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The POU homeodomain protein UNC-86 and the LIM homeodomain protein MEC-3 are essential for the differentiation of the six mechanoreceptor neurons in the nematode Caenorhabditis elegans. Previous studies have indicated that UNC-86 and MEC-3 bind cooperatively to at least three sites in the mec-3 promoter and synergistically activate transcription. However, the molecular details of the interactions of UNC-86 with MEC-3 and DNA have not been investigated so far. Here we used a yeast system to identify the functional domains in UNC-86 required for transcriptional activation and to characterize the interaction of UNC-86 with MEC-3 in vivo. Our results suggest that transcriptional activation is mediated by the amino terminus of UNC-86, whereas amino acids in the POU domain mediate DNA binding and interaction with MEC-3. By random mutagenesis, we identified mutations that only affect the DNA binding properties of UNC-86, as well as mutations that prevent coactivation by MEC-3. We demonstrated that both the POU-specific domain and the homeodomain of UNC-86, as well as DNA bases adjacent to the proposed UNC-86 binding site, are involved in the formation of a transcriptionally active complex with MEC-3. These data suggest that some residues involved in the contact of UNC-86 with MEC-3 also contribute to the interaction of the functionally nonrelated POU protein Oct-1 with Oca-B, whereas other positions have different roles.
POU domain transcription factors are characterized by their bipartite DNA binding domain, consisting of a helix-turn-helix POU-specific domain (POUS) and an adjacent POU homeodomain (POUHD) (for reviews, see references15 and 28). Both protein domains contact DNA and are necessary for high-affinity DNA binding (15). POU class IV is comprised of the mammalian Brn-3-encoding genes, the Drosophila I-POU/acj6gene, and the Caenorhabditis elegans unc-86 gene (28). These are all expressed exclusively in the nervous system, but their expression is not limited to one specific neuronal cell type. In vitro studies have shown that POU class IV proteins bind very similar DNA sequences (12, 24). Therefore, it has been proposed that any differences in target gene activation are due to modulatory protein interactions (28). Most of the data for determination of the promoter specificity of POU proteins are derived from the nonrelated human POU class III protein Oct-1 (13, 41).
POU homeobox gene unc-86 is expressed in 57 neurons in adultC. elegans. These neurons comprise one-fifth of the animal's nervous system and represent 27 different functional classes (10). Consequently, null alleles of unc-86 result in several behavioral defects affecting mechanosensation, egg laying, chemosensation, and thermosensation and cause an uncoordinated phenotype (5, 10, 18, 26, 37). The diversity of neuron types that require UNC-86 raises the possibility that in different cell types, UNC-86 targets the promoters of different genes. This is supported by the fact that in at least two unc-86 mutants, only a subset of the unc-86-mediated behaviors is affected (S. Röhrig and R. Baumeister, unpublished observations). How UNC-86 exerts these selective effects on gene regulation in several distinct neural cell types remains unknown. Interactions with various other proteins are one way to achieve selectivity. For UNC-86, the only binding partner known so far is the LIM homeodomain protein MEC-3 (39).
During the development of the nervous system, UNC-86 functions in 10 neuroblasts to determine their correct cell lineage and subsequently in the development and differentiation of the six mechanoreceptor cells involved in body touch sensing (9). In these cells, UNC-86 binds to at least three regulatory sequences, CS1, CS2, and CS3, in themec-3 promoter (35, 40, 42). A minimalmec-3 promoter that contains 311 bp including CS1, CS2, and CS3 is sufficient to direct reporter gene expression in the six mechanoreceptor cells. The LIM protein MEC-3 then cooperates with UNC-86 to maintain its own expression in a manner that is dependent on the presence of CS1-3 (38). Both proteins together regulate the expression of additional downstream genes (5, 8) that encode functionally crucial components of the mechanosensory neurons (39).
In order to investigate the DNA binding of UNC-86 and the protein interaction of UNC-86 with MEC-3, we developed an in vivo assay with the yeast Saccharomyces cerevisiae. For this purpose, we coexpressed unc-86 and mec-3 in the presence of a bona fide UNC-86 target site. We show here that upon binding to amec-3 promoter site, UNC-86 activates transcription in yeast. We localized the activation domain (AD) and showed that upon MEC-3 interaction, transcriptional activation is strongly enhanced. By mutational mapping of the contact surface, we identified amino acid residues in UNC-86 that specifically interfere with the synergistic coactivation by MEC-3. These results are discussed with respect to data about other POU complexes.
Yeast media and methods.All S. cerevisiaestrains were propagated by standard techniques. Yeast strains were grown at 30°C in liquid or on solid SC medium (synthetic medium) (30). For X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside)-containing plates, SC plates were supplemented with X-Gal at 400 μg/ml and buffered with 10× BU salt (0.26 M Na2HPO4, 0.25 M NaH2PO4). Transformants (11) were allowed to grow on SC media lacking the amino acids required for plasmid selection before further analysis. Plasmid isolation fromS. cerevisiae was performed as previously described (36). Liquid β-galactosidase assays using chlorophenolred–β-d-galactopyranoside (Roche) as a substrate were performed as previously described (7).
AD mapping.UNC-86–LexA fusions were generated by inserting the partial or full-length cDNA of unc-86 (3) into plasmid pBTM116 (2). Full-length unc-86(nucleotides [nt] 1 to 1059) was inserted to generate plasmid pBY436,unc-86 (nt 425 to 1059) was inserted to generate plasmid pBY166 (UNC-86 amino acids [aa] 135 to 342), unc-86 (nt 130 to 1050) was inserted to generate plasmid pBY165 (UNC-86 aa 37 to 342), and unc-86 (nt 1 to 333) was inserted to create plasmid pBY135 (UNC-86 aa 1 to 104). All fusions were analyzed in yeast strain L40 (MATα his3Δ200 trp1-901 leu2-3,112 ade LYS::(lexAop )4-HIS3 URA::(lexAop )8-lacZ GAL4 gal80) (19).
Construction of S. cerevisiae strains RB101 and RB102.Plasmid pBY180 was generated by inserting three tandem copies of 5′-GCATTCGAAATGCATTGCCCATAATG-3′ into plasmid pLacZi (Clontech), which contains a lacZ reporter gene under the control of the PCYC1 minimal promoter. Chromosomal integration of linearized pBY180 reconstitutes the ura3locus of yeast strain RH1533 (Mat-α lys2-801 leu2-3,112 suc2-Δ9 ura3-52 MCL his3-Δ200 trp1-Δ901) (14) and was selected for by plating of the transformants on SC medium without uracil. The resulting strain, RH1533 URA::pBY180, was named RB101. Strain RB102 was constructed analogously using 5′-AATTGCATTCGAAATGAGCTGCCCATAATG-3′.
Plasmid constructions.In order to generateunc-86 yeast expression vector pBY175, unc-86cDNA was inserted into a modified pGBT9 vector (2) from which the GAL4 DNA binding domain was deleted. A partialunc-86 cDNA (nt 425 to 1059) was inserted into the modified pGBT9 vector described above in order to generate plasmid pBY4:40 encoding UNC-86ΔN (aa 135 to 342). unc-86 alanine substitution mutants were constructed by site-directed PCR mutagenesis (17). mec-3 AD yeast expression vector pBY117 was constructed by inserting the mec-3 cDNA derived from plasmid pTU47 (43) into plasmid pGAD10 (2). Plasmid pBY1107 was constructed by subcloning a partial mec-3 cDNA that lacks the regions coding for the LIM domains and the acidic domain into the pET-32a vector (Invitrogen).
PCR mutagenesis.Mutations introduced into theunc-86 cDNA were named by the following scheme: the one-letter abbreviation of the amino acid, followed by its position in the protein, followed by the abbreviation of the amino acid substitution. POUS and POUHD ofunc-86 were mutagenized using the following PCR conditions. A mixture of 2 mM each dATP, dTTP, and dCTP; 10 mM dGTP; 500 mM primers; 200 ng of pBY531 (identical to pBY175 but containing aBamHI restriction site introduced by silent mutagenesis at position 428) template DNA; 10 mM Tris-HCl; 50 mM KCl; 0.8% Nonidet P-40; and 2.5 mM MgCl2 was subjected to 2 min at 92°C, followed by 35 cycles of 30 s at 92°C, 30 s at 45°C, and 1 min at 72°C and a final elongation for 5 min at 72°C.
Introduction of unc-86 mutants into yeast via homologous recombination.Gel-purified, mutagenized PCR products (500 ng) were transformed, together with 200 ng of linearized vector pBY531, into yeast strain RB101 expressing mec-3–GAL4 AD (GAD). Homologous recombination (27) between the PCR fragment and pBY531 was selected for on plates without leucine.
In vitro translation.For in vitro translation of mutant, truncated, or wild-type unc-86, pCITE-4a (Novagen)-based constructs were used. In vitro transcription and translation were performed in rabbit reticulocyte lysate with the TNT T7 Expression System (Promega) and [35S]methionine in accordance with the manufacturer's instructions. The amount of protein was normalized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography and PhosphorImager quantification.
Purification of recombinant MEC-3 protein. E. coli was transformed with plasmid pBY1107 encoding a MEC-3 protein lacking the LIM domains and the acidic tail fused with six His tags to thioredoxin. After induction with 1 mM IPTG at 37°C for 4 h, recombinant MEC-3 was purified from the soluble fraction using Ni2+nitrilotriacetic acid-agarose (Qiagen) in accordance with the instructions of the manufacturer.
Gel retardation assay.One hundred nanograms of oligonucleotide RB135 (5′-GCATTCGAAATGCATTGCCCATAATG-3′) was labeled with 30 μCi of [γ-32P]ATP (3,000 Ci/mmol; Amersham)–2 μl of 10× polynucleotide kinase buffer–1 μl (5 U) of polynucleotide kinase (MBI Fermentas) in 20 μl and incubated at 37°C for 60 min. The reaction was inactivated by heating at 65°C for 20 min, and excess [γ-32P]ATP was removed by passing the reaction over a G-25 column (Roche). The volume activity was determined in a scintillation counter; subsequent hybridization of complementary oligonucleotide RB136 was performed in a 10-fold excess by heating the reaction mixture to 95°C for 2 min and then cooling it to room temperature. Gel retardation experiments were performed for 30 min at room temperature as previously described (43). Equal amounts of mutant, truncated, and wild-type in vitro-translated UNC-86 protein were included in the reaction mixtures. After incubation, reaction mixtures were loaded onto a 6% polyarylamide acid gel in 0.5× TBE (45 mM Tris-borate, 0.5 mM EDTA) and subjected to electrophoresis at 150 V for 3 h at room temperature.
UNC-86 DNA and protein interactions can be analyzed in yeast in vivo.In order to facilitate the analysis of DNA binding and protein contacts of UNC-86, we developed an in vivo system to characterize UNC-86 mutants in S. cerevisiae. For this purpose, we constructed yeast strain RB101, which contains alacZ reporter with a yeast minimal promoter under the control of three copies of the first 26 bp of mec-3 promoter element CS1 (5′-GCATTCGAAATGCATTGCCCATAATG-3′) (Fig.1A; Table1). It has been shown previously that this segment is sufficient for both UNC-86 binding and UNC-86-dependent binding of MEC-3 (25). The resulting S. cerevisiae strain, RB101, did not activate the lacZreporter per se, as colonies on minimal plates did not turn blue even after 10 days of incubation in the presence of X-Gal (data not shown). In contrast, constitutive expression of unc-86 in RB101 resulted in light-blue colonies after a minimum of 3 days, indicating moderate lacZ reporter gene expression (Fig. 1B, row 1; Table 1). This expression was strongly enhanced (32-fold) by coexpression of the C. elegans mec-3 cDNA (Table 1). An even stronger level of activation was obtained (49-fold) whenmec-3 was fused to a heterologous GAL4 AD (mec-3–GAD) (Fig. 1B, row 3; Table 1). In order to obtain maximum lacZ reporter gene expression, we performed all consecutive experiments with this mec-3–GAD variant. Activation is dependent on the presence of UNC-86 protein, since yeast colonies expressing only mec-3–GAD showed nolacZ expression (white colonies; Fig. 1B, row 2; Table 1). This indicates that UNC-86 can function as a transcriptional activator and that association with MEC-3 on DNA further increases transcriptional activation.
It has been proposed that UNC-86 binds to a spaced recognition site, CATtcgAAAT, in CS1 (35). In order to evaluate the contribution of bases flanking this motif to ternary complex formation, we constructed yeast strain RB102. This strain is genetically identical to RB101, except that we mutated the GCAT bases immediately 3′ of the proposed UNC-86 binding site to GAGC (CS1AGC) (35). These nucleotide exchanges did not affectlacZ reporter gene activation in the presence of UNC-86 alone. However, activation with UNC-86 and the MEC-3 AD together was only 3-fold, compared to 49-fold in RB101 (Table 1). By electrophoretic mobility shift assay (EMSA), we confirmed that the mutation does not affect the DNA binding of UNC-86 but severely impairs the interaction of MEC-3 with UNC-86 and/or CS1 DNA (Fig.2A).
Next, we determined the domains of UNC-86 required for DNA binding and activation. For this purpose, we deleted the amino-terminal third of the UNC-86 protein (UNC-86ΔN) and performed EMSAs with CS1 DNA and MEC-3 protein. Neither binding to DNA nor the interaction of UNC-86 with MEC-3 was impaired by this amino-terminal deletion (Fig. 2B). In contrast, expression of UNC-86ΔN resulted in approximately eightfold lower activation of lacZ reporter gene expression in vivo (12% of wild-type activation; Table 1); however, the fold increase in activation obtained with MEC-3–GAD was similar to that obtained with wild-type UNC-86 (47-fold compared to 49-fold; Table 1). This strongly suggests that the amino-terminal deletion in UNC-86 eliminates a potential AD but does not compromise UNC-86 interaction with either MEC-3 or DNA. Interestingly, the synergistic activation by UNC-86ΔN and MEC-3 is strongly reduced (from 32-fold to 3-fold) whenmec-3 is expressed without the Gal4p AD (Table 1).
UNC-86 contains an amino-terminal transactivation domain.Our in vivo results obtained so far indicated that the ability of UNC-86 to activate transcription in yeast is dependent on the integrity of both the DNA binding domain and the amino terminus of the protein. In order to map the AD independently of DNA binding, we fused different fragments of the unc-86 cDNA to a portion of thelexA gene encoding the LexA DNA binding domain (aa 1 to 202) (Fig. 3A). lacZ reporter gene activation was determined quantitatively in S. cerevisiaestrain L40, which contains a lacZ reporter gene controlled by a minimal promoter with multiple lexA operator sites (19). In this strain, the transcriptional activation of UNC-86 does not depend on its intrinsic DNA binding properties. The expression of a full-length unc-86–lexA fusion resulted in strong lacZ reporter gene activation, as determined by β-galactosidase assays (Fig. 3B). Removal of the 35 aa from the amino-terminal end of UNC-86 reduced lacZ reporter gene activation approximately 10-fold. Deletion of all of the amino acids amino terminal to the POU domain (UNC-86ΔN) resulted in complete loss of lacZ reporter gene expression. In contrast, a fusion containing only the first 104 aa of UNC-86 activated reporter expression 13-fold more strongly than did full-length UNC-86. From these data, we conclude that the amino terminus of UNC-86, but not the POU domain, is able to activate PolII-dependent transcription in yeast. In contrast, the POU domain is both necessary and sufficient for DNA binding and MEC-3–GAD interaction in our yeast model, as was suggested previously by in vitro studies (43).
Both POUS and POUHD are involved in interactions with MEC-3.The molecular nature of the UNC-86–MEC-3 interaction had not been analyzed previously in detail. Therefore, we conducted a screen to identify mutations in UNC-86 that interfere with the formation of the ternary complex. For this purpose, random PCR mutagenesis of POUS and POUHD ofunc-86 was performed (see Materials and Methods).
Transformants from a screen for POUS and POUHDmutants were randomly selected and streaked on plates containing X-Gal. The transformants were assigned to three different groups by distinguishing among white, light blue, and dark blue colonies. From a number of light blue and white colonies, we isolated theunc-86-encoding plasmids, retransformed them into yeast with and without the mec-3–GAD plasmid, and sequenced the respective unc-86 genes. The β-galactosidase activities obtained with unc-86 mutants containing single-site missense mutations in the presence or absence of the MEC-3–GAD were determined in a quantitative assay (Table 2). The differences in β-galactosidase activity were not due to protein levels, as determined by Western blot analyses (data not shown). The positions of the substituted amino acids in the respective mutants are depicted in Fig. 4. The UNC-86 mutants that result in light blue yeast colonies can be divided into two classes based on this analysis. The first (class I) is comprised of mutants L197A, M202L, and K206E, with amino acid substitutions in the fourth helix of POUS; mutant R242G, with a mutation at the amino terminus of POUHD; and F297S, with an amino acid substitution in the carboxy-terminal amino acid of POUHD(Fig. 4). All five mutants activated lacZ to an extent similar to that of wild-type UNC-86. Since these results suggested that these mutations do not influence the DNA binding of the respective mutants significantly, we next tested them in an EMSA with CS1 DNA (Fig. 5A and 6A). L197A, M202L, K206E, and F297S each formed a detectable DNA complex with CS1 with an intensity similar to that seen with wild-type UNC-86. No DNA complex was obtained with R242G.
All five mutations strongly affected the enhancement of lacZreporter gene expression in the presence of the MEC-3–GAD (Table 2). The MEC-3–GAD-dependent lacZ activation of the respective mutants (4- to 17-fold activation) was greatly reduced compared to the 49-fold activation seen with wild-type UNC-86 (Table 2). In EMSAs, the L197A and M202L mutants did not form ternary complexes with MEC-3 and DNA (Fig. 6A) and the K206E mutant resulted in ternary complexes with MEC-3 and DNA represented by weaker bands than that observed with wild-type UNC-86. The complex formed with the mutant F297S was indistinguishable from the wild type. The mutant R242G behaved differently. Even though no DNA complex was obtained for this UNC-86 variant alone, a ternary complex with MEC-3 was formed. The data suggest that the residues at positions 197, 202, 206, and 297 are involved in the interaction and/or activation of UNC-86 with MEC-3 in the ternary complex but do not contribute to the DNA binding of UNC-86.
The second class of UNC-86 mutants (class II) that displayed reducedlacZ reporter gene expression includes I189V, with an amino acid substitution in the third α helix of POUS, and K250D, L253P, Q255P, F257L, and Q259P, with substitutions in the first α helix of POUHD (Fig. 4). When we expressed these variants without a mec-3–GAD gene in yeast, only very weaklacZ reporter gene expression was observed (Table 2). This suggests that these amino acid substitutions reduce the affinity of the respective UNC-86 proteins for the CS1 DNA element. In contrast, the increase in lacZ reporter activation after additional expression of mec-3–GAD was in the same range as that seen with wild-type UNC-86 (27- to 108-fold) (Table 2). In vitro DNA binding assays confirmed that these mutants have reduced DNA binding capacities (Fig. 5B and 6B). Although they were not able to bind CS1 alone, all but one of these mutants formed a detectable ternary complex with MEC-3 on CS1. We conclude that although the overall DNA binding affinity of these UNC-86 mutants is reduced, their ability to mediate MEC-3–GAD-dependent activation of transcription is not significantly diminished. We therefore suggest that these mutations do not interfere with binding of MEC-3–GAD to UNC-86.
From the white transformants, three members of a third class of mutants harboring single amino acid substitutions (class III) were identified: K156E, Q166R, and T188A with mutations in POUS (Fig. 4). These mutants showed a decreased ability to activate lacZreporter gene expression compared to the wild-type UNC-86 protein (Table 2). In vitro DNA binding assays confirmed that these mutants display a decreased affinity for CS1 DNA (Fig. 5C). But in contrast to class II mutants, no increase in lacZ reporter gene expression upon addition of MEC-3–GAD was observed (Table 2). As these effects are not due to variations in protein levels (data not shown), these mutations may induce severe structural alterations in the protein, thereby rendering it nonfunctional. Alternatively, these mutations could also influence the ability of the protein to interact both with DNA and with MEC-3–GAD protein. The latter can be assumed for the I189R R242G double mutant that we isolated from this screen as a white transformant. As shown above, the single-site mutation R242G did not have a strong effect on transcriptional activation but eliminated MEC-3–GAD coactivation whereas the I189R mutation probably affects the reporter gene activation of UNC-86 itself (as does I189V) but not MEC-3–GAD interaction. We therefore conclude that the double mutant is defective for both functions.
UNC-86 and Oct-1 use similar residues to interact with their respective protein partners.Our yeast screen has identified several residues in UNC-86 that may contact MEC-3 directly. In the POU protein Oct-1, two of these residues, L344 (corresponding to L197) and M349 (corresponding to M202), are part of the extended surface that interacts with the Oct-1 cofactor Oca-B. In order to further test the conservation of the contact interfaces, we substituted four additional residues in UNC-86 with alanine at positions that, in Oct-1, are involved in protein interactions (1, 21). We tested the ability of each mutant to activate transcription either alone or in conjunction with MEC-3–GAD in yeast strain RB101 (Table 2). The alanine substitution at position R295 resulted in a nonfunctional UNC-86 protein (class III) that did not activate the lacZreporter gene, either alone or with MEC-3–GAD. Mutants E149A and D296A behaved similarly to wild-type UNC-86 both in the presence and in the absence of the MEC-3 AD. The R146A mutation, however, only reducedlacZ reporter gene activation of UNC-86 weakly but reduced coactivation by the MEC-3 AD from 49-fold to 15-fold. Consistent with a role of this position in the MEC-3 contact interface, the R146A mutant bound in an EMSA to CS1 element-like wild type UNC-86 but revealed reduced affinity for MEC-3 and DNA in the ternary complex, as indicated by a band weaker than that observed with wild-type UNC-86 (Fig. 6A).
The interaction of UNC-86 with MEC-3 on DNA can be monitored in yeast.We wanted to understand how the transcriptional activation properties of UNC-86 are modulated by protein interactions. We therefore developed an in vivo system with S. cerevisiae. In contrast to mammalian cells, S. cerevisiae does not contain genes encoding POU proteins which would compete for artificially introduced DNA binding sites (33). Therefore, yeast provides an ideal system for studying POU protein interactions. It allows us to analyze ternary complex formation involving UNC-86, its interacting partner MEC-3, and single DNA binding motifs of the mec-3promoter in vivo.
We have found that UNC-86, in contrast to the human POU protein Oct-1 (14), activates transcription in yeast. The data from the LexA–UNC-86 fusion constructs (Fig. 3) suggest that activation is mediated by the amino terminus of UNC-86. This region contains a 29-aa region termed the POU IV box, which is the only sequence element outside the POU domain that is conserved between UNC-86 and the homologous Brn-3 proteins (34). The POU IV box has already been implicated in transcriptional activation by the Brn-3 proteins (4, 31). We found no evidence of the presence of a strong activator region in the UNC-86 POU domain, as has been suggested for Brn-3a (4).
MEC-3 incorporation into the ternary complex is dependent on the DNA context.In order to contribute to lacZ reporter gene activation, MEC-3 has to be recruited to DNA by the UNC-86 POU domain. UNC-86 binding to CS1 is a prerequisite of MEC-3 activation, since MEC-3–GAD could not activate lacZ expression on its own.
The first 26 bp of the CS1 mec-3 promoter element used in our study contain both a CATnnnAAAT motif and an overlapping octameric consensus motif (AAATGCAT) for UNC-86 binding (35, 40, 42). Both motifs are repeated in promoter element CS2, which, together with CS1, has been shown to be required both for UNC-86-dependent establishment and UNC-86- and MEC-3-dependent maintenance of mec-3 expression in C. elegans (35, 40, 42). Here we show that UNC-86 can still activate transcription in vivo on the CS1agc element in which the nucleotides CAT in the octamer motif are changed to AGC. We suggest that the nucleotides CAT are not a prerequisite for UNC-86 binding to CS1, either in vivo or in vitro (our results and reference35). Therefore, UNC-86 most likely interacts with the CATtcgAAAT motif, as suggested previously (39, 46). We show here that the CAT bases 3′ adjacent to this motif are crucial for MEC-3-dependent activation in yeast and for the incorporation of MEC-3 into the UNC-86–DNA complex. A similar requirement for DNA target site-dependent recruitment has also been suggested for the formation of the Oct-1–Oca-B complex (6, 23). Whether MEC-3 interacts directly with DNA or induces a conformational change in UNC-86 that results in an extension or shift of its DNA binding surface has to be the subject of future studies. The importance of the octamer motif in CS1 and CS2 is further supported by the evolutionary conservation of this sequence among C. elegans, C. briggsae, and C. remanei(40, 42).
MEC-3 enhances the DNA binding of UNC-86.We identified several mutations that strongly reduced the affinity of UNC-86 for DNA in vitro (Fig. 6B), resulting in very weak transcriptional activation in vivo (Table 2). However, coexpression of mec-3–GAD along with these unc-86 variants in the yeast system was still found to cooperatively increase lacZ reporter gene activation by a factor similar to that obtained with the wild type (Table 2). All but one of the mutant proteins were able to form a ternary complex with the MEC-3 protein in vitro (Fig. 6B).
None of the positions affected by these mutations have previously been reported to directly contact DNA in other POU proteins. The K250D mutation alters the charge of a solvent-exposed residue, while three of the other mutations that cluster in the first α helix of POUHD introduce prolines (Fig. 4), which may result in a kink in this helix. A slightly altered structure of POUHDcould impair the DNA binding of these mutants. Interestingly, mutations that affect DNA binding but not ternary complex formation have not been identified in previous mutational analyses of POU proteins (1, 29). From these data, we conclude that MEC-3 interaction may enhance the binding affinity of UNC-86, either by inducing a conformational change in the protein or because MEC-3 itself contributes to DNA binding.
Amino acids in both POU subdomains of UNC-86 influence the interaction with MEC-3.Five amino acid substitutions in POUS and POUHD of UNC-86 (Fig. 4) result in mutants that bind DNA in vitro like wild-type UNC-86 and also activate transcription in vivo to a similar extent. However, when coexpressed with mec-3–GAD, these mutants did not display an increase in lacZ reporter gene activation as seen with wild-type UNC-86 (Table 2). This suggests that we have identified amino acids in both POUS and POUHD of UNC-86 that are required for the interaction with MEC-3 or for the synergistic activation of the UNC-86–MEC-3 heterodimer.
The structures of the POU domains of Oct-1 and Pit-1 are strikingly similar, even though they display only 52% amino acid identity (16). No structural data are available for UNC-86, but from the 45 and 44% sequence identity of the UNC-86 POU domain with those of Oct-1 and Pit-1, respectively, one could assume that it folds very similarly to the POU domains of these proteins (16). Based on these similarities, we compared the locations of mutations in UNC-86 with their respective positions in Oct-1 and Pit-1 (21, 22).
The residues corresponding to R146, L197, and M202 in Oct-1 and F297 in Pit-1 (compare Fig. 4) have been implicated in direct protein contacts (1, 13, 21, 29). Alanine substitutions at E286, L344, and M349 in Oct-1 result in loss of interaction with Oca-B (1, 13, 21), and K60 (F297) is involved in protein contacts in the Pit-1 crystal structure (21). Consistent with a similar function, L197A and M202L did not form ternary complexes in an EMSA and the ternary complex formation of R146A appeared weaker than that of wild-type UNC-86. As both the leucine (position 197) and methionine (position 202) are conserved in the functionally nonrelated UNC-86, Pit-1, and Oct-1 proteins (Fig. 4) (16), one might hypothesize that they are part of a general protein docking surface conserved among POU proteins. K206E may also be part of this docking surface, since this residue is located close to positions 202 and 197 in the structure of the POU domain, and ternary complex formation of the K206E mutant was weaker than with the wild type. There are no data available from Oct-1 or Pit-1 POU complexes that link this position to protein interactions.
F297S, on the other hand, did not affect DNA and MEC-3 binding of UNC-86 in an EMSA, but in vivo coactivation with MEC-3–GAD was reduced from 49-fold to 3-fold. This suggests that F297S is an important discriminator of protein interactions and coactivation. It is consistent with such a role that this position is distinct in the different POU proteins and is located in the vicinity of other residues that, in both Oct-1 and Pit-1, affect DNA or protein interactions. In contrast, E289A and I437A in Oct-1 prevented the interaction with Oca-B (1), whereas the equivalent substitutions in UNC-86 (E149A and D296A) affected neither DNA binding nor MEC-3 interactions. We conclude that Oct-1 and UNC-86 share some of the residues of their surface for protein interactions, but other positions are obviously functionally distinct.
An unexpected result was obtained for position 242. In the cocrystal structures of Pit-1 and Oct-1, this conserved arginine is involved in minor-groove base contacts (21, 22). Consistent with a similar role in UNC-86, R242G did not bind to DNA in vitro, although in vivo, this mutant only showed twofold reduced transcriptional activation. Even more strikingly, R242G was capable of ternary complex formation like the wild type whereas our in vivo data suggest fivefold reduced coactivation with MEC-3–GAD. We have no explanation for this discrepancy. We cannot exclude the possibility that the DNA binding of UNC-86 R242G is enhanced by intrinsic yeast proteins. Alternatively, the R242G mutation could have a negative effect on the DNA binding of UNC-86 and at the same time lead to strong enhancement of the amino-terminal transactivation domain of UNC-86. The latter case would suggest a novel role for the POU domain in transcriptional regulation. While the POU domain does not possess activating properties, it may participate in the regulation of the amino-terminal AD of UNC-86.
Synergistic activation between UNC-86 and MEC-3 may require an extensive interaction surface.The cooperative binding of MEC-3 and UNC-86 strongly enhanced transcriptional activation in yeast, corroborating the previously reported results of in vitro transcription assays (25). However, a comparison of our in vivo and in vitro data clearly demonstrates that the affinity of UNC-86 and MEC-3 in the ternary complex with DNA in vitro does not always correlate with the transcriptional activity of the complex in vivo. For example, binding of UNC-86 to MEC-3 is not significantly affected by deletion of the amino-terminal half of UNC-86. This led us (this study) and others (43) to propose that UNC-86 binding to MEC-3 is mediated by the POU domain only. However, we found that the UNC-86ΔN protein, which lacks all residues N terminal to the POU domain, has almost lost its capability for synergistic transcriptional activation with MEC-3 in vivo whereas, strikingly, this synergism with the MEC-3 AD is not affected. This suggests that under in vivo conditions, UNC-86 and MEC-3 are involved in more complex interactions than previously thought. These require binding of MEC-3 to the UNC-86 POU domain but obviously also involve additional interactions between the UNC-86 amino terminus and an as yet unidentified domain of MEC-3. The latter interaction can be replaced in our yeast system by providing a heterologous AD (Gal4p) with the MEC-3–GAD fusion. The nature of the synergistic activation of UNC-86–MEC-3 is unknown and has to be the subject of future studies.
We thank W. Hammerschmidt for the modified pGBT9 vector and the yeast strain RH1533, M. Chalfie for pTU47, and R. Grosschedl and the members of the Kolanus, Meisterernst, and Baumeister labs for helpful suggestions and comments on the manuscript.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft to R.B.
- Received 23 August 1999.
- Returned for modification 29 November 1999.
- Accepted 5 April 2000.
- Copyright © 2000 American Society for Microbiology


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We know it's an unfair question, but then we love being naughty (and not nasty). So, without further ado, Das ka Dum by Dr Bhaskar Das
Q. Did you have a role model when you started working at BCCL. Who was it and what did you learn from her/him?
A. My role model of life has always been a driver named Krishna. He taught me 18 sutras of serving. It worked for me in BCCL and has been working even now. And that role model got never changed.
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subset: commoncrawl


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Jarvis, D. (2013, September 26). Tizoc Stone. Ancient History Encyclopedia. Retrieved from https://www.ancient.eu/image/1442/
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Kuala Lumpur, April 2 2013 – U Mobile Sdn Bhd, considered to be Malaysia's most dynamic and innovative 3G mobile operator officiated a partnership with SAS Malaysia, world leading business analytics software and service provider. The partnership will consist of the integration of SAS' Visual Analytics solution into U Mobile's operations, here in Malaysia.
Present at the signing ceremony was Jaffa Sany Ariffin, Chief Executive Officer of U Mobile who kept his speech short and simple, exclaiming that the partnership and its deployment will provide its customers a "better value proposition" and adding no more when asked of future possibilities. He also mentioned that SAS' Visual Analytics will aid in determining consumer trends, provide more useful, versatile and adaptive methods of controlling collected data in real time, which is a clear advantage over their competitors.
Also present at the ceremony was Andrew Tan, Managing Director of SAS Malaysia whom represented SAS Malaysia during the signing. As part of his speech, Mr. Andrew Tan explained SAS' Visual Analytics and it's advantages.
This would be a brave and absolutely brilliant step for U Mobile, embracing new technologies that could greatly affect how well they can serve their consumers. U Mobile has always been ahead in this regard, not only serving one of the best mobile and data plans in the market (if not THE best) but also the most innovative payment methods to date. The power of the SAS Visual Analytics solutions is undeniable, it's user friendly interface and real time data crunching is a huge advantage when used appropriately.
Not sure what 'power' I'm referring to? If you're interested in SAS' Visual Analytics, you can go to our previous post here, which includes a microsite with a demo.
[spoiler title="About U Mobile"]
[spoiler title="About SAS Malaysia"]
[spoiler title="Official Press Release"]
Images courtesy of cohn&wolfe XPRasia


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Web Reference: #25949
This asset is a plot in Dromolaksia-Meneou, Larnaca.
The asset has an area of 847sqm.
The immediate area of the asset comprises of residential developments and undeveloped parcels of land.
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The property falls into residential planning zone Kα6 with 90% building density, 50% coverage, 2 floors and a maximum height of 10m.
There are 3 more plots available for sale. Area 732 sq.m.- 785 sq.m.


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Why context matters
Achieving a strong set of school or college grades is key to earning a place at our university. We know that can be challenging and that grades are influenced by things like where you live, where you go to school and other factors. That's why each year we look at each student application in detail. We note circumstances that may have impacted your potential. We treat everyone fairly and admit the brightest and best students, regardless of background.
Advice and support is always available to all future students, but we need to know about you and your context to help you fully. We take certain factors into account when we look at your grades and these factors may generate a contextual support flag to highlight who is eligible for additional help.
What factors do we consider?
The factors we use to generate a contextual flag are:
If you live in a Scottish Index of Multiple Deprivation (SIMD) 20% zone you will get a flag.
You can find your postcode category at: http://simd.scot/
- Type your postcode in the "area finder" box on the left,
- Click "Go" and then hover over the yellow marker for your house and the box at the right of the screen will describe your postcode area, including "Decile "A", Quintile "B".
If Quintile B for your address states "Quintile 1" you will be allocated a contextual flag.
Schools are key to helping pupils achieve the best grades and progress to university and beyond. We have prioritised some partner schools working hard towards Scottish average in this area which, as at autumn 2019, are as set out below.
|Local Authority
|Arbroath Academy
|Auchenharvie Academy
|North Ayrshire
|Baldragon Academy
|Dundee City
|Bannockburn High School
|Beath High School
|Braeview Academy
|Dundee City
|Castlebrae Community High School
|Edinburgh, City of
|Castlemilk High School
|Glasgow City
|Craigie High School
|Dundee City
|Craigroyston Community High School
|Edinburgh, City of
|Doon Academy
|East Ayrshire
|Drumchapel High School
|Glasgow City
|Eastbank Academy
|Glasgow City
|Forrester High School
|Edinburgh, City of
|Govan High School
|Glasgow City
|Gracemount High School
|Edinburgh, City of
|Inverclyde Academy
|Inverness High School
|Irvine Royal Academy
|North Ayrshire
|Kinlochleven High School
|Langholm Academy
|Dumfries & Galloway
|Levenmouth Academy
|Liberton High School
|Edinburgh, City of
|Linwood High School
|Lochend Community High School
|Glasgow City
|Lochgelly High School
|Lochside Academy
|Aberdeen City
|Maxwelltown High School
|Dumfries & Galloway
|Newbattle High School
|Northfield Academy
|Aberdeen City
|Port Glasgow High School
|Smithycroft Secondary School
|Glasgow City
|St Machar Academy
|Aberdeen City
|St Margaret Mary's Secondary School
|Glasgow City
|St Mungo's Academy
|Glasgow City
|St Paul's High School
|Glasgow City
|St Paul's RC Academy
|Dundee City
|Tynecastle High School
|Edinburgh, City of
|Wester Hailes Education Centre
|Edinburgh, City of
|Whitehill Secondary School
|Glasgow City
If your personal circumstances have been especially challenging we care, especially if you are or have been:
- looked after person (Care experienced)
- someone who cares, unpaid, for a friend or family member who due to illness, disability, a mental health problem or an addiction cannot cope without their support
- Government refugee or asylum seeker
Please disclose all your circumstances when you complete your UCAS application.
You then need to verify these circumstances via our Applicant Portal - you will find a link in your University of Dundee Application Acknowledgement Letter. This second stage evidence upload is crucial to allow us to fully consider your achievement in context.
If I get a contextual flag, what happens next?
If you are allocated a contextual flag we will either make you an offer or, for subjects that interview, prioritise you for an interview.
We will also offer you a further route to qualify and prepare for university. Around 1,000 applicants each year are offered such pre-entry Summer support. Options include Access Summer School and Online Summer School, which both run each June and July. Once you have applied through UCAS we will let you know if you qualify for such supported entry. Contextual flag candidates need to keep these months free so they can join us and benefit fully.
We want all students entering Dundee to have a great student experience. We recognise preparing for study / achieving a good balance in your life is part of this. We want to help you in every way we can.
Maximising your success
We want you to succeed and so commit our support to you long before your degree starts. That is why Dundee is such a great place to study.
Crucially, we will never use anything you disclose in a negative way. So as you complete your UCAS application, please encourage your referee to tell us about your circumstances.
We want you to have a great time at Dundee, enjoy our award winning student experience and earn a great degree. If you want to know more or have any questions, email email@example.com
How else might I get a contextual flag?
As our Selectors read your application they may feel that other factors have impacted your study to date and that in combination you deserve a contextual flag. Applicants meeting the criteria below will be given priority for offer or interview wherever appropriate.
Beyond the factors above, our Admissions and Applicant Experience Team will look for further evidence of your path to us being made more difficult by circumstances outside your control. At their discretion they may allocate a contextual flag for factors such as:
- you applied from an SIMD Quintile 2 postcode – see Your home postcode information above
- attended a School where grades and progress to university are above those of our key partner schools but still low
- are an adult returner who has been out of school for five years and out of education for three years or who have a relevant accreditation of prior learning or work record or have been made redundant
- have indicated other factors including having declared a disability, declared a mental health issue, faced disruption to your education assessed as attending five or more Primary and Secondary schools or residing more than 40 miles from their Secondary School, declared other adverse circumstances, being transgender, estranged, are in a vulnerable group (e.g. declared a Criminal Conviction or a Children's Panel enquiry) or have taken part in the Reach or ACES or SWAP programmes (with demonstrable engagement)
Putting everything in context gives us the biggest and clearest picture about you.


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Up for Sale is a Vintage James Avery Sterling Silver Brooch. Measures 1 3/8″ x 2″. Weighs 13.1 Grams. In Very Nice Condition, Some Minor Tarnish. I'm Happy to Polish at Buyers Request. No Box or Pouch Included. Please See Photos/Send Questions for More Detail. International Buyers Please Note. The item "Vtg James Avery Sterling Silver Reindeer Brooch Pin Deer Christmas Retire Signed" is in sale since Sunday, January 12, 2020. This item is in the category "Jewelry & Watches\Fine Jewelry\Fine Pins & Brooches\Precious Metal without Stones". The seller is "dragonpez" and is located in Edmonds, Washington. This item can be shipped worldwide.
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Dozens of artists will take the stage this weekend as the Treasure Island Music Festival returns to Oakland.
The 11th-annual festival will be hosted at the Middle Shoreline Park and will feature artists like A$AP Rocky, Pusha T and Santigold, among others.
In additional to the musical lineup, food vendors and artist will showcase their work.
There will be no on-site vehicle or bike parking available so attendees will be required to ride the free festival shuttle from the West Oakland BART station or get there via ride share apps.
General Admission tickets are on sale for both single day and 2-day general admission along with VIP tickets at treasureislandfestival.com.


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Ramadan is traditionally a time to think of others less fortunate than oneself, so you will find that Malaysian companies often have special Social Responsibility activities during this time of year. This year, Sunway Group partnered with the National Committee for Community Services, Malaysian Red Crescent, to help install an Automated Teller Machine (ATM) for the distribution of rice in the entrance to the Masjid India in Johor Bahru. Sunway have installed these devices in other states in Malaysia, but this is their first in Johor. This Rice ATM can hold 200 kg of rice and Malaysians who qualify for help receive a special card and can collect 2 kg of rice per week. The machine also has a donation compartment for the purchase of more rice. Currently 100 recipients will receive this free supply of rice and it is hoped that donations will keep the supply of rice going throughout the whole year.
In addition, volunteers from Sunway and the Malaysian Red Crescent organised the production and distribution of 1,000 containers of burbur lambuk (flavoured rice porridge) which is traditionally distributed to the poor and needy at mosques for the breaking of the fast during Ramadan.
Dato' Mohd Redza Shah Abdul Wahid is both the CEO of Bank Muamalat and also Head of the Masjid India, indeed it was his grandfather who first established this mosque for the Indian Muslim community here in Johor Bahru. He was here today to give Zakat funds from Bank Muamalat to the needy of Johor Bahru via the hands of Y.A.M. Tunku Idris Iskandar Alhaj ibni Sultan Ibrahim, Tunku Temenggong Johor, who is Chairman of the National Committee for Community Services, Malaysian Red Crescent.
So, let me show you some photos as the afternoon's events unfolded. The main event was the arrivial of the Prince (Y.A.M. Tunku Idris Iskandar Alhaj ibni Sultan Ibrahim, Tunku Temenggong Johor) and people had turned up well in advance, even more than originally planned for.
There was much organising to be done by the red-shirted Sunway people and the red-and-white-shirted Malaysian Red Crescent. If you are unfamiliar with traditional Malaysian male attire, do take special note of the outfits worn here. The baju melayu is the loose tunic warn over trousers and covered by a sarong wrapped around the hips. Each state in Malaysia will have subtle differences in attire. So, Johorean males will wear a tunic with a simple round neckline, and no collar. And their sarong may tell of family heritage as well as location. You can see some esquisite examples of textiles like these at the Johor Weaving Gallery here in Johor Bahru.
Volunteers from Sunway registered the media, of which there were many, while others made sure they knew how to use the Rice ATM. In the meantime, volunteers were busy preparing the burbur lambuk outside the mosque.
Here are five of the people responsible for today's events: from left to right, Dato' Haji Mohd Rashidi bin Haji Mohd Noor, Deputy Chairman of the National Committee for Community Services, Malaysian Red Crescent; Dato' Mohd Redza Shah Abdul Wahid, CEO of Bank Muamalat; Mr Gerard Soosay, CEO of Sunway Iskandar; Lekha Nandey, committe member of the National Committee for Community Services, Malaysian Red Crescent; and the gentleman in blue who's name I did not get.
Right on time at 3:30 pm, the Prince arrived and was greeted by Dato' Haji Mohd Rashidi bin Haji Mohd Noor and other officials. The Prince was immediately put to work by adding some ingrediants to the pot of burbur lambuk , giving it a regal stir, and then pouring some into the pots for distribution.
He then moved into the entrance of the mosque where he emptied a bag of rice into the Rice ATM and then was joined in the anteroom by the officials. Here he and Dato' Mohd Redza Shah Abdul Wahid said a few words to start the proceedings.
The Prince received the Zakat from Dato' Mohd Redza Shah Abdul Wahid and a gift from Mr Gerard Soosay.
Following tradition, prayers were said while the needy waited patiently for their turn in this afternoon's ceremony.
I was impressed by the patience of the Prince (Y.A.M. Tunku Idris Iskandar Alhaj ibni Sultan Ibrahim, Tunku Temenggong Johor) as he insisted on personally giving out the gifts to all the recipients. In particular, he was so thoughtful to give respect to this elderly lady.
Finally the formal photo taking before the Prince left. My photo only shows the group of people to the right of the Prince as this includes the Malaysian Red Crescent volunteers who I was here to support.
Back outside, Sunway volunteers continued to fill pots with burbur lambuk and people started making their way home. Another successful event for the Malaysian Red Crescent.
Click here to return to Travels in Malaysia 2019.
Click here to return to Helen Gray's homepage.
Click here to see more about the activities of the Malaysian Red Crescent.


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Cetaceans Quotes
Collection of top 7 famous quotes about Cetaceans
The word 'impossible' ain't in my dictionary. — Jessica Maria Tuccelli
By its very nature and design, Network Marketing is a strikingly Fair, Democratic, Socially Responsible system of generating wealth. — Robert Kiyosaki
They got rid of bottled water. We got thermoses that say 'Grey's Anatomy Season 4' with our names on it. Cool. — Brooke Smith
To Dogmatism the Spirit of Inquiry is the same as the Spirit of Evil. — Ambrose Bierce
Man is a passion which brings a will into play, which works an intelligence. — Henri Frederic Amiel
I'm afraid there is evidence to refute this claim of human uniqueness. Cetaceans are one example that are known to share experiences. — John Pappas


text:
The experiment of permitting public consumption of alcoholic beverages on the Atlantic City Boardwalk is only a few days old, but lawmakers in Trenton approved a bill Tuesday that would make open containers a permanent fixture at the seaside resort.
The Assembly Budget Committee unanimously voted in favor of a bill allowing open containers in designated tourism zones in Atlantic City. The bill is scheduled for a vote by the full Assembly on Thursday.
Last week, Atlantic City Mayor Marty Small Sr. signed an executive order temporarily permitting public consumption of alcohol on the Boardwalk, the Orange Loop and nonresidential areas of Gardner's Basin. The order went into effect Friday and will expire in November or whenever Gov. Phil Murphy lifts COVID-19-related restrictions on bars and restaurants, whichever comes first.
Under the state legislation, sponsored by Assemblymen Vince Mazzeo and John Armato, both D-Atlantic, the Casino Reinvestment Development Authority will determine areas in Atlantic City's Tourism District where open containers of alcohol can legally be consumed and carried.
ATLANTIC CITY — Contrary to popular belief, drinking alcoholic beverages on the world's most…
"This is another way for businesses in the Tourism District to promote themselves," Mazzeo said, adding "the goal ... has been to make this a permanent law in an effort to continue revamping Atlantic City for a newer generation."
Other high-profile tourist destinations, such as Las Vegas and New Orleans, permit public drinking by adults of legal age in controlled areas.
Armato said the Atlantic City Boardwalk "makes it easy" for someone to move between casinos, bars and restaurants, but if they do so with an open container of alcohol, they can be issued a citation, with penalties that include fines, community service or jail.
"This practice is becoming outdated as more cities across the nation are relaxing liquor laws in tourist areas," Armato said. "It's time we revamp Atlantic City's own laws to match this trend while continuing to keep the tourism area a safe and welcoming place to visit."
The outdoor dining plan for Angelo's Fairmount Tavern in Atlantic City and just about every …
City officials, business owners and Atlantic County's state representatives have been trying to implement an open container law for several years in an effort to create a more tourism-friendly Atlantic City. Last December, City Council narrowly approved a resolution urging the state Legislature to act on a bill that would permit open alcohol containers in certain areas of the city.
Similar legislation has been introduced in prior years and cleared committees, but never made it to a full vote of the state Legislature.
State Sen. Chris Brown, R-Atlantic, reintroduced companion legislation in February. The bill has been referred to the Senate Law and Public Safety Committee.
Local officials were confident the legislation introduced early this year would have been approved before Memorial Day, but COVID-19 caused a drastic shift in priorities.
Nicholas Huba and David Danzis discuss the week that was in Atlantic City, including when Tr…
State emergency and public health orders issued by Murphy in response to the coronavirus provided an opportunity for the city to issue the temporary measure, according to officials.
"We've been supportive of open containers for years," said Michael Chait, president of the Greater Atlantic City Chamber. "We brought (the topic) up during the Atlantic City policy group meeting this year and requested that the city act as quickly as possible."
The city's gaming industry has supported efforts to legalize open containers at the state level for several years as well.
During the mayor's news conference to announce the temporary order, Steve Callender, president of the Casino Association of New Jersey and senior vice president of Eastern regional operations for Tropicana Atlantic City's parent company, Eldorado Resorts, said it was a "great first step."
"Hopefully, as we go through this process, people that make decisions can see that we handle it properly," Callender said last week. "We do it the right way, and we follow all the rules, so that we can make it permanent in the future. It's great for our community."


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Bowel cancer
Breast cancer
Liver cancer
Lung cancer
View all cancers >
Cancer symptoms
What causes cancer?
Stages of cancer
Patient information
Four T.I.P.S. to improve your experience
Support for health professionals
Learn about cancer costs
Lifestyle cancer risks
Skin cancer prevention
Stopping smoking
Breast cancer screening
Cervical screening NSW
Bowel screening
Working with primary care
Patient-reported measures (PRMs)
Reporting for Better Cancer Outcomes (RBCO)
Specialist Cancer Centres
Grant opportunities
Research Grant schemes and resources
Previous grants funded
Taking part in a clinical trial
How do clinical trials progress?
Our clinical trials program
Cancer statistics NSW
NSW Prostate Clinical Cancer Registry
Publications using our data
TCRC program achievements
Funded TCRCs
Reducing tobacco use
Preventing skin cancer
Healthy lifestyles
Increasing early detection
Improving outcomes
Building cancer research capacity
Fostering research translation
Enhancing the experience of people with cancer
Primary liver cancer
Aboriginal communities
Multicultural communities
Patient-centred quality cancer care
Primary health care
Community and Consumer Advisory Panel
Aboriginal Governance and Engagement
Media enquiries
Media releases
Writing about cancer guidelines
Upcoming events
NSW Premier's Research Awards
Innovations Conference
View more >
People with compromised immune systems or pre-existing medical conditions, including cancer, may be at increased risk of contracting coronavirus (COVID-19) and increased risk of more severe infection.
"Our knowledge about COVID-19 is changing rapidly. If you are receiving cancer treatment that suppresses your immune system and you develop a fever or respiratory symptoms, call your oncologist or a member of your treatment team, as you usually would if you develop a fever while on treatment. Be sure to follow their guidance on actions to take," said Professor Dorothy Keefe, CEO, Cancer Australia and medical oncologist.
Everyone should practice good hygiene to protect against infection, including people who have had cancer in the past.
Good hygiene includes:
For people at increased risk of getting sick from COVID-19, such as people receiving treatment for cancer, and people living with metastatic cancer
As well as practicing good hygiene:
If there is a COVID-19 outbreak in your community, stay home as much as possible to further reduce your risk of being exposed.
For family, friends and carers of people at increased risk of getting sick from COVID-19
Follow advice for good hygiene to protect against infection, including:
Avoiding contact with people in the community who are unwell.
There is no vaccine for COVID-19 yet. However, this year, it is important to have your influenza vaccination as early in the flu season as possible.
Useful COVID-19 information links:
Coronavirus Health Information Line
Call this line if you are seeking information on novel coronavirus. The line operates 24 hours a day, seven days a week. Phone: 1800 020 080.


text: Because YOU demanded it, film critics William Bibbiani and Witney Seibold are reviewing all of the POLICE ACADEMY movies on this, the last-ever episode of THE TWO-SHOT! That's not just on the Movie House network, but anywhere! Film critics William Bibbiani and Witney Seibold are saying goodbye to this long-running podcast by making a HUGE announcement about what their next project is, which you can only listen to at The Critically Acclaimed Network, along with their other shows: CRITICALLY ACCLAIMED, CANCELED TOO SOON, THE IRON LIST and WE'VE GOT MAIL! Check out their website at www.criticallyacclaimed.net and head on over to www.patreon.com/criticallyacclaimednetwork to contribute to their shows and get tons of exclusive content!


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Forgive me if you see me next and I no longer have any hair… this maybe normal for a 5th year medic at this point who is caught up at the moment in a flurry of "foundation application", "elective organisation" and "general this is the last year drama". I'll be talking a lot more about those these next few weeks but right now I do feel another list is coming on.
Before you ask the Foundation Application is where 5th year medics sit down and look at the whole of the UK and ask themselves "Where do I want to work in the next 2 years?" A question which has many sub questions such as "Where would I like to live?" and "What is the town centre like?" and "Does this place have a good vibe" and "Will it be a good deanery to learn in?" Several of these questions where answered when sitting down with Dad and finding out exactly where 'Staffordshire' actually is (being a lorry driver he is such an expert on places in the UK we call him Dadnav!)
I've said it before and I'll say it again to any new students, returning students or people in general- whenever everything seems overwhelming make a list, break down everything and just start getting the tick boxes checked off and you'll realise everything is getting done a lot easier and more manageable!
Particularly if you're just about to start at University where you're swamped with paper work and registration and can't decide between University hall registration or shopping for a saucepan!
I promise that I will get more interesting after more of these boxes have been ticked (it is my birthday this month after all)!
Till then,
P.s. I should probably point out that when I say that "I've said it before and I'll say it again" I must admit that the more accurate quote is "Mum's said it before"!


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Seven Oakes Motel - Kingston44.25778, -76.551
Seven Oakes Motel is a 2-star family venue near Christ Church Cataraqui Parish Centre. The property features 44 air-conditioned rooms.
Lake Ontario Park is 2.8 miles from Seven Oakes Motel, while Fort Henry National Historic Site is 4.9 miles from the hotel. The venue boasts location within 2.4 miles from Kingston city center. In the vicinity of the property there is St. Marguerite Bourgeoys Catholic School.
A short drive takes guests to Kingston airport.
The accommodation offers rooms with a view of garden as well as individual climate control, a personal safe and a sitting area. For your comfort, you will find a shower, a hairdryer and a drier in the bathroom.
Eat & Drink
Other dining options include Cavelier Room, Smitty's Family Restaurant and Cafe la Salle located nearby.
Facilities that will make your stay enjoyable include an outdoor seasonal pool, a basketball court and a play area.
Wireless internet is available in the hotel rooms for free.
Guest Parking
Public parking is possible on site for free.
Number of rooms: 44.
Free Wi-Fi in rooms
On-site parking lot
24-hour reception
24-hour security
Flat-screen TV
- Breakfast
- Fax/Photocopying
Room view
- Garden view
Room features
- Free Wi-Fi in rooms
- Air conditioning
- Heating
- Sitting area
- Free toiletries
- Flat-screen TV
- AM/FM alarm clock
Room decor
- Carpeted floor
Rooms and availability
Bed options:
- 2 Queen-size beds
- 2 Double beds
- 1 Queen-size bed
Max occupancy:6 persons
Bed options:
- 2 Queen-size beds
Max occupancy:5 persons
- Nearby
- Restaurants
- City landmarks
Rating by category:
Guest rating
Booked the motel, after getting to the room, noticed mold on the ceiling and water damage. When I went to the front desk, was told they had no other rooms and that they would not return my money. Ended up staying at another hotel. Do Not Stay Here, management is very rude, the place should be shut down by the health department.
If you want to get sick, stay here, your room will have mold.
Mold Fill Rooms.
Very good
CLean room, comfort bed, free wifi internet connection and free refrigerator.
Guest rating
The outdoor pool was nice and clean…that's about it.
This hotel was one of if not the dirtiest hotels I have ever stayed in. There was at least an inch of dust on the head board, walls where dirty and trim was falling apart…room looked like it had not been maintained for the last ten years. The blankets smelt like dust and there was no clock, the tv looked like it was from the late 80s. I would rather sleep in my car before staying there again.
Very good
obviously had been doing renovations, new windows, new ac unit redone the bath/shower area around the tub. moved me to a different room with great access for the dog very quiet clean, funishings just looked a little worn new mattresss, on both beds!
door was very questionable, needs to be replaced light did not work in one of the bathrooms playground, not really so much, needs to be updated before you can advertise you have one
Quiet and relaxing. I would highly recommend getting a room with a whirlpool, well worth it. A/c is quiet, and the water pressure in the shower is strong. The fridge works great and tv has alot of channels, and the rooms have free Wi-Fi. We will definitely stay there again on our next weekend trip to Kingston!
Pour la modique somme de 85$, nous avons eu une chambre avec 2 lits queen qui était très propre et confortable, de plus l'hotel est bien situé et facile à trouver et l'acceuil était chaleureux.
L'hotel serait peut être due pour quelques rénovations. Le panneau d'alarme incendie indiquait une panne. Il n'y avait pas de séchoir à cheveux ni de boîte de kleenex dans la chambre.
Very good
Extremely clean room and comfortable beds
While a caa rate was advertised, it bEcame clear in speaking with the manager that this was a euphemism for "lowest rate". As a caa member, I don' t think it's right that caa is advertised but that the owners don't work with caa at all and instead use it as a marketing ploy. Take the caa off the signage!
Great welcome! Reception came across more as family rather than motel proprietor. I was offered a free upgrade as soon as I arrived. Great value for money, rooms larger than I thought and great location for the thousand island cruise. Free WIFi, discount vouchers(saved $30), swimming pool and car parking adds to the experience. Though advertised as a 2* this is a great choice for a short stay and a base for outdoor activities
Not close to the Kingston waterfront so need a car. Not an issue for us!
Very good
The Staff was so nice, a real family run hotel! The pool was very nice after a long day of driving. I would stay at the hotel again, but feel I cannot reccomend it to my friends.
It was a little outdated and run down. Only a couple of very thin towels, no kleenex in the kleenex drawer, mold on the bathroom ceiling, and older bedding!
Budget motel located beside good grocery store. Very friendly staff! We had a brand new, very comfortable mattress. We slept very well as it was also very quiet (despite being beside a very busy, major street). Bathroom was very small but the second sink area outside of it is a very handy feature. Great prices but they also are a reflection of some of my points below.
Though quite clean, items such as doors, windows, fixtures, etc. all need replacing due to being verrrry outdated, rusty, worn etc. We did look beyond those things and really appreciated why we were there. Comfort, quiet, good prices but others may not be so kind. I appreciate that it takes time and money to accomplish these things and I wish the owners much luck. They are very pleasant people and seemed to appreciate my advice / comments to them. We will stay there again :)
Guest rating
The tap in the shower was coming out of the wall. In general if I had known the motel already I wodn't have stayed there
Easy to find, plenty of parking, direct access to room from the parking lot, large rooms, clean.
Would have been nice to have more towels and a box of Kleenex in the room.
Everything was very good. The room, the beds were comfortable and even the owners, they were helpful.
Very good
Friendly staff, clean and comfortable. Facilities and grounds could use some updating.
Went to Fort Henry and Gananoque!!! Had a wonderful stay!! The fridge in the room was put to good use as was the lovely, crystal clear pool. Awesome shower too! Bed was very comfortable


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Former WWE Wrestler Tyler Reks Reveals WWE Pay Structure Details
Former WWE Wrestler Tyler Reks recently spoke publicly about how WWE pay works.
Since WWE wrestlers are not actual employees but rather independent contractors, much of their expenses are not included.
Here is what Reks, real name Gabe Tuft, had to say"
Here's a perfect example: I hate to spill my salary on the internet, but when I left I got a bump to $100,000 a year. But a third of it goes to road expenses. The only thing they pay for is your flight. You pay for your own hotel, and car, and food. Could you imagine trying to eat out five times a day? As a body guy, you have to maintain your physique and that means eating five times a day. Spending all your money trying to maintain that? Good luck. Then Uncle Sam takes 20%-30%. You guys do the math and see how much I walked away with, which was next to nothing. I was making more money fresh out of college as an engineer fresh out of college in an entry level position than being on TV. That's gotten worse from what I hear. They do a ton of tours, and some guys would be making $30,000-$40,000 in the day. I went to Europe and I think I brought home $5,000. Then there was one tour I flew overseas and did a ten day tour and made less than $2,000. I made less than $500 a show. The video games are getting worse – guys used to get paid up to $100,000 for being in the video game and now it's much, much less. The reading audience will think that's a lot of money, but when you're on the road it's not. We don't all have busses like John and Punk and Show. You're breaking your body to barely pay the bills.
Check out the whole interview at wrestlinginc.com
Tags: Tyler Reks


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Don't you just love this little creepy guy? When I saw the Boo To You Stamp Set in the holiday catalog, I knew I had to have it. I fell in love with the creepy images and could see it would make some fun cards that kids would love.
For this card, I chose Basic Gray as the card base and layered it with a piece of Smoky Slate embossed with the new Stylish Scroll Embossing Folder. I used the #5 Stitched Rectangle Die to cut out the Granny Apple Green layer and used the #4 Stitched Rectangle Die to cut out the Whisper White layer that I stamped the creepy guy and the sentiment. I colored the creepy image with the Light Granny Apple Green Stampin' Blends and the Mango Melody Stampin' Blends Combo.
Before I placed the Granny Apple Green stitched rectangle layer , I wrapped a piece of the Granny Apple Green Textured Weave Ribbon around the middle of the embossed layer. I then adhered the Granny Apple Green stitched rectangle with Multipurpose liquid glue. The whisper white layer with the creepy image and sentiment were popped up with stampin' dimensionals. To make the image pop, I added 2 googly eyes from the Assorted Googly Eyes embellishment. A must have for your Halloween creepy cards.
The inside is just a layer of Granny Apply Green cardstock and I stamped the sentiments and cat with Tuxedo Black Memento Ink. The Happy Halloween and the cat are from the Wonderfully Wicked stamp set.
I'm so pleased with the way this card turned out and I think it's a cute little creepy card. I will definitely be making more creepy cards with the Boo To You stamp set for some little kids that I know will get a kick out of them.
- Basic Gray Card Base – 5 1/2″ x 8 1/2″ scored at 4 1/4″
- Smoky Slate Layer – 4″ x 5 1/4″
- Granny Apply Green for the Stitched Rectangle Layer – 2 3/4″ x 4″. Inside Layer – 4″ x 5 1/4″
- Whisper White for the Stitched Rectangle Layer – 2 1/2″ x 2 7/8″
- Granny Apply Green Textured Weave Ribbon approximately 4 3/4″
You can purchase the products used to create this creepy Halloween card by clicking on the product images below ↓ . For all other Stampin' Up! products, CLICK HERE and you will directed to my online store where you can shop 24/7 from anywhere in the United States. Thank you!


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The South Pole Telescope
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If you have a question about this talk, please contact Dr Hardip Sanghera.
The South Pole Telescope (SPT) is a 10-m, off-asix Gregorian telescope equipped with a ~1k-pixel, 3-colour, millimetre-wave bolometer camera. The telescope has an unblocked aperture, shielded beams and co-moving ground shields to minimize systematic errors due to scattering. SPT has just started a large survey of galaxy clusters using the Sunyaev-Zel'dovich effect. The goal is to set constraints on dark energy by measuring the redshift evolution of the density of clusters.
This talk is part of the Institute of Astronomy Extra Talks series.
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COEUR D'ALENE — Sometimes it takes the Cape Canaveral launch to get out of bed.
5-4-3-2-1 …
Rain patters the roof like migrating lemmings and the morning is black as an iron skillet.
The woods are sodden, and not something a hunter looks forward to. The inclines are steep and slippery and the cold will eventually sneak, snakelike under soaking wet garments.
By then, the pickup truck with its thermos of coffee is parked miles away and the gray light of dawn opens like an eye as the surrounding forest comes into focus under a smoky sky.
It was one of those mornings that John Partridge of Coeur d'Alene killed his first bull elk with a bow.
It would have been easy to stay in bed, to go back to sleep and blame the weather, but Partridge, a Coeur d'Alene real estate agent, went the other route.
He launched himself out from under the covers, however methodically, and placed his feet on the floor.
By the kitchen light, he made sure he had all he needed for a day in the woods, then walked outside in the dark and drove into the Coeur d'Alene Mountains.
He wasn't alone. His archery equipment was at his side, and all the advice he had heard for more than a decade about hunting bulls with a bow bounced around in his head as the wheels of his pickup left the blacktop.
"I just said to myself, you've got to go," Partridge recalls. "You've got to do this."
When he arrived at the spot he'd been hunting for several years, he parked the pickup truck and went for a hike.
Most of it was downhill.
Since moving to North Idaho a decade ago, Partridge had not killed an elk or a deer with a bow, relying instead on his rifle to put meat in the freezer. Harvesting a bull elk, one that he lured with a mouth call, or a bugle, one whose white-tipped antlers he would see first as the bull quietly zeroed in on his calling; a sneaky, big-shouldered bull elk, the top quarry of Panhandle hunters, was still the game animal that topped Partridge's bucket list.
To harvest a bull with a bow was etched at first place.
After many unsuccessful seasons he had given up on archery for a while, returning to rifle hunting, then within the last couple months again picked up his bow.
"I had let the bowhunting thing flip," he said.
He started pegging a target last summer and generally began to mentally prepare to kill an elk during the archery season.
As he headed downhill that morning earlier this month to a series of benches where elk often browsed or bedded, Partridge recalled the advice of fellow hunters.
"Don't worry about making noise," one hunter had told him years ago. "Elk make noise."
There was the perfunctory sitting in the dark as eyes adjusted to the blackness like a cat.
He used his cow call, waited, then let out a squeal on a bugle.
"Don't bugle big, you may spook away smaller bulls, or even big ones," another hunter once told him.
Partridge had gone through these motions many times before, following the advice of others, using a call to chuckle and squeal and mew.
"So that's what I did," he said. "The rain was coming down, and I was soaked."
And then a bull answered back.
"Close the distance," was the advice of the hunters he knew.
And he did that too. He worked his way closer to the bull and then as day broke, the wind changed.
"It was blowing uphill, in may favor," he said.
The bull responded quietly to Partridge's calls and then fell silent.
Partridge waited. He teased the bull with some whispers.
"You'll usually see antlers first," a hunter had told him.
When he heard something sneaking, popping brush, he questioned the noise. It wasn't a big animal, or was it? The rustling seemed too dainty a noise for a big, hoofed bull elk.
"He wasn't loud. He was quiet," Partridge said. "And then I saw antler tips over the top of the brush and I knew I was in business."
Partridge drew his bow in anticipation and remembered that when bulls sneak in, they often have a hunter pinpointed, knowing the exact location of the source of the calls they have followed.
When that happens, Partridge knew, a bull elk will stare down a hunter.
When the 5x5 nosed its way into the opening, it turned to Partridge and their eyes met.
"He looked at me," Partridge said. "It was a stare down, and I didn't have a shot."
He expected the bull to grunt and jump, turn tail and in a huff of spraying leaves vaporize into the emerging morning.
Instead, something wonderful happened.
Another bull faintly bugled, just loud enough to distract the 5x5 and it stepped into an opening at 25 yards and turned, quartering away from the drawn bow and razor head arrow.
Partridge squeezed the release.
"It was a double lung shot," Partridge said.
The bull still did not run, but walked, leisurely, back downhill toward the bugler and Partridge slipped his cellphone out and called his wife.
"She didn't believe me," he said. "She thought I was razzing her because I was bored."
And then he called his pals and learned if your best friends aren't elated when you tell them you killed a bull, it's because they know you're asking them to get their boots on, and step away from whatever plans they had that day.
"The next four or five hours we spent packing meat up the mountain," he said.
Checking an item off a bucket list can be anticlimactic. It can bring with it the realization that sometimes chasing a goal is more rich than meeting it.
Not for Partridge.
Harvesting the 5x5 was sort of an end game. If he is not able to kill another bull, he's fine with that.
"I don't think I can top that experience," he said.


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On Saturday 21st September the Somali terrorist group Al Shabaab attacked the Westgate Shopping Mall in Nairobi, leading to a 4 day siege on the shopping centre in which at least 39 civilians were killed. The tragic loss of so many lives has shaken Kenya and the international community. Such a profound, international tragedy has led to much speculation and discussion over the motives for and consequences of the attacks.
Where to get the news on why Westgate was a target:
The Other Casualty In The Nairobi Mall Massacre Is Kenya's Economic Progress | The Huffington Post
Nairobi Westgate Mall Siege: Jewish and American Stores Made Shopping Centre Al-Shabaab Target | International Business Times
Why Kenya was targeted:
Why did al-Shabaab target Kenya? | World Bulletin
Why Kenya Is An Inviting Target For Terrorists | NPR
For news on casualties and victims:
Kenya shopping mall attack: Stories of the heroes and the victims- From a boy who took on terrorists to the off-duty SAS soldier | The Independent
Kenya mall attack: dozens more bodies believed buried under rubble | The Guardian
Information on Al Shabaab:
Al-Shabaab leader's ambitions appear to be as complex as his personality | The Washington Post
Al-Shabaab's Global Menace | Wall Street Journal
The 'White Widow' Speculations:
Kenya widens mall attack probe, alert for UK 'White Widow' | Reuters
'White Widow' Samantha Lewthwaite: Interpol Issues Red Notice On Behalf of Kenya | Huffington Post
Who is the 'white widow' Samantha Lewthwaite? | BBC News


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To Quit or Not to Quit [my job]?
As an entrepreneur there comes a point when you have to make the most important decision of your business. Those tough decisions start with none other than the universal "To Quit or not to Quit?" This decision is not based on leaving a task unfinished instead it's a decision to focus on your desires, passions, and dreams. To do what makes you happy and not what just pays the bills.
With this theme I will be referring to entrepreneurs/business owners who have to make that striking decision of whether or not they should quit their jobs.
- Aim to be fulfilled by what you do each and everyday -
When my (now) husband and I started our first company together he was working full-time in Corporate America for an IT company. Right from the beginning in our own company we networked heavily in our community, overdrove the door-to-door sales, pushed our name out there, and started gaining clients rather quickly. In our 2nd year of business we took off in Sales and grew over 600% in gross revenue. We knew that this was more than just a hobby. At this point I made the decision to quit my part-time job to focus 100% on the business. I will never forget the conversation I had with him that year. Our business needed a full-time leader and I was going to need him to focus on the business full-time with me and quit his job. The work load he was under in Corporate America doubled with his leadership position and continual promotions. Forcing him to juggle the tasks of his day job as well as our business growth. He was performing great but it had come to that point where we really had to choose one or the other.
The decision of leaving his job wasn't the biggest issue, figuring outwhen to leave was the greatest debacle.
After much counsel, praying and fasting we planned our next step-we decided that Jamison was putting in his two weeks at his job in December 2013. YES! It was liberating, however very daunting. (Meanwhile we had about 5 months left until our wedding date.) It was definitely a BIG RISK.
I can specifically remember how we both felt in making that decision; it's bittersweet.
On one hand - we were thrilled and excited. We knew this was the right move. (We planned everything to the T to ensure that we had backup plans after backup plans. We made triple sure all pennies were accounted for, etc).
On the other hand - it was scary, it was risky, there were several unknowns, and we kept going back to the drawing board - over and over again - and reviewing our plans. It got overwhelming in a matter of minutes.
Ultimately, through many mental battles, we overcame and we made the firm decision. We were never to go back to Corporate America or work as an employee for anyone else.
For many of you who are juggling both - a full time job and growing a business - you may be wondering - "how do I know when it is the right time for me?" The aim of this blog post is to provide you with a guide to help you make the right decision for you and your family.
**DISCLAIMER: what worked for me and my family may or may not work for other families. This is in no way a guarantee that this will work for any one. This is a simply a guide that could help lead you to making your own decision.**
1. Pick a Date
Don't worry just yet about all of the expenses and incomes. Start simple and PICK A DATE. You might initially start out with several dates in mind, and that's okay. Go ahead and pick one, write it down on a sheet of paper in large letters, and post it somewhere you and your partner will see it every day. And then work towards it.
Once you pick a date, you're next step would be to develop an action plan (see next step).
2. Have a Plan
Now you have a date - this is a great first step. Without a date in mind, your exit from your job will never happen. Now the most important step is to develop a plan to make that happen in reality.
My husband and I created a plan and broke down month by month all of our expenses (business/personal) as well as income (from our jobs, from parents, from savings, from business, etc.)
Then we wrote down when we were expecting each payment, as well as each expense.
Our ultimate goal was to figure out how long we could go without any income (i.e. that steady 9 to 5 paycheck) and give us clarity as to where we really were in terms of finances. I do recommend that you seek a financial advisor or someone you trust to help you break these things down.
TIP: The reason why you should break this down month by month is because some months have significantly more expenses than others for your business and/or your family. For example: December may be higher in expenses vs. March due to Holiday Season. Also, the same goes for income. Depending on your type of business (especially if it's seasonal) you will know which months produce more revenue than others. This way you can really get an understanding of what your financial forecast looks like. I am not a financial planner, but this will give you an idea of where you are in terms of money and how flexible you can be. The cool thing is that once you map this out, some of you may be surprised to see that you may be in better shape than you think. Perhaps you could quit today and not even know it.
3. Stick to the Plan
We had our date and we had our plan - are you ready to go? Now the hard part. Stick to the plan, believe, and have faith.
The hardest part of it all wasn't quitting our jobs, it was sticking to the plan. Things can get tough and plans could change. We were afraid of failing and losing everything. I'm sure many of you can relate to this. However in the end we reminded ourselves why we were doing this in the first place. I encourage you all to stick to the plan, you'll be glad that you did. In times where you are in need of help and support - seek a mentor or a trusted family member. It's important to stay encouraged and uplifted, and of course, surround yourselves around those types of people are encouraging and uplifting. Trust me, you can never run out of that!
Remember - No Risk, No Reward.
Remember this too - The only people who really fail are those who give up. This will definitely be a future blog post, as I have many stories to share.
Entrepreneurs and readers of all types - thank you for reading this post. I hope you find useful and practical information which enriches your lives.
Courtney Gavin