a winning atmosphere here that we're just going out there and doing our job and not looking at it from an individual perspective but as a full unit," reliever Shawn Kelley said. "We're getting big hits when we do and we're rolling well as a team. That helps as a team. We all kind of feed off each other."
That actually means that when Murphy or Harper or Scherzer aren't doing what those three do, the Nationals still have a chance. The team has six pinch-hit home runs, including two on Friday from Stephen Drew and Chris Heisey. And that, Harper said, is key.
"I think [a big part of bench production is] just having faith in the guys and just throwing everybody out there and having them think they're going to do damage when they get up there," hesaid. "We have a very confident team and a very confident bench and I think that's huge."
With the exception of Daniel Murphy and catcher Wilson Ramos, the Nationals' offense has yet to kick into gear. The batting averages for the regulars who are likely to face Noah Syndergaard in the first game of the series:
Ben Revere CF .093
Anthony Rendon 3B .227
Bryce Harper RF .265
Daniel Murphy 2B .400
Ryan Zimmerman 1B .234
Jayson Werth LF .208
Wilson Ramos C .350
Danny Espinosa SS .214


text:
Please note that not all databases access online sources to the current date.
L'express international
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© 2015. LBIS, Kenyon College
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Meet the School Councelors:
Phone: 843-903-6300
Degrees and Certifications:
Mrs. Shan Going Francis
My name is Shan Goings Francis and I joined the River Oaks Elementary family in 2015. I'm excited to be a part of such a wonderful school and community!
I'm a native of Myrtle Beach and graduated from Myrtle Beach High School. I received my Bachelor of Arts degree in Psychology from Coastal Carolina University, my Master of Arts in Counselor Education and Educational Specialist degrees from South Carolina State University. What I love most about the elementary level is the genuine curiosity students have in their learning and the world around them. I also appreciate the opportunity to provide early intervention support that can have a positive impact on a child.
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I moved to the area with my husband in 2006. We enjoy spending time with our 2 sons and traveling. I look forward to working with the students and families this year. Please call or email me any time you have have a question or concern.


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February 25, 2011
ROBERT K. BROWN, APPELLANT (PLAINTIFF),v.CITY OF CASPER AND OFFICER ERIC E. WALTERS, INDIVIDUALLY, APPELLEES (DEFENDANTS).
Appeal from the District Court of Natrona County The Honorable W. Thomas Sullins, Judge
The opinion of the court was delivered by: Kite, Chief Justice.
Before KITE, C.J., and GOLDEN, HILL, VOIGT*, and BURKE, JJ.
KITE, C.J., delivers the opinion of the Court; GOLDEN, J., files a specially concurring opinion; VOIGT, J., files a dissenting opinion.
*Chief Justice at time of oral argument.
NOTICE: This opinion is subject to formal revision before publication in Pacific Reporter Third. Readers are requested to notify the Clerk of the Supreme Court, Supreme Court Building, Cheyenne, Wyoming 82002, of any typographical or other formal errors so that correction may be made before final publication in the permanent volume.
[¶1] Robert K. Brown was injured when the vehicle he was driving was struck by a vehicle driven by Casper Police Officer Eric E. Walters. Mr. Brown presented a notice of claim to the City of Casper (City) and subsequently filed a complaint in district court. The district court dismissed the complaint, finding that it lacked subject matter jurisdiction because Mr. Brown failed to allege that he complied with the constitutional requirements for maintaining an action against a governmental entity. We reverse.
[¶2] Mr. Brown presents the issues for this Court's consideration as follows:
1. The District Court abused its discretion in refusing to allow [him] to amend his complaint to correct a deficiency in an allegation and to conform to the undisputed facts of the case.
2. The District Court erred in ruling that the allegations contained in the Complaint, when viewed in the light most favorable to [him], did not sufficiently allege the prerequisites for the District Court's subject matter jurisdiction.
3. The judicially created rules for pleading a governmental claim lead to injustice and are against public policy and should be abolished.
The City and Officer Walters assert the district court correctly concluded that it lacked subject matter jurisdiction to proceed, denied the motion to amend and dismissed the case with prejudice.
[¶3] On April 28, 2007, Mr. Brown was traveling northbound on Wolcott Street in Casper, Wyoming. Officer Walters was driving westbound on East 12th Street. At the intersection of the two streets, Officer Walters failed to stop for a red light and collided with Mr. Brown's vehicle. Officer Walters was on duty at the time of the accident and was acting within the scope of his employment.
[¶4] Mr. Brown presented a notice of claim to the City on April 25, 2008, within one year of the collision. On April 16, 2009, less than two years after the collision, Mr. Brown presented an amended notice of claim, more fully setting forth the damages he sustained in the collision. On April 23, 2009, less than one year after presenting his notices of claim, Mr. Brown filed a complaint against the City and Officer Walters in district court. In his complaint, Mr. Brown alleged that "all requirements of the Wyoming Governmental Claims Act, Wyoming Statute § 1-39-101 et. seq. ., have been complied with, and a claim in conformity therewith was served upon the City of Casper, pursuant to Wyo. Stat. Ann., 1999 §§ 1-39-113(c) . on April 25, 2008 and an Amended Claim . delivered . to the City of Casper . . . on the 16th day of April, 2009." Although Mr. Brown alleged that a copy of the notice of claim was attached to the complaint as exhibit A, there is no attachment to the complaint contained in the appellate record.
[¶5] The City and Officer Walters answered the complaint. In its answer, the City denied Mr. Brown's allegation that he had met the requirements of the Wyoming Governmental Claims Act (WGCA) and asserted as an affirmative defense that he had failed to comply with those requirements. On July 16, 2009, both the City and Officer Walters filed motions for judgment on the pleadings, claiming they were entitled to judgment as a matter of law because Mr. Brown had not properly invoked the district court's jurisdiction within one year of presenting his notice of claim. Specifically, they asserted the allegation in the complaint was insufficient to invoke jurisdiction in that it did not allege compliance with the requirements of the Wyoming Constitution, Art. 16 §[¶6] On July 17, 2009, the day after the City and Officer Walters filed their motions and supporting memoranda, Mr. Brown filed a motion to amend his complaint to reflect that the notice of claim he had presented to the City complied with the constitution. The amended complaint attached to his motion alleged that "all requirements of Article 16, § 7 . . . have been complied with." Mr. Brown also attached the amended notice of claim he had presented to the City on April 16, 2009. The City and Officer Walters filed memoranda opposing Mr. Brown's motion, asserting the district court had no jurisdiction to consider it.
[¶7] After further briefings by the parties, the district court convened a hearing on the motions. Following the hearing, the district entered an order converting the W.R.C.P. 12(c) motions for judgment as a matter of law to Rule 56 motions for summary judgment and granting summary judgment for the City and Officer Walters. The district court's order states:
[Mr. Brown]'s complaint fails to contain an allegation that he complied with the Wyoming Constitution and, therefore, this Court lacks subject matter jurisdiction under Wyoming Supreme Court precedent. See McCann v. City of Cody, Wyoming, 2009 WY 86, 211 P.3d 1078 (Wyo. 2009); and see Beaulieu v. Florquist, 2004 WY 31, ¶ 14, 86 P.3d 863, 868 (Wyo. 2004) (Beaulieu II) . . . . Without subject matter jurisdiction, the Court FURTHER FINDS it lacks any authority to grant [Mr. Brown]'s motion to amend his Complaint. See Gose v. City of Douglas, 2008 WY 126, ¶ 19, 193 P.3d 1159, 1164 (Wyo. 2008) . . . . The only action the Court can take is to deny [Mr. Brown]'s motion to amend his Complaint . . ..
Finding that it was not possible at that point for Mr. Brown to sue the City or Officer Walters within the time period specified in § 1-39-114 of the WGCA, the district court dismissed the complaint with prejudice. This appeal followed.
[¶8] The district court ruled as a matter of law that Mr. Brown's complaint must be dismissed for lack of subject matter jurisdiction. The existence of subject matter jurisdiction is a question of law that we review de novo. Cantrell v. Sweetwater County School Dist. No. 2, 2006 WY 57, ¶ 6, 133 P.3d 983, 985 (Wyo. 2006).
[¶9] The district court concluded it lacked subject matter jurisdiction over this governmental claim case such that it could not consider a motion to amend the complaint because binding precedent dictated that conclusion. While there is no question that presentation of a notice of claim is constitutionally and statutorily required before an action can be brought against a government entity, the rule this Court understandably but mistakenly has attributed to a statement made in Board of Trustees of UW v. Bell, 662 P.2d 410, 415 (Wyo. 1983) that district court jurisdiction is not invoked unless the complaint alleges compliance with the constitution and statute is inconsistent with prior precedent as well as statutory and constitutional provisions granting jurisdiction over these cases to the district courts. We resolve the inconsistency with prior precedent and hold that subject matter jurisdiction is invoked upon the filing of a complaint alleging a claim against a governmental entity. We continue to require that complaints alleging claims against governmental entities must also allege compliance with the statutory and constitutional provisions governing notices of claim. See § 1-39-113(d) (LexisNexis 2009 & Supp. 2010). However, we clarify that in cases where a notice of claim has been properly presented but the complaint fails to allege that fact, district courts have the discretion to allow amendment of the complaint to cure the failure.
[¶10] Having performed a lengthy and thorough analysis of Wyoming legal principles relating to subject matter jurisdiction and governmental claims, we conclude Bell has mistakenly been interpreted to mean that courts have no power to act in a case against a governmental entity unless the complaint alleges presentation of a notice of claim. Contrary to that interpretation, Bell had to have meant that dismissal was appropriate in that case because it did not appear the condition precedent to suit had been met. In order to understand this distinction, it is appropriate to begin with a discussion of the law preceding Bell.
1. Wyoming Law Before Bell
[¶11] Article 5, § 1 of the Wyoming Constitution provides:
The judicial power of the state shall be vested in the senate, sitting as a court of impeachment, in a supreme court, district courts, and such subordinate courts as the legislature may, by general law, establish and ordain from time to time.
Article 5, § 10 provides further:
The district court shall have original jurisdiction of all causes both at law and in equity and in all criminal cases, of all matters of probate and insolvency and of such special cases and proceedings as are not otherwise provided for. The district court shall also have original jurisdiction in all cases and of all proceedings in ...
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