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Taf14 is required for the stabilization of transcription pre-initiation complex in Saccharomyces cerevisiae
Kadri Peil1,
Henel Jürgens1,
Johanna Luige1 nAff2,
Kersti Kristjuhan1 &
Arnold Kristjuhan ORCID: orcid.org/0000-0002-5842-49521
The YEATS domain is a highly conserved protein structure that interacts with acetylated and crotonylated lysine residues in N-terminal tails of histones. The budding yeast genome encodes three YEATS domain proteins (Taf14, Yaf9, and Sas5) that are all the subunits of different complexes involved in histone acetylation, gene transcription, and chromatin remodeling. As the strains deficient in all these three genes are inviable, it has been proposed that the YEATS domain is essential in yeast. In this study we investigate in more detail the requirement of YEATS domain proteins for yeast survival and the possible roles of Taf14 YEATS domain in the regulation of gene transcription.
We found that YEATS domains are not essential for the survival of Saccharomyces cerevisiae cells. Although the full deletion of all YEATS proteins is lethal in yeast, we show that the viability of cells can be restored by the expression of the YEATS-less version of Taf14 protein. We also explore the in vivo functions of Taf14 protein and show that the primary role of its YEATS domain is to stabilize the transcription pre-initiation complex (PIC). Our results indicate that Taf14-mediated interactions become crucial for PIC formation in rpb9Δ cells, where the recruitment of TFIIF to the PIC is hampered. Although H3 K9 residue has been identified as the interaction site of the Taf14 YEATS domain in vitro, we found that it is not the only interaction target in vivo.
Lethality of YEATS-deficient cells can be rescued by the expression of truncated Taf14 protein lacking the entire YEATS domain, indicating that the YEATS domains are not required for cell survival. The YEATS domain of Taf14 participates in PIC stabilization and acetylated/crotonylated H3K9 is not the critical target of the Taf14 YEATS domain in vivo.
The YEATS domain is a highly conserved protein domain present in more than 100 proteins from yeast to human [1]. Together with bromodomain, the double PHD finger (DPF), and the double pleckstrin homology (PH) domain proteins, it belongs to the family of the acetyllysine readers [2,3,4,5]. There are three YEATS domain proteins in budding yeast Saccharomyces cerevisiae: Taf14, Yaf9, and Sas5. All of them are subunits of different complexes involved in histone acetylation, gene transcription, and chromatin remodeling. Disruption of all YEATS-containing proteins is lethal in yeast, while single deletions of these genes cause relatively mild phenotypes [1, 6,7,8,9]. Although the YEATS domain was identified for more than a decade ago, several in vitro studies proposing its targets have been published just recently. It has been shown that Yaf9, the subunit of NuA4 and SWR1 complexes, binds primarily to acetylated histone H3 with a high preference for H3K27ac [10]. Another study showed that the human YEATS domain-containing protein Gas41 as well as yeast Yaf9 displayed strong binding affinity toward the succinylated H3K14, H3K56, H3K79, and H3K122 peptides in vitro, while Yaf9 did also bind the succinylated H4K12 and H4K31 [11]. Taf14, the subunit of TFIIF, TFIID, INO80, SWI/SNF, and NuA3 complexes, was first shown to have the strongest interaction with H3K9ac peptides [12], but subsequent studies specified crotonylated H3K9 (H3K9cr) as its preferred binding target [13]. Structural analysis of the Taf14 YEATS domain revealed that Phe62 and Trp81 residues of the protein form an aromatic cage, which is required for Taf14 binding to the acetylated H3K9. Mutation of Trp81 to alanine in the YEATS domain is sufficient to abolish this interaction completely [12].
The function of the YEATS domain has been just as enigmatic as its targets. As the YEATS proteins are subunits of various chromatin-modifying, or transcription-regulating complexes, it has been proposed that YEATS domains target these complexes to designated chromatin regions. For example, inactivating mutation of the Yaf9 YEATS domain was shown to impair the function of the SWR1 complex, leading to decreased deposition of H2A.Z into the PHO5 promoter region [10]. Cells lacking Taf14 protein display reduced growth rate, sensitivity to DNA damage, and elevated temperatures, although the YEATS mutants of Taf14 cause only a slight increase of sensitivity to DNA damaging agents [12]. Furthermore, taf14Δ phenotype can be rescued by expression of the Taf14 C-terminal part that lacks the entire YEATS domain [14].
In this study, we tested the significance of YEATS domains for S. cerevisiae viability. We show that the yeast strain lacking all YEATS domain proteins, but expressing the C-terminus of Taf14, has only mild growth defect compared to wt cells. Further investigation of the Taf14 YEATS domain functions revealed that it is needed for the stabilization of the transcription pre-initiation complex (PIC) formation on gene promoters.
Taf14 C-terminus rescues lethality of YEATS-negative cells
To explore the functions of YEATS domain-containing proteins in cells, we deleted SAS5, YAF9, and TAF14 genes in all combinations in yeast. Of single gene deletions, only taf14Δ cells displayed reduced growth phenotype in standard conditions (Fig. 1b). Additional deletion of SAS5 had no synthetic effect in combination with deletions of YAF9, or TAF14 (Fig. 1b), while double-knockout of YAF9 and TAF14 was lethal (Additional file 1: Fig. S1A). Previous studies have revealed that typical taf14Δ phenotype (reduced growth rate, sensitivity to DNA damaging agents, and elevated temperatures) can be rescued by expression of truncated Taf14 protein that lacks its YEATS domain [14]. Based on these observations, we hypothesized that the cause of lethality for mutant yeast strain lacking both TAF14 and YAF9 was not the absence of the YEATS domain as such, but rather the inability of taf14Δ strain to cope with extra stress resulting from deletion of YAF9. To test this, we replaced full-length TAF14 in its genomic locus with genes encoding YEATS-deleted (taf14ΔYEATS), or YEATS-mutated (taf14W81A) versions of Taf14 (Fig. 1a) and combined them with deletions of YAF9 and SAS5 genes. As expected, taf14ΔYEATS was viable in all combinations with YAF9 and SAS5 deletions (Additional file 1: Fig. S2), moreover, both taf14ΔYEATS and taf14W81A were able to rescue the strain in which all YEATS-containing proteins were deleted (Fig. 1c). This shows clearly that the lethality of yaf9Δtaf14Δ strain was not caused due to the lack of the YEATS domains, but rather due to the absence of the C-terminal part of Taf14.
Highly conserved YEATS domain is non-essential for viability in yeast. a Schematic of wt and mutant Taf14 proteins used in this study. Wt Taf14 protein contains a highly conserved YEATS domain in its N-terminus. Mutant Taf14 protein without the YEATS domain (Taf14ΔYEATS) lacks amino acids 6–121 and mutant Taf14 protein with a non-functional YEATS domain (taf14W81A) has substitution of Trp81 to alanine. b Tenfold serial dilutions of cells with single or double-knockout of TAF14, YAF9 and SAS5 were spotted onto SC plates and grown at 30 °C for 2 days. c Tenfold serial dilutions of taf14ΔYEATS or taf14W81A cells combined with YAF9 and SAS5 deletions. Cells were spotted onto SC plates and grown at 30 °C for 2 days
Synthetic phenotype of RNAPII and Taf14 YEATS mutants
To explore the functions of the Taf14 YEATS domain in more detail, we tested whether the YEATS mutants can tolerate various stress conditions (lower and higher temperature, different carbon source, osmotic stress, DNA damage). taf14Δ strain was sensitive to all conditions tested, while taf14ΔYEATS and taf14W81A cells responded to the majority of stresses in the same way as wt strain. The only exception was growth at 16 °C, where YEATS mutants displayed intermediate phenotype between wt and taf14Δ strains (Fig. 2). However, in contrast to our results, it has been reported that the taf14W81A cells were temperature and MMS sensitive in the BY4741 strain background [12]. The discrepancy between our and previous results suggests that the requirement of the Taf14 YEATS domain might depend on strain background, or assay conditions. In previous studies, the plasmid-based expression system of Taf14 mutants was used, while our assays were performed with genomic replacement of TAF14 with taf14W81A in its native locus.
The lack of functional Taf14 YEATS domain does not cause any substantial growth defects in different growth conditions in yeast. Tenfold serial dilutions of taf14ΔYEATS or taf14W81A strains were spotted onto SC plates and grown at different temperatures for either 2 days (at 30 °C and 37 °C) or 5 days (at 16 °C). Cells spotted onto minimal medium (MIN) plate and cells spotted onto SC plate with galactose as a different carbon source were grown at 30 °C for 2 days. Cells spotted onto SC plate containing 0.9 M NaCl for osmotic stress were grown at 30 °C for 3 days. For DNA damage response analysis cells were spotted onto SC plates containing indicated concentrations of MMS or treatment with ionizing radiation (45–85 Gy) on SC plates was used and cells were grown at 30 °C for 2 days
As all Taf14-containing protein complexes are involved directly or indirectly in gene transcription, we tested whether Taf14 YEATS mutants may have more evident effects on regulation of RNAPII-dependent transcription. To make cells more vulnerable for minor disturbances in transcription, we used rpb4Δ and rpb9Δ cells to test Taf14 YEATS mutants. In yeast, Rpb4 and Rpb9 are non-essential subunits of RNAPII, although in the absence of either protein cells grow slower and display several defects in transcription initiation [15,16,17,18,19]. We found that the deletion of TAF14 was lethal in rpb4Δ and rpb9Δ background, while Taf14 YEATS mutants were viable (Fig. 3 and Additional file 1: Figs. S1B–E). However, the YEATS mutants had synthetic phenotype with rpb4Δ and rpb9Δ, underlining the substantial role of the Taf14 YEATS domain in these strains (Fig. 3).
Taf14 YEATS domain is important in yeast strains with mutant RNAPII. a Tenfold serial dilutions taf14ΔYEATS strain combined with either RPB4 or RPB9 deletion were spotted onto SC plates and grown at 30 °C for 2 days. b Tenfold serial dilutions of taf14W81A strain combined with either RPB4 or RPB9 deletion were spotted onto SC plates and grown at 30 °C for 2 days
H3K9 is not the only target of the Taf14 YEATS domain in vivo
Recent studies have indicated that the Taf14 YEATS domain interacts specifically with acetylated and crotonylated N-terminal tails of H3 [12, 13]. Although modification of several different lysine residues in H3 N-terminal peptides supported the recruitment of Taf14 YEATS domain, and multiple modifications on the same peptide had a cumulative effect in these assays, the acetylated/crotonylated H3K9 was identified as the primary target of Taf14 YEATS domain in vitro [12, 13, 20]. The synthetic phenotype of Taf14 YEATS mutants with rpb4Δ and rpb9Δ strains suggests that recognition of modified H3K9 by Taf14 may become more critical in the absence of these proteins. To test whether H3K9 is the primary target of Taf14 also in vivo, we compared the growth rates of rpb9Δ strains carrying either taf14W81A, or K9R mutation in histone H3, which does not allow its modification. If acetylated or crotonylated H3K9 is the primary target of Taf14 in vivo, the H3K9R strain should express the same level of genetic interaction with rpb9Δ as does the taf14W81A strain. While taf14W81A displayed a clear synthetic phenotype with rpb9Δ, the H3K9R mutation did not distinguish from rpb9Δ strain carrying wt H3 (Fig. 4a). An identical result was obtained when Rpb9 was removed from the nucleus by the anchor-away technique (Fig. 4b). The latter approach allows to pre-grow cells in wt conditions and remove Rpb9 just before the assay, thus avoiding possible adaptation of the strain for growth in the absence of Rpb9. These results show that acetylated/crotonylated H3K9 is not the critical target of Taf14 YEATS domain in vivo, suggesting that other modified lysine residues in histone tails, or in non-histone proteins can compensate the lack of H3K9, providing the alternative docking sites for Taf14 binding. Our previous study showed that none of the single lysine residue mutations in the H3 N-terminal tail had synthetic phenotype with RPB9 deletion [21], supporting the idea that H3 tail modifications are functionally redundant. Unfortunately, we cannot test combined mutations of multiple H3 N-terminal lysines in the rpb9Δ background, as this leads to genomic instability and inviability of the cells due to inefficient activation of DNA damage checkpoint response pathway [21].
Modified H3K9 is not the only target of the Taf14 YEATS domain. a Tenfold serial dilutions of rpb9Δ cells expressing either wt H3 or mutant H3K9R histones and rpb9Δ cells expressing taf14W81A were spotted onto SC plates and grown at 30 °C for 2 days. b Tenfold serial dilutions of Rpb9 anchor-away cells expressing either wt H3 or mutant H3K9R histones and Rpb9 anchor-away cells expressing taf14W81A were spotted onto SC plates in the absence (−RAPA) or presence of rapamycin (+RAPA) and grown at 30 °C for 2 days. Strain without a functional anchor-away system (AKY1159) was used as a control
Taf14 YEATS is necessary for the stabilization of the pre-initiation complex
Taf14 is a subunit of two basal transcription factors, TFIID and TFIIF that are both required for PIC formation. Considering that Taf14 YEATS mutants were not distinguishable from wt cells upon exposure to genotoxic stress (Fig. 2), but had synthetic phenotype with RNAPII subunits (Fig. 3), we decided to test whether Taf14 YEATS mutants affect the efficiency of PIC assembly. We used Rpb9 anchor-away strain carrying either wt TAF14 or taf14W81A and measured the relative amounts of RNAPII, TFIIF, and TFIID complexes on promoters of two highly expressed genes FBA1 and RPS8A. Among the PIC components, TFIIF is the most obvious common target of Taf14 and Rpb9. Taf14 is a subunit of TFIIF and on the other hand, in vitro interaction between purified RNAPII and TFIIF complexes is strongly reduced in the absence of Rpb9 [22]. Although there was a slight reduction of TFIIF on promoters in taf14W81A cells compared to their wt counterparts, exclusion of Rpb9 from cell nucleus had a more significant effect on the recruitment of TFIIF, regardless of the status of Taf14 (Fig. 5a). This confirms that Rpb9 is the major interaction partner of TFIIF, although some of TFIIF is recruited to the PIC independently of Rpb9. On the other hand, the presence of TFIID on gene promoters was reduced in taf14W81A cells, while removal of Rpb9 had only minor effect for TFIID occupancy. Notably, the reduction of TFIID level was dependent mainly on taf14W81A mutation, as depletion of Rpb9 did not lead to further loss of TFIID in taf14W81A cells (Fig. 5b). This suggests that the most likely role of Taf14 is to stabilize PIC components on gene promoters via YEATS-mediated interactions. We observed a relatively low amount of TFIID on FBA1 promoter in all conditions, which is in accordance with previous findings that the RPS8A promoter is strongly dependent on TFIID, while the FBA1 promoter is not [23, 24]. A relatively small reduction of TFIID occupancy in taf14W81A cells is in concordance with the mild phenotype of the Taf14 YEATS mutant strains and indicates supportive, but not critical role of Taf14 in TFIID stabilization. In contrast, the occupation of RNAPII was reduced by depletion of Rpb9 and inactivation of the Taf14 YEATS domain, and a combination of both factors had a cumulative effect in the reduction of RNAPII on promoters (Fig. 5c). Collectively, these results suggest that cells can tolerate moderate instability of either TFIID or TFIIF on promoters, but a simultaneous weakening of both interactions results in substantial loss of RNAPII recruitment to the PIC, which in turn is reflected in the synthetic phenotype of rpb9Δ taf14W81A cells.
The simultaneous absence of Rpb9 and inactivation of Taf14 YEATS has a cumulative effect in the reduction of the relative levels of RNAPII on promoters. The relative amount of TFIIF (a), TFIID (b), and RNAPII (c) at the highly expressed FBA1 and RPS8A gene promoters in the indicated strains was analyzed with ChIP and qPCR. Rpb9 was removed from the cell nucleus by the anchor-away technique in the presence of rapamycin. Tfg2 (TFIIF) and Taf2 (TFIID) were C-terminally tagged with FLAG tag, Rpb3 (RNAPII) was C-terminally tagged with E2-tag. Non-coding region in the right arm of ChrVI telomere was used as an internal control. Error bars represent the standard deviation of at least four independent experiments. d Tenfold serial dilutions of indicated anchor-away cells were spotted onto SC plates in the absence or presence of rapamycin and grown at 30 °C for 2 days. ** indicates p ≤ 0.01 and *** indicates p ≤ 0.001
Although these results suggest that Taf14 is required primarily for TFIID stabilization, it has to be considered that Taf14 is also a subunit of TFIIF, and therefore, taf14W81A mutation affects both of these complexes. To evaluate whether the synthetic phenotype of taf14W81A in the absence of Rpb9 was primarily caused by the impediment of TFIID, or TFIIF, we tested the effect of Rpb9 depletion in the taf2ΔC strain background. In this strain, the last 147 amino acids from Taf2 C-terminus are deleted, which disrupts Taf14 interaction with TFIID [25]. In taf2ΔC cells, fully functional Taf14 is expressed and incorporated into all Taf14-containing complexes, except TFIID. When Rpb9 was depleted in this stain background, the growth of cells was similar to the Rpb9-depleted taf14W81A cells, suggesting that Taf14 interaction with TFIID becomes critical for cell growth in the absence of Rpb9 (Fig. 5d). However, we cannot rule out that the deletion of Taf2 C-terminus does not affect other functions, or interactions of TFIID that may lead to the synthetic phenotype with Rpb9 depletion independently from TFIID–Taf14 interaction. In fact, when taf14W81A was combined with the deletion of the C-terminus of Taf2, the synthetic phenotype with Rpb9 depletion was even stronger than by either mutation alone (Fig. 5d).
It has been proposed that the YEATS domain is essential for cells, as the deletion of all three YEATS-containing proteins is lethal in yeast [1, 9]. This assumption would imply that in the absence of one YEATS domain protein, other YEATS domain proteins could substitute its function. However, this scenario is rather unlikely, as the complexes containing either Yaf9, Sas5, or Taf14 are required for different functions in the cell. To explore the roles and requirements of YEATS proteins in vivo, we deleted all the three genes in yeast and found that the lethality of yaf9Δsas5Δtaf14Δ triple-knockout strain can be fully rescued by the expression of a truncated version of Taf14 protein, which lacks the entire YEATS domain. Therefore, the YEATS domain is dispensable for viability of budding yeast, if the C-terminal part of Taf14 is expressed in cells. Previous studies have demonstrated that Taf14 C-terminus is required for its incorporation into TFIID, TFIIF, SWI/SNF, INO80, and NuA3 complexes [14]. On the other hand, the integrity and enzymatic activity of these complexes were not substantially affected in the absence of Taf14, suggesting that it was not required for the basic functions of the complexes [25,26,27,28,29,30]. This observation is also supported by the fact that Taf14 is the only non-essential subunit in TFIID and TFIIF, and inactivation of catalytic subunits in SWI/SNF, or INO80 complexes leads to far more severe phenotypes than deletion of TAF14 gene [31, 32]. Based on these observations, it has been proposed that the main role of Taf14 is to recognize histone modifications via its YEATS domain and target the protein complexes to the designated chromatin regions. However, as the YEATS-less Taf14 can compensate for the lack of all YEATS-containing proteins, it suggests that YEATS-independent roles of Taf14 may have been underestimated and the functions of Taf14 in protein complexes are not restricted to its YEATS domain.
It has been shown that Taf14 YEATS interacts with acetylated and crotonylated histone H3 N-terminal peptides [12, 13]. Modifications of H3K9 residue have been identified as the primary targets of Taf14 in vitro, although these studies also revealed that Taf14 interaction with polyacetylated H3 peptides was even better than with mono-acetylated H3K9ac peptide [33]. However, the specificity of Taf14 interactions has not been confirmed in vivo. The main obstacle for testing the Taf14 target sites in vivo is that the strains harboring Taf14 YEATS mutations display only very mild phenotypes [12, 14]. To enhance the influence of Taf14 YEATS domain mutations in vivo, we were searching for the synthetic phenotype of Taf14 YEATS mutants with other transcription factors and found that the strains lacking either Rpb4 or Rpb9 subunits of RNAPII displayed severe growth defect in taf14ΔYEATS and taf14W81A background. This opened a unique opportunity to test the possible targets of the Taf14 YEATS domain in vivo. We assumed that if H3K9 is the only target of Taf14, then elimination of any possible modification of H3K9 in rpb9Δ cells should copy the phenotype of Taf14 YEATS mutants in the rpb9Δ background. Surprisingly, we found no synthetic phenotype of H3K9R mutation with rpb9Δ, indicating that acetylated/crotonylated H3K9 cannot be the only binding target of Taf14 in vivo. This suggests that the Taf14 YEATS domain can fulfill its functions also through alternative interaction sites in chromatin, if H3K9 modifications are not available. It is also possible that lysine modifications of non-histone proteins may provide alternative binding sites for the Taf14 YEATS domain in vivo.
Although the Taf14 protein is present in many chromatin- and transcription-related protein complexes, its role in transcription regulation has remained obscure. Considering that Taf14 YEATS mutants had synthetic phenotype with RNAPII subunits, we tested whether these mutants affect the efficiency of PIC assembly. The formation of PIC requires coordinated recruitment of approximately 60 proteins [34, 35] that are stabilized by multiple, and often redundant, interactions between its components. Elimination of some of these interactions does not necessarily cause the dramatic failure of complex formation, however, the abolition of multiple interactions may lead to cumulative effect and destabilization of the whole complex. Our results indicate that the YEATS domain of Taf14 is one of those factors that contribute to the formation and stabilization of PIC. While its effect alone is rather minor, it becomes more prominent, when the recruitment of TFIIF to the PIC is hampered. Although the levels of all tested PIC components (TFIID, TFIIF, and RNAPII) were slightly reduced in the taf14W81A strain background, the strongest reduction was seen in the recruitment of TFIID. Stoichiometry of purified TFIID indicates that it contains multiple copies of Taf14 protein and also at least two Taf14 binding domains have been identified in the C-terminus of Taf2 [25, 36]. Therefore, the stability of TFIID in the PIC may be more dependent on Taf14-mediated interactions than the stability of other PIC components. Remarkably, the deletion of Taf2 C-terminus and taf14W81A mutation had a similar synthetic phenotype with depletion of Rpb9, suggesting that Taf14 is primarily required as a subunit of the TFIID complex in Rpb9-deficient cells. However, we saw that taf14W81Ataf2ΔC strain had an even stronger synthetic phenotype with Rpb9 depletion than either mutation alone. This suggests that the C-terminus of Taf2 may have more functions in TFIID than providing a binding domain for Taf14, and therefore it may affect PIC stability also independently from Taf14. For example, a recent study of Komagataella phaffii TFIID structure revealed that Taf14 binds to Taf2 side-by-side with the Taf8 subunit [37], suggesting a possible role of Taf2 C-terminal domain in orchestrating the correct arrangement of these subunits in TFIID. Also, it has to be considered that the deletion of the Taf2 C-terminal domain fully eliminates incorporation of Taf14 to the TFIID complex, while the Taf14 YEATS domain mutants are expected to interact with all Taf14-containing complexes. Moreover, as the expression of the YEATS-less Taf14 can rescue the phenotypes of taf14Δ cells, the YEATS-independent functions of Taf14 may be as important as its interactions through the YEATS domain. At least two possible mechanisms can explain this phenomenon. First, although the lack of Taf14 from different protein complexes seems not to affect the activities of these complexes, it may stabilize the active conformation of these complexes. For example, the interaction of Taf14 with Taf2 can compensate some of the taf2-ts mutants in vivo, although these mutations are located outside of the Taf14-binding sites on Taf2 [25]. Second, Taf14 can form dimers in vitro [38], suggesting that it may facilitate inter-complex interactions via Taf14–Taf14 dimerization. In this respect, the C-terminal domain of Taf2 might serve as "a landing platform" for the various Taf14-containing complexes during the PIC formation.
Neither Taf14 YEATS mutations, nor the deletion of Taf2 C-terminus causes any remarkable phenotype when present alone, or combined. However, all these mutations have synthetic phenotype with inactivation of Rpb9. The most obvious common target of these mutations is the TFIIF complex, which contains Taf14 as its subunit, and its interaction with RNAPII is strongly dependent on Rpb9 [22]. Therefore, it is likely that the diminished recruitment, or stabilization of TFIIF into the PIC is the major reason for the synthetic phenotype. First of all, in the absence of Rpb9, the interaction between TFIIF and RNAPII is weak and the stable binding of both factors to the PIC becomes more dependent on other protein–protein interactions. We propose that in this situation the Taf14-mediated interactions become critical for efficient PIC formation. Taf14 can stabilize the PIC by providing TFIID and TFIIF additional modules for interaction with chromatin (via the YEATS domains) or with each other (via Taf14–Taf14 dimerization). When one of these supporting interactions is disabled in Rpb9-deficient cells either due to the inactivation of the Taf14 YEATS domain, or by the deletion of the Taf2 C-terminus, the synthetic phenotype appears. When both these interactions are abolished in the taf14W81Ataf2ΔC strain, the synthetic phenotype becomes even more severe than by either mutation alone.
Interestingly, all the major components of this network are found in yeasts, but not in higher eukaryotes. In metazoans, the Taf2 proteins do not contain the C-terminal domain that is found in yeasts and concordantly, the TFIID complex does not contain the Taf14 subunit. Also, the metazoan TFIIF contains two subunits, lacking the third, Taf14, which is present in yeast TFIIF. In addition, deletion of Rpb9 is lethal in higher eukaryotes, while yeasts can survive without this subunit of RNAPII. Collectively, these observations suggest that yeasts may have a Taf14-based back-up system for recruitment of TFIIF to gene promoters, while metazoans rely mostly on direct interactions between TFIIF and RNAPII. The benefits of this back-up system are not clear, although it might allow faster and more flexible responses of unicellular organisms to the changes in their growth environment, for example in response to the availability of nutrients, or fluctuations of temperature.
In this study, we show that the highly conserved YEATS domain is not required for cell viability in budding yeast. Although the Taf14 YEATS domain interacts specifically with acetylated and crotonylated histone H3 K9 in vitro [12, 13], our data indicate that it is not the critical target of the Taf14 YEATS domain in vivo. Furthermore, we show that Taf14 YEATS domain supports the formation of PIC on gene promoters by stabilizing TFIID and TFIIF binding to the complex.
Yeast strains, plasmids, and antibodies
All Saccharomyces cerevisiae strains were derived from the W303 background [39] and are listed in Additional file 1: Table S1. Strains AKY1027 (rpb9Δ) and 1158 (Rpb9 anchor-away) were used in plasmid shuffling assays. These strains express wild type copies of HHT2 and HHF2 from a URA3-based plasmid (YCp50:HHT2-HHF2) as a sole source for histones H3 and H4. Histone H3K9R point mutation was made in HIS3-based plasmid (pRS413-H3H4-3F12). Either wt or H3K9R plasmid was transformed into AKY1027 or AKY1158, and counter-selected on 5-fluoroorotic acid (5-FOA) plates (1 mg/ml) to obtain strain with a wt or a mutant H3. Rpb9 anchor-away strains were derived from strain HHY168 (Euroscarf) [40], where RPB9 locus was replaced with rpb9-FRB-hphMX expression cassette. In the presence of rapamycin Rpb9-FRB is depleted from the nucleus by conditional tethering to the "anchor" Rpl13a protein, containing C-terminal FKBP12-tag. In control strain (AKY1159) Rpb9 protein is still C-terminally tagged with FRB-tag, but RPL13A locus lacks the FKBP12-tag. We confirmed that in these strains both wt Taf14 and Taf14W81A proteins were expressed in equal levels before and after the removal of Rpb9 from the cell nucleus (Additional file 1: Fig. S3). Yeast strains expressing Taf14ΔYEATS (lacking amino acids 6-121) and Taf14W81A were generated by replacement of genomic TAF14 locus with either spHIS5-taf14ΔYEATS or spHIS5-taf14W81A expression cassette. The intron sequence of Taf14 was present in these expression cassettes. RNAPII Rpb3 subunit was C-terminally tagged with E2-tag and detected with 5E11 antibody (Icosagen), Tfg2, and Taf2 were C-terminally tagged with FLAG tag and detected with M2 antibody (Sigma-Aldrich). Taf14 was detected with an anti-Taf14 antibody (A278, antibodies.com). For Western blot, cell extracts were prepared as described [41] and protein samples were separated on SDS–polyacrylamide gel.
Yeast growth assays
Culture density was measured with Z2 Cell and Particle Counter (Beckman Coulter). For spot test assays, tenfold serial dilutions of cell suspensions were made and 5 µl of each dilution was spotted onto plates with synthetic complete (SC) selective medium. Indicated concentrations of methyl methanesulfonate (MMS) in SC plates were used to test the viability of cells. Cells were also treated with ionizing radiation (45–85 Gy). In experiments with Rpb9 anchor-away strains, 1 µg/ml rapamycin (Cayman Europe) in 0.1% DMSO as a final concentration was added to the cultures (0.1% DMSO was used for controls). Plates were incubated for 2 days at 30 °C, unless otherwise stated.
Chromatin immunoprecipitation (ChIP) assay
Yeast cultures were inoculated into 25 ml fresh YPD media at density 8 × 106 cells per ml. After incubation 120 min in a shaker at 30 °C, either DMSO (0.1% final concentration) or rapamycin (1 µg/ml) in 0.1% DMSO was added and cells were cultured for another 120 min. Cells were fixed in 1% formaldehyde for ChIP assay. ChIP assays were performed as described previously [42]. Whole-cell extract from 1 × 107 cells was used for ChIP assays with antibodies directed against anti-E2 tag or anti-FLAG tag. Co-precipitated DNA was analyzed by quantitative real-time PCR using LightCycler 480 Real-Time PCR System under standard conditions (40 cycles, 95 °C for 15 s + 60 °C for 1 min). Maxima SYBR Green/ROX qPCR master mix (Thermo Scientific) was used. PCRs were performed with primer pairs covering the promoter regions of FBA1 and RPS8A. Non-transcribed region in chromosome VI right arm telomere was used as an internal control and for normalization of ChIP results. The primer sequences used in these analyses are as follows: FBA1algF 5′GAGAAAGACCGGTGTCATCGTTGG3′; FBA1algR 5′CCTTACCAGCGAAGTAAGCAGCAC3′; RPS8ApromF 5′CAGGACCTCTCTTTGAATGGAATAG3′; RPS8ApromR 5′CTTCTTGTGAAAAACTCGGCGTTTC3′; Tel6RF 5′TAACAAGCGGCTGGACTACTTTC3′; Tel6RR 5′GATAACTCTGAACTGTGCATCCACTC3′. Data were obtained from at least four different experiments. Error bars represent the standard deviation between the biological replicates. Student t test was used when comparing mean differences of two experimental groups. The level of statistical significance was established at a p value of < 0.05, ** indicates p ≤ 0.01, and *** indicates p ≤ 0.001.
All data generated or analyzed during this study are included in this published article and its additional files.
PIC:
Pre-initiation complex
RNAPII:
TFIID:
Transcription factor IID
TFIIF:
Transcription factor IIF
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Pillai B, Sampath V, Sharma N, Sadhale P. Rpb4, a non-essential subunit of core RNA polymerase II of Saccharomyces cerevisiae is important for activated transcription of a subset of genes. J Biol Chem. 2001;276(33):30641–7.
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Klein BJ, Vann KR, Andrews FH, Wang WW, Zhang J, Zhang Y, et al. Structural insights into the π-π-π stacking mechanism and DNA-binding activity of the YEATS domain. Nat Commun. 2018;9(1):4574.
Sein H, Reinmets K, Peil K, Kristjuhan K, Värv S, Kristjuhan A. Rpb9-deficient cells are defective in DNA damage response and require histone H3 acetylation for survival. Sci Rep. 2018;8(1):2949.
Ziegler LM, Khaperskyy DA, Ammerman ML, Ponticelli AS. Yeast RNA polymerase II lacking the Rpb9 subunit is impaired for interaction with transcription factor IIF. J Biol Chem. 2003;278(49):48950–6.
Warfield L, Ramachandran S, Baptista T, Devys D, Tora L, Hahn S. Transcription of nearly all yeast RNA polymerase II-transcribed genes is dependent on transcription factor TFIID. Mol Cell. 2017;68(1):118–29.e5.
Basehoar AD, Zanton SJ, Pugh BF. Identification and distinct regulation of yeast TATA box-containing genes. Cell. 2004;116(5):699–709.
Feigerle JT, Weil PA. The C terminus of the RNA polymerase II transcription factor IID (TFIID) subunit Taf2 mediates stable association of subunit Taf14 into the yeast TFIID complex. J Biol Chem. 2016;291(43):22721–40.
Dutta A, Sardiu M, Gogol M, Gilmore J, Zhang D, Florens L, et al. Composition and function of mutant Swi/Snf complexes. Cell Rep. 2017;18(9):2124–34.
Sen P, Luo J, Hada A, Hailu SG, Dechassa ML, Persinger J, et al. Loss of Snf5 induces formation of an aberrant SWI/SNF complex. Cell Rep. 2017;18(9):2135–47.
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Martin BJ, McBurney KL, Maltby VE, Jensen KN, Brind'Amour J, Howe LJ. Histone H3K4 and H3K36 methylation independently recruit the NuA3 histone acetyltransferase in Saccharomyces cerevisiae. Genetics. 2017;205(3):1113–23.
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Shen X, Mizuguchi G, Hamiche A, Wu C. A chromatin remodelling complex involved in transcription and DNA processing. Nature. 2000;406(6795):541–4.
Andrews FH, Shanle EK, Strahl BD, Kutateladze TG. The essential role of acetyllysine binding by the YEATS domain in transcriptional regulation. Transcription. 2016;7(1):14–20.
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Kornberg RD. The molecular basis of eukaryotic transcription. Proc Natl Acad Sci USA. 2007;104(32):12955–61.
Sanders SL, Garbett KA, Weil PA. Molecular characterization of Saccharomyces cerevisiae TFIID. Mol Cell Biol. 2002;22(16):6000–13.
Kolesnikova O, Ben-Shem A, Luo J, Ranish J, Schultz P, Papai G. Molecular structure of promoter-bound yeast TFIID. Nat Commun. 2018;9(1):4666.
Kimura M, Ishihama A. Tfg3, a subunit of the general transcription factor TFIIF in Schizosaccharomyces pombe, functions under stress conditions. Nucleic Acids Res. 2004;32(22):6706–15.
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Haruki H, Nishikawa J, Laemmli UK. The anchor-away technique: rapid, conditional establishment of yeast mutant phenotypes. Mol Cell. 2008;31(6):925–32.
Zhang T, Lei J, Yang H, Xu K, Wang R, Zhang Z. An improved method for whole protein extraction from yeast Saccharomyces cerevisiae. Yeast. 2011;28(11):795–8.
Kristjuhan A, Walker J, Suka N, Grunstein M, Roberts D, Cairns BR, et al. Transcriptional inhibition of genes with severe histone h3 hypoacetylation in the coding region. Mol Cell. 2002;10(4):925–33.
We thank Dr. Signe Värv and Dr. Tõnis Org for critical reading and helpful discussion of the manuscript.
This work was supported by the Estonian Research Council research Grants IUT20-23 and PRG757 to AK.
Johanna Luige
Present address: Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark
Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010, Tartu, Estonia
Kadri Peil, Henel Jürgens, Johanna Luige, Kersti Kristjuhan & Arnold Kristjuhan
Kadri Peil
Henel Jürgens
Kersti Kristjuhan
Arnold Kristjuhan
KP—designed the study, performed experiments, analyzed data, and wrote the initial manuscript draft. HJ—performed experiments. JL—performed experiments. KK—performed experiments. AK—designed the study, analyzed data, and wrote the final manuscript. All authors discussed the final manuscript. All authors read and approved the final manuscript.
Correspondence to Arnold Kristjuhan.
Additional file 1: Figure S1
. Deletion of TAF14 is lethal in yaf9Δ, rpb4Δ and rpb9Δ cells in W303 background, but functional C-terminal domain of Taf14 rescues rpb4Δtaf14Δ and rpb9Δtaf14Δ lethality. Tetrad analysis following sporulation of AKY1820+AKY1916 (A), AKY1786+AKY1818 (B), AKY718+AKY1818 (C), AKY1850+1938 (D) and AKY719+AKY1850 (E) yeast strains. The tetrads were dissected on YPD medium and plates photographed after 4 days of growth at 30 °C. Figure S2. Expression of C-terminal domain of Taf14 rescues yaf9Δtaf14Δ double-mutant from lethality. Tenfold serial dilutions of cells, where TAF14 in its genomic locus is replaced with gene encoding YEATS-deleted Taf14 protein (taf14ΔYEATS) and combined with YAF9 and SAS5 deletions, were spotted onto SC plates and grown at 30 °C for 2 days. Figure S3. Western blot analysis of Taf14 (A) and Rpb3 (B) in response to Rpb9 depletion. Rpb9 anchor-away strains with wt Taf14 or mutant taf14W81A were incubated with DMSO (+Rpb9) or rapamycin (−Rpb9) for 2 h. Taf14 was detected with anti-Taf14 antibody, RNAPII Rpb3 subunit was C-terminally tagged with E2-tag and detected with 5E11 antibody. A sample from taf14Δ strain expressing Rpb3 without E2-tag was used as a negative control (N). Table S1. Yeast strains.
Peil, K., Jürgens, H., Luige, J. et al. Taf14 is required for the stabilization of transcription pre-initiation complex in Saccharomyces cerevisiae. Epigenetics & Chromatin 13, 24 (2020). https://doi.org/10.1186/s13072-020-00347-7
Taf14
Pre-initiation complex (PIC)
TFIIF
TFIID


text:
VHS and Videos
The Sbuirrels #5 - "Crime" Talkback
The Sbuirrels #5 - "Crime"
Original Release Date: December 15, 2017
This issue is another Deadfall-inspired one; it's based on a scene where Eddie (Nicolas Cage) and his girlfriend try to do a con in a bar, and do so poorly. The opening scene is based upon a now-lost YouTube video (from c. 2011, basically a shitpost, even though they weren't call that then). It was a series of such videos by someone featuring Nic Cage scenes from Deadfall. I can't recall the original creator at the moment, but I do remember his first name being Travis.
Of all the off-model artwork in the early comics, nothing irritates me more than Mary's hideously damaged arm in the last panel of page 5. But on the bright side, I guess, the first panel of the same page does look pretty fantastic.
The Sbuirrels #2 - "Ed's Card Trick" Talkback
The Sbuirrels #3 - "The Chip" Talkback
The Sbuirrels #4 - "Halloween" Talkback

redpajama_set_name: RedPajamaCommonCrawl


Robert Half Legal is seeking a Pre-Litigation (not Litigation) Case Manager/ Paralegal for our client, a well-known law firm in Phoenix with offices in the GLENDALE and ARROWHEAD areas.
You must have 3+ years of solid Personal Injury Pre-Litigation experience including recent Arizona experience.
Salary of $40-48K DOE + bonus potential + benefits package.


conneXion Dubai was another great success. See why.
Consumer behavior is changing fast, and we have to be nimble and ready to react to change if we want to stay ahead of the game. This was the conclusion for the over 400 retailers, hospitality operators and software providers, who attended conneXion 2015 – the LS Retail and Hospitality conference and expo, which took place in Dubai at Madinat Jumeirah Resort on April 14-17, 2015.
Presenting software solutions that meet the needs of retail and hospitality businesses worldwide.
LS Retail's yearly event kicked off with a "readiness day", which presented a series of technical workshops and training sessions tailored on the interests of retailers and hospitality specialists. Participants had the chance to get a deeper understanding of all technical aspects of their chosen software solution, as well as discuss software- and industry-specific challenges and needs.
Panelists included IT specialists sharing software tips and tricks as well as retailers and restaurateurs discussing trends, issues and successes in their software implementations. Among the retailers and hospitality operators who shared best practices using LS Retail solutions were: Fabrizio Preti, CTO of A. S. Roma; Jonas Carlsson, Director Hotel IT Operation Services at Nordic Choice Hotels; Ashika Charan, Head of IT at Cost Club Ltd – Megamart ; Shubhojit Mahalanobis, Deputy General Manager at Danube Home; Sascha Voelz, Poresy Managing Director; George Mathew, IT Manager at Grandstores.
At the Expo guests got acquainted with the latest software solutions from big names in the field of retail and hospitality business IT. The two Gold sponsors of conneXion 2015, Microsoft and Hewlett-Packard (HP), showcased their products at their booths in the expo. Other exhibitors were: Toshiba, AGR, Cenium, SQL Perform, DK Online, Mergetool, SaaSPlaza, Godrej, aswaaq, Powa, Datavan, 1Click Factory.
conneXion 2015 closed on April 16th with a formal dinner and awards ceremony at the Armani Hotel in Dubai honoring the LS Retail's top-performing partner companies.
We also want to thank all participants, speakers, sponsors, exhibitors and staff members that contributed in making conneXion 2015 a great success. Without your active participation, we could not have made this such a huge accomplishment!
It was a great honor receiving you all and we hope you were inspired, and returned home with a compass, a roadmap and a strategy on how you are going to lead your business forward to greater success.


text:
The Original Picnic at Hanging Rock Is the Dreamiest Nightmare Ever
Filed to: Retro ReviewFiled to: Retro Review
Peter Weir
Miranda, transfixed by the mountain.
Image: Criterion Collection
Starting this Friday, May 25, Amazon will stream a six-episode series based on Australian author Joan Lindsay's 1967 classic novel Picnic at Hanging Rock. But no new adaptation will ever replace Peter Weir's 1975 film version—still one of cinema's most gorgeous and eerie mysteries.
It opens with a title card letting you know exactly where you are and what you're about to see: It's Valentine's Day, circa 1900, and a group of Australian schoolgirls are about to visit the titular rocky mountain... where several of them will "disappear without a trace." That's a lot of exposition to go into the movie with, but Picnic at Hanging Rock is less concerned about its plot, and much more interested in its imagery and tone. Of course, you do get a huge clue about what to expect there, too, thanks to the film's opening line, a slightly bastardized recitation of one of Edgar Allen Poe's most famous poems: "All that we see or seem/is but a dream within a dream." It frames the movie within its own reality, with all the logic of a dream and more than a few night terrors.
The young women of Appleyard College are introduced as a giddy group, but a few students stand out—mainly the intuitive, ethereal beauty Miranda, but also Sara, the wide-eyed orphan who adores her, and chubby tagalong Edith. Sara's being punished, so she's excluded from the excursion to Hanging Rock, but the rest of the girls leave their paper Valentines behind and head out, excited when their headmistress, the stern Mrs. Appleyard, allows them the impropriety of removing their gloves once they've passed through the nearby town. They're unworried, however, about Mrs. Appleyard's fraught warnings about the dangerous ants and snakes they're liable to encounter on their field trip. Still, a sense of malevolence emanates from the natural world. Once the party arrives at Hanging Rock—an ancient, untamed place that juts up from the ground like a row of teeth—pocket watches stop, and time seems to stop, too, as four of the girls, led by Miranda, break away from the group and go exploring.
Watch out for poisonous snakes, though.
These sequences are filmed with a golden hue and gauzy lighting, and the dreaminess is enhanced by a score that leans heavily on the talents of pan flutist Zamfir. (Also adding to the movie's general out-of-body-ness: The fact that several of the girls' voices were later dubbed in by more experienced actresses.) The foursome is spotted from a distance by a pair of young men who are understandably startled to see schoolgirls wearing prim, frilly white dresses hiking through the forest: rich Englishman Michael, who's instantly mesmerized by Miranda, and local kid Albert (notably, played by a young John Jarratt, who's now best-known for his terrifying turn as the psycho in the popular Aussie horror series Wolf Creek).
As the boys stare in awe, the girls move through the forest and begin climbing up the rock formation. There's a fairy-tale quality to their journey as they ascend, and then they start feeling dizzy from the sun and probably also the rock's strange powers. Something compels three of the girls to remove their stockings and shoes (gasp!) and march into a rocky crevasse. Lone holdout Edith gapes in shock before screaming her way back down the mountain, passing a teacher who's mysteriously drawn in the same direction as the girls, and who vanishes along with them.
The saga of missing children—particularly girls, and particularly pretty young white girls—has become an incredibly common theme in crime dramas nowadays, and while Picnic at Hanging Rock hits some of the expected beats (detectives, a search party, rampant rumors and gossip), its central mystery reverberates on a more cosmic level than most police procedurals. There's a repeated line, "Everything begins and ends at exactly the right time and place," and the sense of borders being crossed also repeats—be they between girlhood and womanhood, between teachers and students, between Australia's colonial settlers and the unfamiliar wilderness that surrounds them, or even between life and death. It's a constant theme that goes back to that Poe quote. Is this real, or are we in a dream... or a dream within a dream?
The volcano is dormant, but Hanging Rock is still full of secrets.
While one of the girls is eventually recovered (she remembers nothing about her experience, naturally), the other three missing women, including the luminous Miranda, remain lost. In the emotional aftermath, Mrs. Appleyard's flinty Victorian facade begins to crumble, and as she grows more unhinged, her cruel treatment of Sara escalates to the point of psychological torture. Before the end, both women die mysteriously—their already-troubled relationship pushed to deadly extremes by the Valentine's Day tragedy.
But the biggest takeaway from Picnic at Hanging Rock is realizing we'll never know what actually happened on the mountain—did the women just fall into a hole and die? Were they absorbed by the elements? Did they transform into swans? Are they still out there... somewhere?—and yet still being totally satisfied by the darkly disturbing yet intensely beautiful puzzle the movie leaves behind.
Australian Cult Classic Picnic at Hanging Rock Is Getting a Miniseries Remake
10 Australian Scifi Movies You Need to Watch
The 30 Weirdest Horror Movies of the 1970s
10 Obscure Cult Horror Movies Everyone Should Watch (and Re-Watch)
The Best Scifi Movies to Watch When You're Seriously Stoned
10 Banned, Censored, and Controversial Movies That Are Now Cult Classics
io9 News Editor


James Madison, the fourth President of the United States, lived at Montpelier plantation until he died in 1836. In 1901, the DuPont family acquired the property which they owned until 1985. In 1910 William DuPont financed the construction of the train station to provide both freight and passenger service to the Montpelier community. Passenger service was discontinued in the 1960s and the freight depot was closed in 1974. Today Norfolk-Southern freight trains pass by the depot, but rail service to Montpelier is a thing of the past. The station was built to a Southern Railway standard architectural plan.
Montpelier is now owned by the Montpelier Foundation and the station, has been restored. Part of the station houses a small museum and one end serves as the U.S. Post Office for Montpelier Station, Virginia. The active Norfolk-Southern tracks are just out of view on the right of the picture above.
Also on the grounds is a small freight house. There was once a siding that ran in front of the building and ended in a coal loading trestle just beyond. Montpelier Station is located in Orange County, Virginia. All photos were taken in December, 2015.


Ala Carte operates an on-demand, delivery-only virtual food hall. Its fulfillment centers pre-pare, package and ship 16 dif-ferent brands, all from the same location. Want fried chicken butyour partner wants a burrito? Ordering thruAla Carte means one easy order conveniently delivered to you via their partnerships with Post-mates, Uber Eats, Yelp and more.


While we are sure your cat is really awesome, we ask you to leave him at home. All of our cats arrive with a clean bill of health from MEOW Foundation and are socialized to the environment for the comfort of both the kitties and our guests.
What if I'm allergic to cats?
Not a problem! We have a full-service café with lots of comfortable seating and a great view of the Kitty Kingdom. The two areas have separate ventilation for your comfort. No kitties are ever transferred through the cafe, so you should not have any allergy issues in our cafe.
Why is there a fee to visit the kitties?
While our primary goal is to provide you with a unique environment to fall in love with your next 'fur baby', we do charge a nominal fee to help pay for the beautiful space and care for the cats.
How do I adopt one of the resident kitties?
We work closely with MEOW Foundation to ensure our kitties find the perfect home. Adoption applications are available online at http://www.meowfoundation.com/adopt/application-form/. Just fill out the general application, and then send an email to adopt@meowfoundation.com letting them know which kitty you have fallen in love with. A MEOW Foundation adoption specialist will aim to contact you within one business day, and adoptions are on a first come first serve basis after meeting the cats.
We would love to help you celebrate your PURRthday! If you bring a group of 10 or more, the birthday person will receive a complimentary kitty visit. Reservations can be booked online for up to 12 guests. If you would like to book up to 16 guests (the full room), please make a note of it in your reservation comments. We also have delicious cupcakes that are PURRfect for a party and can special order some for your group with a few days notice. We kindly ask that guests not bring in outside food or drinks. We do ask birthday parties with young guests have one adult for every 3-4 children present in the Kitty Kingdom with them. Please note that we are unable to guarantee a table in the cafe as reservations are for the Kitty Kingdom only, but often tables become available once the next group of guests goes in to the Kitty Kingdom.
Looking for the PURRfect event for your bachelorette party, team building, or milestone celebration? We can offer semi-private events that will be sure to delight all your furriends.
Kitty Visit - Book the whole Kitty Kingdom for you and your guests for a private visit with the kitties (up to 16 guests) $150/45 minutes.
Cats & Yoga - Morning or evening yoga in the Kitty Kingdom with our aMEOWzing yoga instructor. $250 for up to 10 guests. Guests must be 18+.
Includes a one-hour gentle hatha yoga session, a quick kitty visit, and a complimentary small brewed coffee or loose-leaf tea for each guest.
Kitty Paint Parti in Partnership with Raw Canvas YYC - $45/person for a two-hour painting session. Includes all instruction, supplies, and a quick kitty visit during the break.
Minimum 10 guests, and YOU can choose the painting!
Is the Cafe and Kitty Kingdom wheelchair accessible?
Absolutely! We are fully wheelchair accessible in our Cafe and Kitty Kingdom. It was our goal to create a space that all our furriends could enjoy.
Why do we only partner with MEOW Foundation?
We understand that there are many different rescues throughout Calgary, and we have selected MEOW Foundation as they are a cat focused no-kill foundation dedicated to improving the lives of Calgary cats. By focusing on a single partnership, we are able to manage the health, care, intake, and adoption process smoothly. Our hope is to make a meaningful impact on the homeless cat population, and ultimately make a PAWsitive impact for all Calgary cats.
Can I surrender my cat to Regal Cat Cafe?
Unfortunately, we are not able to take in rescue cats here at Regal Cat Cafe. All our kitties arrive in tip-top health from MEOW Foundation and introduction of an outside cat would put them at risk. We recommend reaching out to the local Calgary rescues to see who is able to accept your kitty for surrender.
Why are the cats separate from the cafe?
We have worked closely with Alberta Health and Safety to design a space that is safe and healthy for our guests. This is why we have a completely separate area called the Kitty Kingdom for our cats to relax and play. We also have special doors for intake and adoptions so no kitty will go through our cafe at any point. We strive to have the cleanest and healthiest cafe for our guests, and this helps ensure that all food and drink is free from kitty hair.


text:
Rhene daitarensis est une espèce d'araignées aranéomorphes de la famille des Salticidae.

Distribution
Cette espèce est endémique d'Odisha en Inde.

Étymologie
Son nom d'espèce, composé de daitar[i] et du suffixe latin , « qui vit dans, qui habite », lui a été donné en référence au lieu de sa découverte, Daitari.

Publication originale
 Prószyński, 1992 : Salticidae (Araneae) of India in the collection of the Hungarian National Natural History Museum in Budapest. Annales zoologici, Warszawa, , .

Liens externes

Notes et références

Salticidae
Espèce d'araignées (nom scientifique)
Faune endémique d'Odisha

redpajama_set_name: RedPajamaWikipedia


text:
using JumpAndRun.Content.Blocks;
using Microsoft.Xna.Framework;
using System;
using SkiaSharp;

namespace JumpAndRun.Content.BlockDefinitions
{
    public class SandBlockDefinition : BlockDefinition
    {
        public SandBlockDefinition()
        {
            _textures = new[] { GameLib.Helper.GetBitmapFromPath("sand.png") };
        }

        public override Type GetBlockType => typeof(SandBlock);

        public override BoundingBox[] GetBoundingBoxes()
        {
            return new[] { new BoundingBox(new Vector3(0, 0, 0), new Vector3(1, 1, 1)) };
        }

        public override string Name => "JumpAndRun.SandBlockDefinition";
    }
}

redpajama_set_name: RedPajamaGithub


text:
Air photograph of Tell Abu al-Kharaz (state October 2014)
Storage jars, bowl and jug in situ from Area 9, Phase IX
The Swedish excavations, directed by Peter M. Fischer from the University of Gothenburg, Sweden, have been carried out at Tell Abu al-Kharaz since 1989. The excavations are financed by the Royal Swedish Academy of Letters, History and Antiquities since 2008.
Tell Abu al-Kharaz is a 12 ha tell in the central Jordan Valley. The current objective is to shed further light on the Iron Age occupation of this city, first settled around 3200 BCE, which corresponds to the conventional Early Bronze Age IB. The Iron Age occupation lasted from the 12th century BCE until 732 BCE, when the city was conquered by the Neo-Assyrians. From 2009 to 2012 excavations in Area 9 revealed an exceptionally well-preserved two-storey compound dating from Iron Age I (local Phase IX), that is around 1100/1050 BCE. The stone compound was exposed for a length of 46 m (not counting the western annex of additional 20 m) and consists of 21 rooms, with walls still standing to a height of more than 2 m. Several hundred complete vessels and other objects point to the extensive contacts of a fairly rich society. Direct and indirect contacts with Phoenicia, the Aegean and Cyprus were ascertained. At the end of the 2012 season of excavation the eastern limit of the compound was reached. In 2013 and 2014 complementary excavations were carried out to the north and east of the compound and – for the first time – in central parts of the tell. The eastern extension revealed a defence system, which was originally built in the Early Bronze Age IB/II around 3100 BCE, but which was reused as a part of the Iron Age I defence structures. Trenches in hitherto unexplored areas of the city in the centre and along the city wall attested remains dating mainly from the Late Bronze and the early and late Iron Ages. Due to publication obligations, the excavations rest at present. Resumed excavations are planned in the near future.
Phasing of Tell Abu al-Kharaz
Duration BC
EB IB
IIA–B
EB II
IIIA–B
EB III–MB II/III
IV/1
MB III
MB/LB
LB IA
LB IB
LB IB/C–IC
LB IC–II
LB II
IA IB
1050?–930
IA IB/(IIA)
IA IIA
IA IIA/B
IA IIB
IA IIC
P. M. Fischer and T. Bürge, Tall Abu al-Kharaz. American Journal of Archaeology 120:4, 2016, 640–641.
P. M. Fischer and T. Bürge, The Swedish Jordan Expedition 2014 at Tall Abu al-Kharaz: Preliminary Results from Areas 12 and 13. Opuscula 8, 2015, 157–179.
P. M. Fischer and T. Bürge, The Swedish Jordan Expedition 2013 at Tall Abu al-Kharaz: Preliminary Results from Areas 9, 10 and 11. Opuscula 7, 2014, 129–148.
P. M. Fischer, Primary Early Bronze Age Contexts from Tall Abu al-Kharaz, Jordan Valley, in: F. Höflmayer and R. Eichmann (eds.), Egypt and the Southern Levant in the Early Bronze Age, Orient-Archäologie 31, Rahden 2014, 19–56.
P. M. Fischer, Tell Abu al-Kharaz in the Jordan Valley. Volume III: The Iron Age (Contributions to the Chronology of the Eastern Mediterranean 34) Vienna 2013.
P. M. Fischer, Tell Abu al-Kharaz in the Jordan Valley. Volume I: The Early Bronze Age (Contributions to the Chronology of the Eastern Mediterranean 16) Vienna 2008.
P. M. Fischer, Tell Abu al-Kharaz in the Jordan Valley. Volume II: The Middle and Late Bronze Ages (Contributions to the Chronology of the Eastern Mediterranean 11) Vienna 2006.
P. M. Fischer (ed.), The Chronology of the Jordan Valley during the Middle and Late Bronze Ages: Pella, Tell Abu al-Kharaz and Tell Deir Alla (Contributions to the Chronology of the Eastern Mediterranean 12) Vienna 2006.


text:
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 Upholstered & Leather Sofa Dry, Steam Cleaning Heston Central TW3 | 1st Carpet Cleaning Ltd.
We also provide and other services in nearby areas including: Upholstery Cleaning Heston West, Upholstery Cleaning Heston East, Upholstery Cleaning Heston, Upholstery Cleaning Heston, Upholstery Cleaning Norwood Green, Upholstery Cleaning Cranford, Upholstery Cleaning Cranford, Upholstery Cleaning Southall, Upholstery Cleaning Southall, Upholstery Cleaning Hayes, Upholstery Cleaning Whitton, Upholstery Cleaning Syon, Upholstery Cleaning Osterley, Upholstery Cleaning Isleworth, Upholstery Cleaning Elthorne.
Suitable for hotels, hostels, hospitals and everything that has beds, Mattress Cleaning + Bed Bug Mattress is an offer you should definitely add to upholstery cleaning service Heston Central TW3. You will get a discount for the second service. Domestic mattress cleaners destroy and remove dust mites, fungal spores, bacteria and other harmful debris. The procedure works with intensity ultra-violet light and eliminates dirt and debris. This is the best way for you to protect your family from dust allergies, asthma, runny nose, depression /not 100% scientific evidence/, headaches, eczema, itchy red eyes.
Dear customers, if you have an expensive or luxurious upholstery, don`t forget to mention it to our helpful and polite consultants. For such individual cases, the diligent and enthusiastic sofa cleaners in Heston Central TW3 can execute leather and suede cleaning. Leather cleaning utilizes foam leather cleaner, leather rejuvenator, crack filler with different colors and mat leather sanitizing technique. Suede sofa applies delicate dry cleaning treatment and can be suitable for velvet, suede and other delicate materials!
Once you come back home, you will see your sofa 100% pure and as clean as a whistle! We recommend steam cleaning for all synthetic fibres and special dry cleaning technique for delicate materials. We clean the following fabrics: synthetic, wool, cotton, linen, silk, viscose, organic fibre, suede, plush, chenille, nubuck, leather, acrylic. Hovering and deodorizing, as well as sofa stain removal are included.
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Usually what bothers upholstery owners is the availability of organic, hard, stubborn or old stains. We always send the hardworking and experienced upholstery cleaners in Heston Central TW3 for a preliminary examine of sofa. Once we identify stains, we consider the type of the material and the source of the filth to offer the most effective risk-free solution. We clean the following types of upholstery: couch or settee, Traditional, Convertible sofa, Loveseat, Divan, Settee, Sectional, Sofa bed, Hide-a-bed, Couch bed, Sleeper sofa. Note that stain treatment method is 100% secure and non chemical. We employ only kid-friendly and natural remedies.
Rather of losing time in unnecessary efforts or money for suspicious sofa cleaning detergents, better lean on the trusted professional sofa cleaning company in Heston Central TW3! We offer you a fantastic deal, wonderful final outcome and great client support services 24/7.
The nearest tube station serving TW3 Heston Centralis Hounslow West.


text:
The 11 Best Apple Orchards Near NYC
If you don't pick some apples, it's not really Autumn.
By Meg Donohue and Katie Robinson
S847Getty Images
As autumn approaches, dreams of apple crisps and colorful leaves are already taking ahold. While autumn in New York City is undoubtedly a sight to see, it's also a great time to get out of the city, if only for an afternoon, and stroll through a bucolic orchard to pick your own apples–truly a classic fall activity. Fret not, the perfect autumnal escape is never far from Manhattan. The Hudson Valley, Connecticut, and New Jersey all boast scenic spots to go apple picking within driving distance. Below, find some of the best farms and orchards to visit this fall.
1 Love Apple Farm
Love Apple Farm
Located in the Hudson Valley, Love Apple Farm has all the makings for a perfect autumn outing: cider donuts, a farm animal petting zoo, an art exhibition, and of course, apple picking. Plus, you can't beat the 99 cent per pound deal on your apple bags.
FROM NYC: About 2 hours, 12 minutes (125 miles) from NYC by car.
1421 State Route 9H, Ghent, NY 12075; (518) 828-5048
More: The Best Places to Go Fruit Picking in NYC
2 Terhune Orchards
A post shared by Terhune Orchards (@terhuneorchards) on Oct 3, 2017 at 5:32pm PDT
Looking for some variety in your fruit bowl? Terhune Orchards has 34 types of apples. They also have pears, peaches, corn, herbs, flowers, and more, so you can fully stock up. The orchard is especially well-suited for families; they have dwarf apple trees that make picking easy for even the smallest farm-goers. Afterwards, drop off the kids at the corn maze or the petting zoo and cozy up at the onsite winery.
From NYC: About 2 hours, 35 minutes by public transport. Take an NJ Transit train from Penn Station to Princeton JCT, then cab to the farm. Or, about 1 hour, 35 minutes by car.
330 Cold Soil Road, Princeton, NJ 08540; (609) 924-2310
3 Alstede Farms
A post shared by Alstede Farms (@alstedefarms) on Sep 23, 2017 at 5:18am PDT
You can get more than just your fill of apples at Alstede Farms. They grow pumpkins, beans, peppers, raspberries, and sunflowers available too. Check out their handy guide detailing what produce is available and how much each costs. Open daily, the farm has hayrides, a petting zoo, farmer's market, and fresh homemade goodies like ice cream and pie.
FROM NYC: Take the NJ Transit train from Penn Station to Gladstone Station. From there, you'll be able to hop aboard their free shuttle to the farm. Or, about 1 hour, 8 minutes by car.
1 Alstede Farms Lane, Chester, NJ 07930; (908) 879-7189
4 Applewood Orchard & Winery
A post shared by Applewood Winery (@applewoodwinery) on May 26, 2017 at 9:36am PDT
Let's be real, apple picking takes about 25 minutes tops. To make your journey outside New York worthwhile, come to Applewood and enjoy the onsite winery's homemade wine and award-winning cider, which is made with fresh honey instead of refined sugars. Translation: no sugar-induced headache if you happen to overindulge.
FROM NYC: Take the 197 Port Authority bus line to Warwick Park & Ride (about 1 hour, 50 minutes). Call a local taxi service to take you the rest of the 4 mile leg.
82 Four Corners Road, Warwick, NY 10990; (845) 986-1684
5 Hillview Farms
Rosmarie WirzGetty Images
This family-owned 53-acre farm has been growing delicious fresh produce for 150 years. Apple picking season begins in September and continues through October, when pumpkins are also available. Make sure to stop at Hillview's Farm Market, where you can buy freshly made honey, rhubarb, cider, pies, fresh free-range eggs, and much more.
FROM NYC: About 48 minutes by car.
223 Meyersville Rd., Gillette, NJ 07933; (908) 647-0957
6 Wilkens Fruit & Fir Farm
Apple picking simply isn't enough for Wilkens. The farm also offers peach picking and pumpkin picking in the fall, and a Cut-Your-Own Christmas tree farm starting the Saturday after Thanksgiving. Wilkens also boasts three different markets where you can get your fill of all apple-related pastries, and wine at their onsite winery.
FROM NYC: Take the Metro-North (Hudson Line) train to Croton-Harmon or Peekskill station (about one hour), then taxi 15 minutes to the farm.
1335 White Hill Rd, Yorktown Heights, NY 10598; (914) 245-5111
7 Stuart's Fruit Farm
A post shared by Charles Pritchard (@charliegriffon) on Sep 15, 2017 at 10:48am PDT
Stuart's Farm has been a Westchester County staple since 1828. Apple picking season begins September 6th and is open daily through November. Take in the stunning fall foliage, enjoy a tractor ride through the orchards, and wait in line at the bakery for those pies and cider donuts (they're well worth it). Stop by the farm store for other fresh produce, flowers, pumpkins, and more.
From NYC: Take Metro-North to the Croton Falls station and cab the rest of the way.
62 Granite Springs Road, Granite Springs, NY 10527; (914) 245-2784
8 Sun High Orchard
A post shared by Mimi O'Connor (@themimioconnor) on Oct 1, 2017 at 6:50pm PDT
For a full farm experience complete with much more than just produce, head to Sun High Orchard. Kids will love all the petting zoo animals, including sheep, rabbits, a donkey, and even an alpaca. After you've visited all the furry friends, hit the fields to pick apples, peaches, flowers, zucchini, and tomatoes.
From NYC: About one hour by car from Manhattan.
19 Canfield Ave, Randolph, NJ 07869; (973) 584-4734
9 Fishkill Farms
A post shared by Fishkill Farms (@fishkillfarms) on Nov 8, 2018 at 9:54am PST
Owned by the same family for over 100 years, Fishkill Farms is known for their sustainable and ethical farming practices. But beyond that, they also boast a 270-acre orchard with breathtaking views of the Catskill mountains. During peak apple picking season in mid-October, they host weekend Harvest Festivals with music, hayrides, cider, and donuts. It's an autumn dream come true
From NYC: Around two hours by train on the Hudson line from Grand Central Station.
9 Fishkill Farm Road, Hopewell Junction, New York 12533
10 Greig Farm
A post shared by Greig Farm (@greig_farm) on Aug 21, 2019 at 2:54pm PDT
Get active with your apple-picking! Greig Farm has walking trails winding throughout 3.5 miles of orchards and pastures, so you can get those steps (and views) in. At Grieg, there's also a community garden, shop, and a farmhouse with two bedrooms that can be booked through AirBnB. Why not make it a weekend getaway
From NYC: Two hours by car from Manhattan.
227 Pitcher Lane, Red Hook, NY, (845) 758-8007
11 Soons Orchards
A post shared by Soons Orchards (@soonsorchards)
Soons Orchards is a one-stop shop. U-pick season reopens Labor Day weekend and continues until late October. Farm-goers can pick from more than 50 varieties of apples (including one that is unique to their orchards!) and 20 different types of peaches and nectarines. Check their detailed schedule to find out when certain varieties are available. Before heading out, be sure to hit the store, as well as the Tap Room at Orchard Hill Cider Mill, which serves lunch, dinner, and weekend brunch. Cider donut bread pudding, anyone?
From NYC: About 1 hour, 19 minutes by car from Manhattan.
23 Soons Circle, New Hampton, NY; (845) 758-8007
More From Everything You Need for Fall
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Fall Scarves for Every Style
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Tasty Apple Cider Cocktails to Make This Fall
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The 7 Best Places to See Fall Foliage in New England
11 Classic Fall Films to Watch Now
11 Elegant Ways To Decorate With Pumpkins This Fall
10 Real-Life Haunted Hotels in the U.S. You Can Visit
12 Of The Best Quotes About Autumn
The Best Places to Visit in October 2019


text:
Can You Get HIV From Having Sex With Someone Who Has AIDS?
HIV/AIDS also can pass from mother to child during pregnancy, childbirth, or breastfeeding.
To reduce your risk of getting HIV/AIDS if you are sexually active:
Use a condom every time you have sex (including vaginal, oral, or anal sex).
Get tested for HIV and make sure all partners do too.
Have fewer sexual partners.
Get tested and treated for STDs (sexually transmitted diseases); having an STD increases the risk of HIV infection.
Consider taking a medicine every day (called PrEP or pre-exposure prophylaxis) if you are at very high risk of getting infected (for example, if you are in a sexual relationship with someone who has HIV/AIDS).
It's also important to:
not inject drugs or share any kind of needle
not share razors or other personal objects that may touch blood
not touch anyone else's blood from a cut or sore
*Names have been changed to protect user privacy.
Reviewed by: Robyn R. Miller, MD
Date reviewed: October 2018
STDs (Sexually Transmitted Diseases)
How to Get Tested for HIV
How Do People Get AIDS?
Talking to Your Partner About Condoms
© 1995- The Nemours Foundation. All rights reserved.


text:
Jack Haley, former UCLA and Lakers center, dies…
Jack Haley, former UCLA and Lakers center, dies at 51
Former UCLA and Lakers center Jack Haley, seen at an NBA playoff game between the Lakers and Phoenix Suns in 2006, died Monday at the age of 51. (Stephen Shugerman/Getty Images)
By Jack Wang | jwang@scng.com | Daily News
PUBLISHED: March 17, 2015 at 6:12 p.m. | UPDATED: August 28, 2017 at 7:08 a.m.
Former UCLA and Lakers center Jack Haley, who parlayed his size into a nine-year NBA career, died Monday at the age of 51.
A statement released by the family through the Lakers said Haley died as a result of heart disease. The Orange County Coroner Division of the Orange County Sheriff's website said Haley, of Seal Beach, died at Los Alamitos Medical Center.
"It is with great sadness that the Haley family announces the passing of our beloved father, son, and brother," the Haley family said in the statement. "Our hearts are broken by this sudden, unexpected loss. Jack was honored and grateful for the opportunity to play in the NBA for nine years, alongside world-class athletes with the Los Angeles Lakers, Chicago Bulls, San Antonio Spurs and New Jersey Nets. He cherished every moment and gave 100% whether on or off the court. And more than anything, Jack dearly loved his family. We are humbled and grateful for the outpouring of love, support, and prayers from around the country."
• PHOTOS: Former UCLA and Lakers center Jack Haley dies at 51
Born in Long Beach and an alumnus of Huntington Beach High, the 6-foot-10 big man had a decade-long professional career in the United States and abroad, bouncing everywhere from the Chicago Bulls to the Los Angeles Lakers to AEK Athens to the LaCrosse Bobcats of the Continental Basketball Association.
In the NBA, he played for four teams, spending two stints with the Bulls and the New Jersey Nets as well as two seasons with the San Antonio Spurs.
In his lone season with the Lakers in 1991-92, he scored 76 points and grabbed 95 rebounds in 49 appearances.
"Jack was a hard worker and always very professional," Lakers general manger Mitch Kupchak said in a statement. "He was proud to wear the Lakers uniform, and he was always a credit to our organization and the Lakers family. Our condolences go out to his family and friends at this time."
Haley was a part of the Bulls' 1995-96 NBA title run, although he famously missed all but seven minutes of the season due to tendinitis in his left knee. He subbed in during Chicago's final regular-season game against Washington, and ended up with five points.
"Member of Bulls 95-96 team of ages," tweeted former Bull coach Phil Jackson, the first to publicly report the news of Haley's death. "RIP."
Former Chicago teammate Scottie Pippen also expressed his condolences on Twitter: "Very sorry to hear my old teammate, Jack Haley, has passed away. Great guy who I stayed in touch with over the years. RIP."
Haley also became known as somewhat of a caretaker for the enigmatic Dennis Rodman, although he disliked the label.
"This year, I specifically go out of my way not to speak to the press when they ask, 'Hey, can we talk to Dennis?' or 'Can you get an interview with Dennis?'" he told the Chicago Tribune in 1996. "That's not my job. Call Dennis Rodman, don't ask me."
Jack's father, Jack Haley Sr., a world class surfer who won the first U.S. Open of Surfing in 1959 at Huntington Beach, opened the popular Captain Jack's restaurant in Sunset Beach in 1965. The restaurant is still owned by the family.
Haley played at UCLA for three seasons (1984-85 to 1986-87) and won a college championship as part of UCLA's 1985 NIT title squad, which was honored at Pauley Pavilion in January. He was drafted by Chicago in the fourth round of the 1987 NBA Draft with the 79th overall pick.
After his playing career ended in 1998, Haley was an assistant in New Jersey and worked as a broadcaster on pregame shows for the Lakers on Fox Sports Net and Prime Ticket.
"Jack Haley, who played for me and was an assistant when I was coaching with the Nets, passed away too young," tweeted John Calipari, now the head coach at Kentucky.
Haley and his wife, Stacey, had two sons, Jack — a walk-on guard at UCLA in 2010 — and Jeff.
Jack Wang | reporter
Jack Wang covers the Chargers, the latest NFL team to relocate to Los Angeles. He previously covered the Rams, and also spent four years on the UCLA beat, a strange period in which the Bruins' football program often outpaced their basketball team. He is a proud graduate of UC Berkeley, where he spent most of his time in The Daily Californian offices in Eshleman Hall — a building that did not become earthquake-safe until after his time on campus.
jwang@scng.com
Follow Jack Wang @thejackwang


Over the course of the 1850s, the world was reshaped by technology, trade, mass migration, and war. The global economy expanded fivefold, millions of families emigrated to the ends of the earth to carve out new lives, and technology revolutionized communications, while steamships and railways cut across vast continents and oceans, shrinking the world and creating the first global age. In this fast-paced, kaleidoscopic narrative, Ben Wilson re-creates this time of explosive energy and dizzying change, when Great Britain—then at the peak of its power—attempted to determine the destinies of hundreds of millions of people. Along with a chronology, this book includes eight pages of color illustrations and numerous black and white images throughout.


-BASE | Want to participate in the 'Pioneers' climate innovation program?
Climate-KIC is an initiative by the EU to stimulate climate innovations.
Climate-KIC is an initiative by the EU to stimulate climate innovations. They have a special talent program called 'Pioneers', which might interest you.
Pioneers is Climate-KIC's professional mobility programme, developed especially for people who want to challenge themselves and explore their innovative ideas for climate change solutions. Pioneers put into practice their expertise to create new products and services in the field of the climate change, or to join an existing project and provide their insight.
During the programme, pioneers are offered a four-to-six week placement in one of the participating international regions, working on leading low-carbon projects with host organisations that are looking to collaborate on interesting projects or help you develop your own. Besides this international exchange, they work together in a group with Dutch pioneers to address a regional challenge, focusing on real-world challenges with tangible results! We prepare them for this exciting exchange with tailored mentoring and training, workshops and e-learning.
In 2017, Pioneers will be running in 15 European countries! Participating countries are: Cyprus, Estonia, Germany, Helsinki, Hungary, Italy, Latvia, Malta, the Netherlands (encompassing Belgium and Luxembourg as well), Poland, Romania, Serbia, Slovenia, Spain, and the United Kingdom.
· Excellent English, at least B2. Other languages are a plus, especially Spanish, Hungarian, German, Polish, and Italian!
· Have a University degree related to the field of climate change and/or sustainability OR other equivalent degree in another field plus at least two years of working experience in a field related to climate change and/or sustainability.
· Available and committed to attend the key dates of the programme.
Climate-KIC is the EU's main climate innovation initiative and brings together researchers and businesses across Europe to find solutions to climate change, creating sustainable growth and jobs in the process. Climate-KIC is one of the Knowledge and Innovation Communities (KICs) founded through the European Institute of Innovation and Technology (EIT), a body of the EU.


Total Posts: 0 We had a guy doing PSK-31, he got well over 100 contacts. It works quite well.
I did JT65 for a couple hours, it was tilting at windmills. I made a few contacts, but they didn't count. The problem is that there's no protocol for getting the state and category, and it's impossible to explain what you want 16 characters at a time.
Total Posts: 0 PSK-31 worked well for me last few FDs.I assume FT8 will be popular next year. If you reset the GRID location to reflex the class and section, it should be a work around the limited character issue.


Feb. 8, 2018 – Washington Co., WI – On Wednesday evening, Feb. 7 at 10:28 p.m. a concerned family member contacted the Washington County Sheriff's Office to report his father, an 85-year-old Town of Polk man, had left a friend's home in the City of West Bend and did not arrive home.
The man was last seen at 8:20 p.m. when he stopped and asked for directions at a Village of Jackson gas station. The family went out searching for the man and contacted authorities.
The sheriff's office made contact with relatives who responded to the scene and the gentleman was turned over to family.