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Fks1, with orthologs in nearly all fungi as well as plants and many protists, plays a central role in fungal cell wall formation as the putative catalytic component of β-1,3-glucan synthase. It is also the target for an important new antifungal group, the echinocandins, as evidenced by the localization of resistance-conferring mutations to Fks1 hot spots 1, 2, and 3 (residues 635 to 649, 1354 to 1361, and 690 to 700, respectively). Since Fks1 is an integral membrane protein and echinocandins are cyclic peptides with lipid tails, Fks1 topology is key to understanding its function and interaction with echinocandins. We used hemagglutinin (HA)-Suc2-His4C fusions to C-terminally truncated Saccharomyces cerevisiae Fks1 to experimentally define its topology and site-directed mutagenesis to test function of selected residues. Of the 15 to 18 transmembrane helices predicted in silico for Fks1 from evolutionarily diverse fungi, 13 were experimentally confirmed. The N terminus (residues 1 to 445) is cytosolic and the C terminus (residues 1823 to 1876) external; both are essential to Fks1 function. The cytosolic central domain (residues 715 to 1294) includes newly recognized homology to glycosyltransferases, and residues potentially involved in substrate UDP-glucose binding and catalysis are essential. All three hot spots are external, with hot spot 1 adjacent to and hot spot 3 largely embedded within the outer leaflet of the membrane. This topology suggests a model in which echinocandins interact through their lipid tails with hot spot 3 and through their cyclic peptides with hot spots 1 and 2.
The predominant cell wall component in ascomycetous yeasts such as Saccharomyces cerevisiae and Candida albicans is β-1,3-glucan, and it remains essential even in fungi such as Aspergillus fumigatus and Cryptococcus neoformans, in which other polysaccharides predominate (8, 15, 25, 26, 44). In addition to this structural role, β-1,3-glucan has roles in innate immunity (12) and in fungal diagnostics (35) and as biofilm matrix (32). Moreover, β-1,3-glucan synthesis has emerged as a critical therapeutic target. Until recently, the antifungal agents available for treating invasive candidiasis or aspergillosis were limited to amphotericin B and azoles such as fluconazole. Amphotericin B is fungicidal but toxic; azoles are better tolerated, but their activity is fungistatic (6). Furthermore, resistance represents a serious limitation to azole use, particularly in Candida glabrata (37). Thus, the introduction of β-1,3-glucan synthesis-inhibiting echinocandins, beginning with caspofungin in 2001 followed by micafungin and anidulafungin, represented an important clinical development (6, 7). Echinocandins are well tolerated and exhibit potent fungicidal activity against most Candida species, including those that are azole resistant. Their activity against Aspergillus species is similarly potent, although fungistatic. For reasons that remain unclear, echinocandins exhibit little or no activity against other pathogenic fungi, including C. neoformans (8, 36, 44).
Echinocandins noncompetitively inhibit incorporation of substrate UDP-glucose into β-1,3-glucan (9). Since the substrate is cytosolic and the product is external, this glycosyltransferase reaction is topologically complex and, predictably, involves a membrane protein complex. Although it has resisted full purification, this complex appears to be limited to a small number of proteins, including Fks1, a large (ca. 1,900-residue) integral membrane protein and putative catalytic component, and Rho1, a small GTPase and putative regulatory component (9, 11, 28, 36, 38). C. albicans and related yeasts, including S. cerevisiae, encode Fks2 and Fks3 paralogs of Fks1; however, with few exceptions, these are minimally expressed under normal growth conditions (28, 30), and most molds, including Aspergillus species, encode only a single Fks1 ortholog. In all fungi examined, including intrinsically resistant C. neoformans, gene disruption studies have demonstrated that Fks1 (or Fks2 in fks1Δ strains of S. cerevisiae) is essential to viability (10, 15, 28, 30, 36, 44). In addition to enrichment of Fks1 in partially purified β-1,3-glucan synthase preparations, evidence for their equivalency derives from the fact that mutations conferring high-level echinocandin resistance map exclusively to this protein, specifically to a region known as hot spot 1 (residues 635 to 649 in S. cerevisiae Fks1) or, less commonly, hot spot 2 (residues 1354 to 1361) or the recently described hot spot 3 (residues 690 to 700) (9, 18, 36). Moreover, specific mutations within these hot spots confer differential echinocandin resistance (e.g., 16-fold-decreased caspofungin but unchanged micafungin susceptibilities), consistent with direct interaction between echinocandin and Fks1 (18).
Beyond this, however, our understanding of Fks1 structure, its role in β-1,3-glucan synthesis, and the mechanism by which echinocandins inhibit this activity is very limited. An initial attempt to directly map the echinocandin binding site on Candida albicans Fks1 by cross-linking with an azido derivative was unsuccessful (13, 39). A similar attempt to map the binding site for the substrate UDP-glucose using partially purified β-1,3-glucan synthase from Neurospora crassa also suffered from low specificity; however, among the cross-linked peptides were four clustered between residues equivalent to 1070 to 1273 in S. cerevisiae Fks1 (41). More recently, the characterization of temperature-sensitive S. cerevisiae mutants implicated specific Fks1 residues in β-1,3-glucan synthase activity, as well as other phenotypes, including polarized growth and endocytosis (33).
Here we explore one important aspect of Fks1 structure: its membrane topology. Initial in silico hydrophobicity analysis of the Fks1 sequence led to a model incorporating 16 transmembrane helices (TMH) divided into two clusters flanking a central region believed to represent the catalytic domain (11). While many of the predicted TMH appeared to be robust, others were questionable, and for all, their orientations (cytosolic versus external) were ambiguous. Of most concern with respect to understanding echinocandin action, the hot spots have inconsistent topologies in this initial model, localized either internally (hot spot 1) or externally (hot spots 2 and 3). This is difficult to reconcile with the apparent dominant effect of individual hot spot 1, 2, or 3 resistance-conferring mutations, which is most readily explained by a model in which these hot spots come together to form a single echinocandin binding site. It is similarly difficult to reconcile an internal hot spot with echinocandin structure: cyclic peptide coupled to a lipid tail essential to activity (2, 7). Analogous to the lipopeptide antibiotic daptomycin (1), the lipid tails most likely target echinocandins to the outer leaflet of the membrane.
A widely used approach to experimentally analyze membrane protein topology in S. cerevisiae involves construction of C-terminal fusions to hemagglutinin (HA)-Suc2-His4C, followed by complementary assays of His4C expression (requiring localization of that C terminus to the cytosol) and Suc2 glycosylation (requiring localization to the endoplasmic reticulum and subsequently to the outer leaflet in the case of plasma membrane proteins) (21, 42). This method has been used for topological analysis of numerous membrane proteins, including Pmt1 (43), Stt3 (22), Nce102 (27), and Arv1 (47). We combined this method with in silico analyses to generate a model of S. cerevisiae Fks1 topology. In this model, all three hot spots are external, adjacent to the membrane or partially embedded. The central domain, with newly identified homology to structurally defined glycosyltransferases, is cytosolic.
Strains, media, and reagents.S. cerevisiae strain STY50 (MATa his4-401 leu2-3,112 trp1-1 ura3-52 HOL1-1 suc2::LEU2) and plasmid pJK90 were obtained from J. Nickels (47). S. cerevisiae BY4742 fks1::KANMX was obtained from Open Biosystems (Huntsville, AL). Yeasts were cultured on YPD medium (1% yeast extract, 2% peptone, 2% dextrose) or, where indicated, synthetic defined SD-ura (DOB plus CSM-ura; Sunrise Science Products, San Diego, CA) or SDh-ura-his (DOB plus CSM-ura-his supplemented with 2 mM histidinol; Sunrise Science Products and Santa Cruz Biotechnology, Santa Cruz, CA). DNA primers (see Table S1 in the supplemental material) were obtained from IDT (Coralville, IA) and FK506 from Tecoland (Edison, NJ).
Protein sequences.Fks1 sequences from Saccharomyces cerevisiae (NP_013446), Candida albicans (XP_721429), Aspergillus fumigatus (XP_751118), Cryptococcus neoformans (O93927), Arabidopsis thaliana (NM_123045), Phytophthora infestans (XM_002998508), Phaeodactylum tricornutum (XM_002177407), Chlamydomonas reinhardtii (XM_001691217), and Toxoplasma gondii (XM_002370473) were obtained from GenBank. Fks1 sequences from Rhizopus oryzae (RO3G_10349) and Allomyces macrogynus (AMAG_00576) were obtained from the Broad Institute Fungal Genome Initiative.
Construction of plasmids for topology analysis.All constructs were generated by gap repair homologous recombination. PCR products were amplified with Phusion high-fidelity DNA polymerase (New England BioLabs, Ipswich, MA) as recommended by the manufacturer using S. cerevisiae genomic DNA as the template, with forward primer ScFks1-1TAGF and various reverse primers (e.g., ScFks1-300 TAGR) fusing Fks1 C termini as indicated below to the HA-Suc2-His4C tag. Both primers incorporated 33 bases at their 5′ ends complementary to pJK90 sequences flanking its unique SmaI restriction site. These products along with pJK90 that was SmaI linearized and alkaline phosphatase treated (New England Biolabs) were cotransformed into competent S. cerevisiae STY50 cells using the EZ Yeast Transformation II kit (Zymo Research, Irvine, CA). Transformants were selected on SD-ura. To confirm desired plasmid recombination, yeast lysates (19) were screened by PCR with Taq polymerase (New England BioLabs) and vector primer pJK90F or pJK90R (see Table S1 in the supplemental material) in conjunction with various FKS1 coding region primers (data not shown).
Plasmids were subsequently rescued, purified, sequenced with appropriate primers, and retransformed into STY50 before growth assays and protein analysis. For this, overnight SD-ura cultures (4 ml) were pelleted, resuspended in 2 ml SCE buffer (1 M sorbitol, 0.1 M sodium acetate, 60 mM EDTA [pH 8.0]) supplemented with 2.5 mg Zymolyase (ICN Biochemical, Aurora, OH) and 15 μl β-mercaptoethanol, and incubated at 37°C for 1 h. The resulting yeast spheroplasts were pelleted and processed using a plasmid miniprep kit (Denville Scientific, Metuchen, NJ). Plasmid products were transformed into Escherichia coli DH5α with selection on Luria broth agar containing 20 μg/ml ampicillin, purified from overnight cultures, and retransformed into STY50.
Growth assay of His4C expression.Approximately 1,000 cells in 3 μl sterile water were replica plated on SD-ura and SDh-ura-his. Plates were incubated at 30°C for 72 h before being photographed.
Western blot assay of Suc2 glycosylation.Cells from 5-ml SD-ura overnight cultures were pelleted, washed twice in 5 ml H2O, and resuspended in 150 μl sample buffer (10 mM Tris-HCl [pH 7.4], 140 mM NaCl) supplemented with 5% Triton X-114. Cells were mechanically lysed by addition of 100 μl cold glass beads and by vortexing 4 times in 2-min cycles at 4°C. Lysates were incubated on ice for 2 h and then centrifuged at 100 × g for 5 min at 4°C. Supernatants were collected and layered on top of 0.5 volume of 6% sucrose in sample buffer. These were incubated at 34°C for 20 min, followed by centrifugation at 2,000 × g in a swinging bucket rotor. The resulting crude membrane pellet was resuspended in 75 μl sample buffer. Aliquots of 18 μl each were supplemented with potassium acetate (pH 5.8) to a final concentration of 80 mM, followed by addition of 1 μl endoglycosidase H (Endo H; Roche, Indianapolis, IN) for treated samples or 1 μl H2O for mock controls (21 μl total volume), and incubated at 37°C for 1 h. Paired Endo H-treated and untreated samples were electrophoresed through a 5% SDS-polyacrylamide gel for 2 h, followed by overnight transfer to a nitrocellulose membrane. Membranes were blocked in 5% skim milk-TBST (10 mM Tris-HCl [pH 7.5], 100 mM NaCl, 0.1% Tween 20) and probed with a horseradish peroxidase-conjugated antihemagglutinin antibody (1:20,000 dilution; Santa Cruz Biotechnology).
Construction of plasmids for functional analysis.For site-directed mutagenesis, gap repair with two partially overlapping PCR products was employed. One product was generated using ScFks1-1TAGF and a reverse primer encoding the desired mutation (e.g., ScFks1-D1102AR), and the second was generated using ScFks1-1876*TAGR (incorporating a stop codon before the tag) and a forward primer typically encoding the same mutation (e.g., ScFks1-D1102AF; for the R319A mutation, only the reverse primer encoded the mutation). Both products were cotransformed with SmaI-linearized and alkaline phosphatase-treated pJK90 into competent BY4742 fks1::KANMX with selection on SD-ura. PCR screens, plasmid rescue, purification, sequencing, and retransformation were performed as described above.
To construct C-terminal truncations, PCR products were generated with ScFks1-1TAGF and reverse primers (e.g., ScFks1-1830*TAGR) that incorporate a stop codon following the indicated Fks1 residue from 1830 to 1876. Products were cotransformed with SmaI-linearized and alkaline phosphatase-treated pJK90 into competent BY4742 fks1::KANMX, with selection and processing as described above.
To test the function of mutated or truncated Fks1, ∼1,000 cells were spotted on SD-ura with or without FK506 (1 μg/ml). Plates were incubated at 30°C for 72 h before being photographed.
Computational methods.In silico TMH predictions were generated using TMHMM (http://www.cbs.dtu.dk/services/TMHMM) (23) and PRO-TMHMM (http://topcons.cbr.su.se) (46). Sequences were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2). Homology detection and structure prediction were performed using HHpred (http://hhpred.tuebingen.mpg.de/hhpred) (17).
In silico modeling of Fks1 topology.Significant advances in transmembrane helix (TMH) prediction have been made since the original S. cerevisiae Fks1 model was proposed in 1994 (5, 46). Also, many additional Fks1 sequences are now available from evolutionarily diverse fungi, including representatives of all 4 fungal phyla, e.g., the ascomycetous yeast C. albicans and mold A. fumigatus, basidiomycete C. neoformans, zygomycete Rhizopus oryzae, and chytrid Allomyces macrogynus. Alignment of these sequences using ClustalW2 (see Fig. S1 in the supplemental material) confirms previous observations (9) that conservation is concentrated in the central domain (S. cerevisiae Fks1 residues ∼715 to 1300), with 43% identity among the 6 diverse species. In contrast, the N- and C-terminal domains (residues ∼1 to 715 and ∼1300 to 1876) are relatively divergent, with 13% and 11% identities, respectively.
TMHMM is the most widely used and validated single sequence TMH prediction algorithm (5, 23, 29, 31). For S. cerevisiae Fks1, TMHMM predicts up to 18 TMH (Fig. 1; see also Fig. S2 in the supplemental material), with probabilities ranging from high (1.0) to low (0.4 to 0.6). Seven of these TMH localize to the N-terminal domain between residues 445 to 724, and 11 localize in the C-terminal domain between 1302 to 1822. As an initial test of these predictions, Fks1 sequences from the 5 additional diverse fungi listed above were similarly analyzed by TMHMM (Fig. S2). Predicted TMH locations and their probabilities are listed in Fig. 1. Comparison of these locations (allowing for minor shifts) with the ClustalW2 alignment (Fig. S1) indicated that 16 of 18 TMH predicted for S. cerevisiae Fks1 are similarly predicted for the 5 other fungal Fks1. The exceptions involve the final N-terminal domain TMH and the third C-terminal domain TMH (TMH 702–724 and 1403–1422, respectively) (Fig. 1).
To complement the single sequence-based TMHMM, Fks1 topology predictions were also generated using PRO-TMHMM (46), using S. cerevisiae Fks1 as a query. Analogous to our approach above, PRO-TMHMM incorporates evolutionary analysis, but it does so more comprehensively by generating a consensus sequence. A total of 15 TMH were predicted: 6 in the N-terminal domain between residues 445 and 712 and 9 in the C-terminal domain between 1296 and 1821 (Fig. 1). The PRO-TMHMM predictions coincide with those described above, with the following exceptions: N-terminal domain TMH 675–697 and 702–724 predicted by TMHMM are replaced by TMH 691–712, and C-terminal domain TMH 1402–1422 and 1738–1760 predicted by TMHMM are not predicted by PRO-TMHMM.
Topology of Fks1 N-terminal domain.To directly test these in silico predictions, we employed Fks1 fragment C-terminal fusions to the HA-Suc2-Hic4C topology reporter (21). The C termini were selected to fall within loop regions before or after predicted TMH (Fig. 1). Constructs were generated by gap repair between FKS1 PCR products and SmaI- and alkaline phosphatase-treated plasmid vector pJK90 which were cotransformed into S. cerevisiae STY50 with selection on SD-ura medium. In this his4 strain, cytosolic localization of the C-terminal fusion permits His4C-mediated growth on SDh-ura-his medium (SD-ura-his medium supplemented with histidinol). Alternatively, C-terminal fusions destined for external localization undergo N-linked glycosylation within the endoplasmic reticulum on the Suc2 moiety; this is detected by a band shift following endoglycosidase H (Endo H) treatment in a Western blot with anti-HA antibody. Together, these complementary assays provide a robust system for topological analysis.
S. cerevisiae STY50 expressing Fks1 residues 1 to 300 fused at its C terminus to HA-Suc2-Hic4C (designated Fks1-300 fusion) demonstrated robust growth on SDh-ura-his medium and no detectable band shift upon Endo H digestion, both indicating cytosolic localization (Fig. 2). This was also predicted by TMHMM (see Fig. S2 in the supplemental material), although the localization predictions provided by these algorithms are less reliable than their TMH predictions, as evidenced by external predictions for the N termini of other Fks1 proteins (Fig. S2). For an Fks1-445 fusion, a similar cytosolic localization was determined (Fig. 2), confirming the predicted lack of TMH within this N-terminal region. In contrast, an Fks1-475 fusion grew minimally on SDh-ura-his and on a Western blot exhibited a clear Endo H-mediated band shift, indicating external localization (Fig. 2). This supports formation of predicted TMH 445–467 (Fig. 1).
The growth and band shift assays similarly indicated cytosolic, external, and cytosolic localizations, respectively, for Fks1-531, Fks1-561, and Fks1-610 fusions (Fig. 2). These results support predicted TMH 497–519, 532–552, and 567–589, respectively (Fig. 1).
Importantly, with respect to the region representing echinocandin resistance hot spot 1 (residues 635 to 649), both growth and band shift assays of the Fks1-650 and Fks1-669 fusions indicate external localization (Fig. 2). TMH 621–643, responsible for this localization, was predicted with only low probability in S. cerevisiae Fks1 but with high probability in all other fungi (Fig. 1). Unexpectedly, an Fks1-637 fusion also showed external localization (Fig. 2), suggesting that the actual TMH may be shifted upstream of the predicted TMH. This alignment is supported by the alternative algorithm PRO-TMHMM, which predicts a TMH from residues 614 to 634 (Fig. 1).
The cytosolic localization of the Fks1-715 fusion (Fig. 2) provides support for either TMH 675–697 (predicted by TMHMM) or TMH 692–712 (predicted by PRO-TMHMM). To resolve this, an additional Fks1-685 fusion was constructed; its external localization supports TMH 692–712. Thus, as with hot spot 1, hot spot 3 (residues 690 to 700) is externally located, albeit largely membrane embedded. The final, inconsistently predicted TMH 702–724 of the N-terminal domain (Fig. 1) was not supported, since an Fks1-726 fusion remained in the cytosol (Fig. 2).
Topology of Fks1 central domain.The Fks1-715 and Fks1-726 fusions described above tentatively localized the central domain to the cytosol. This was confirmed with the Fks1-742 and Fks1-1008 fusions, which similarly exhibited robust growth on SDh-ura-his and no band shift following Endo H treatment (Fig. 2).
For Fks1 regions beyond residue 1008, Western blot analysis using fusions initiated at residue 1 was complicated by inadequate resolution of Endo H band shift due to the large size of the resulting protein. Consequently, additional fusions were constructed that initiated at Met residue 1204, located ∼100 residues upstream of the first predicted TMH of the C-terminal domain. Growth on SDh-ura-his, however, was determined for fusions initiated at both residues 1 and 1204.
As expected, an Fks1204–1294 fusion (i.e., initiated at residue 1204 and fused at 1294 to HA-Suc2-His4C) was cytosolically localized as indicated by growth on SDh-ura-his and lack of band shift (Fig. 3), consistent with its inclusion in the central domain. Conversely, an Fks1204–1354 fusion was external (Fig. 3), in support of predicted TMH 1302–1322 (Fig. 1). Combined with fusion Fks1-715, these results define the limits of the cytosolic central domain as residues 715 to 1294.
Topology of Fks1 C-terminal domain.As with Fks1204–1354 above, an Fks1204–1380 fusion was similarly external (Fig. 3). This was unexpected, since TMH 1357–1379 was consistently predicted (Fig. 1), but was confirmed by an Fks1204–1410 fusion which also indicated external location (Fig. 3). Thus, these results demonstrate an external location for echinocandin resistance hot spot 2 (residues 1354 to 1361).
TMH 1403–1422 was also not supported, since an Fks1204–1425 fusion remained external as indicated by both absence of growth on SDh-ura-his and band shift following Endo H treatment (Fig. 3). Indeed, cytosolic localization appears to require extension to residue 1470. This was demonstrated by an Fks1204–1470 fusion which exhibited a clear lack of band shift, although growth on SDh-ura-his was minimal for unclear reasons (Fig. 3). The same pattern was observed with an Fks1204–1493 fusion; however, an Fks1204–1550 fusion provided both strong growth on SDh-ura-his and lack of band shift indicating cytosolic localization (Fig. 3). We interpret these data as supporting TMH 1440–1462 or, in its absence, TMH 1472–1489.
Subsequent TMH 1560–1582 and 1598–1620 are supported by both growth and band shift assays with Fks1204–1590 (external) and Fks1204–1630 (cytosol) fusions (Fig. 3); both are predicted with high probability in diverse fungi (Fig. 1). Predicted TMH 1641–1663, however, was not supported, since an Fks1204–1666 fusion remained cytosolic. This was confirmed by the band shift and growth assays for the Fks1204–1707 and Fks1-1707 fusions, respectively; the growth assay for the Fks1204–1707 fusion was negative for unclear reasons (Fig. 3).
Interestingly, a TMH predicted only in C. albicans involving residues 1718 to 1736, equivalent to S. cerevisiae residues 1706 to 1724 (see Fig. S1 in the supplemental material), appears to be supported by the external location of an Fks1204–1738 fusion. The final two predicted TMH, TMH 1738–1760 and 1801–1822, are supported by fusions Fks1204–1775 and Fks1204–1821, respectively (Fig. 3). Thus, the C terminus of S. cerevisiae Fks1 is external, as confirmed by fusion Fks1204–1876 (Fig. 3).
Functional analysis of selected N- and C-terminal residues.As demonstrated above, both the N-terminal (residues 1 to 445) and central domain regions of Fks1 are cytosolic; however, unlike the latter, the former is evolutionarily divergent, with the exception of an ∼25-residue segment which is conserved in Fks1 homologs from species representing three kingdoms: fungi, plants, and protozoa (Fig. 4A). In particular, several residues are universally conserved, implying functional importance. To test this, we mutated one of these, R319, to A and examined the effect on Fks1 function. This was accomplished by plasmid-based expression of the mutated gene in an fks1::KANMX strain and then blocking of expression of the "backup" gene FKS2 with calcineurin inhibitor FK506. Cells expressing wild-type Fks1-R319 were unaffected by FK506, while those expressing Fks1-R319A exhibited clearly reduced growth in the presence of FK506 (Fig. 4B).
The external C terminus of S. cerevisiae Fks1 (residues 1823 to 1876) is enriched in N, S, and T residues (13 within the final 30 residues; see Fig. S1 in the supplemental material) that could serve as glycosylation sites. This is true as well for the C termini of C. albicans and A. fumigatus Fks1, although their sequences are otherwise divergent (Fig. S1). To test the functional importance of the C terminus, truncations were constructed. In the fks1::KANMX strain background, plasmids expressing wild-type Fks1 or an Fks1Δ1871–1876 truncation were unaffected by FK506, indicating full function (Fig. 4C). However, an Fks1Δ1861–1876 truncation led to reduced growth in the presence of FK506, and further truncation eliminated growth (Fig. 4C).
In silico and functional analyses of the putative catalytic central domain.On the primary sequence level, homology between the Fks1 cytosolic central domain (defined by the topological analysis described above as residues 715 to 1294) and known glycosyltransferases cannot be discerned by conventional search tools such as BLASTP (data not shown). As a more sensitive alternative, HHpred employs profile-hidden Markov model searches at the primary and secondary structure levels to identify distant homologs to proteins with experimentally defined structures (17). Indeed, using the central domain as a query, the top 2 matches returned by HHpred are glycosyltransferases that employ UDP-sugar substrates, i.e., have the same basic catalytic activity (and presumably catalytic mechanism) as β-1,3-glucan synthase. With all other regions of Fks1 as queries, matches were minimal and random (data not shown). The central domain match to glycosyltransferases encompasses two sections of Fks1, residues 825 to 881 and 1079 to 1158 (Fig. 5A). Related glycosyltransferase matches were similarly identified by HHpred using Fks1 central domain regions from other fungi, as well as from distantly related orthologs found in plants and oomycetes (data not shown). The glycosyltransferase region homologous to S. cerevisiae Fks1 residues 1079 to 1158 includes the DXD motif which coordinates Mn2+ interaction with the diphosphate of UDP-glucose substrate, and it is catalytically essential (3, 14). In Fks1, the corresponding sequence is DAN (residues 1102 to 1104 [Fig. 5B; see also Fig. S1 in the supplemental material]). To directly test its role in Fks1 function, a D1102A mutation was generated using the plasmid-based approach described above, with expression in the fks1::KANMX strain. This mutation led to a modest decrease in growth rate relative to the wild-type R319 control and to a complete loss of function in the presence of FK506 (Fig. 5C).
Fks1 belongs to the GT-A superfamily of glycosyltransferases (20) in which the catalytic residue is a D (less commonly E) located ∼100 residues downstream of the substrate-binding DXD motif (4, 40). ClustalW2 alignment of Fks1 orthologs from highly divergent species identified a conserved stretch of six residues from 1192 to 1197 terminating in D (Fig. 5D). Generation of a D1197A mutation at this putative catalytic residue completely blocked Fks1 function, as indicated by a loss of growth on FK506 medium (Fig. 5C).
By combining analysis of sequences from evolutionarily diverse fungi with HA-Suc2-His4C fusion technology in S. cerevisiae, we have defined the membrane topology of Fks1, the putative catalytic component of β-1,3-glucan synthase. This topology (Fig. 6) includes 6 TMH in the N-terminal domain, 7 TMH in the C-terminal domain, a cytosolic N terminus, a cytosolic central domain, and an external C terminus. All 6 TMH consistently predicted for the N-terminal domain were experimentally confirmed, as were 7 of the predicted TMH for the C-terminal domain. On the other hand, the remaining 4 TMH predicted for the C-terminal domain were not confirmed. This emphasizes the importance of experimental verification; similar discrepancies between in silico and experimental topology results have been reported by others (22, 27, 43, 47).
Fks1 has emerged as an important target for antifungals, notably the clinically approved echinocandins (24), as well as investigational agents, including the enfumafungins (16, 34) and recently described piperazinyl-pyridazinone derivatives (48). Since numerous mutations conferring resistance (or in some cases hypersensitivity) to echinocandins have been mapped to three distinct hot spots within Fks1 (residues 635 to 649, 690 to 700, and 1354 to 1361), we reasoned that correlating these mutation sites with Fks1 topology would shed light on the mechanism behind this resistance. Furthermore, since some mutations confer differential resistance (e.g., to micafungin but not caspofungin), implying a direct interaction of an echinocandin-specific side chain with the mutated residue (18), Fks1 topology should also shed light on the echinocandin mechanism of action.
In this regard, a major finding of this work is the observation that all three hot spots are externally located (Fig. 6). This is reassuringly consistent with the echinocandin structure, which includes a common cyclic hexapeptide and a lipid tail essential to activity (2, 7). Specifically, we propose that the lipid tail anchors the drug in the outer leaflet, where it interacts with membrane-embedded regions of Fks1 within hot spot 3 or immediately upstream of hot spot 1. (With regard to hot spot 2, predicted TMH 1357–1379, which would have anchored hot spot 2 in the membrane, was not experimentally supported; however, it is quite possible that these residues associate with the membrane without spanning it.) This positions the peptide moiety for interaction with the externally located hot spot residues. We further speculate that the three hot spots are juxtaposed in the three-dimensional structure of Fks1 to form a single echinocandin binding pocket. While there is no direct evidence for this, it is consistent with the apparent dominant effect (i.e., high-level resistance) of specific mutations at any one hot spot.
A second important finding is the cytosolic location of the conserved central domain (residues 715 to 1294), consistent with its presumed role as the catalytic core of β-1,3-glucan synthase. Until recently, evidence in support of this role has been limited. Four Neurospora crassa Fks1 peptides mapping to this domain, equivalent to S. cerevisiae Fks1 residues 1070 to 1082, 1156 to 2273, 1249 to 1261, and 1262 to 1273, were cross-linked in vitro to an azido derivative of UDP-glucose, the substrate for β-1,3-glucan synthase (41). However, specificity in this cross-linking study was apparently limited, since an additional peptide mapped to the equivalent of residues 650 to 665, demonstrated above to be external, and other peptides mapped to proteins not known to bind UDP-glucose. By characterizing in vitro glucan synthase activity of S. cerevisiae temperature-sensitive Fks1 mutants, Okada et al. (33) implicated multiple central domain residues between 853 and 1047 in catalysis. Most recently, two mutations conferring resistance to a novel, nonechinocandin inhibitor of β-1,3-glucan synthase have been mapped to residues 1175 and 1297 (48).
In light of this mounting evidence, we were encouraged to reexamine the cytosolic central domain for homology to structurally defined glycosyltransferases using HHpred. The most significant Fks1 region identified in this analysis, residues 1079 to 1158, overlaps with sequences corresponding to two of the UDP-glucose cross-linked peptides and includes a variant of the DXD motif involved in binding UDP-glucose substrate in known glycosyltransferases. Mutation of the D1102 residue within this motif abolished Fks1 function. Mutation of a candidate catalytic site residue, D1197, identified on the basis of its location within a highly conserved region ∼100 residues downstream of the DXD motif similarly abolished Fks1 function. Needless to say, this initial mutational analysis is far from exhaustive, but it does lead to hypotheses regarding residues involved in Fks1 catalysis that can be more definitively tested in future studies.
Two additional implications of this work relate to the Fks1 N terminus (residues 1 to 445) and C terminus (1823 to 1876). Although the N terminus is minimally conserved, it has one or more essential functions, as indicated by the effects of R319A mutation described here and the temperature-sensitive mutations (residues 146, 302, 329, and 335) described previously (33). Various roles can be envisioned for the N terminus that are consistent with its cytosolic localization as determined here; for example, it may interact with Rho1. With respect to the C terminus, four different Fks1 fusions (Fks1-1821, Fks1-1876, Fks1204-1821, and Fks1204-1876) demonstrated clear external phenotypes, consistent with previously reported results for Fks2 using the same methodology (15). (This appears to contradict the results obtained with a strain expressing Fks1 with C-terminally fused green fluorescent protein [GFP] [45]; however, the fluorescence associated with GFP fusions to other integral membrane proteins with external C termini such as Stt3 [22; http://yeastgfp.yeastgenome.org] implies that this is not a reliable marker of cytosolic versus external topology.) As with the N terminus, the C terminus has an essential role as indicated by truncation analysis. Within ascomycetes, at least, this C-terminal region is enriched in N, S, and T residues (see Fig. S1 in the supplemental material), which may serve as N- or O-linked glycosylation sites required, for example, for appropriate Fks1 trafficking to the membrane.
In conclusion, the model for Fks1 membrane topology presented here forms a framework for future studies of Fks1 structure-activity relationships. These studies will include detailed analysis of the central domain, since the evidence for its role as the catalytic component of β-1,3-glucan synthase is now compelling. Also, further modeling of the interaction between echinocandin, Fks1, and the plasma membrane can now be pursued, with the goal of designing expanded-spectrum agents with improved antifungal activity.
We thank M. Villasmil, J. Nickels, T. Daly, and J. Alaro for providing reagents and invaluable advice.
This work was supported by National Institute of Allergy and Infectious Disease grant AI075272.
- Received 9 March 2012.
- Accepted 2 May 2012.
- Accepted manuscript posted online 11 May 2012.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/EC.00082-12.
- Copyright © 2012, American Society for Microbiology. All Rights Reserved.


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A More Robust Process for Dealing with Link Removal RequestsBlog Post: August 11, 2014
These are some great points, yet another example of something that a shady competitor could do to easily bring down your SEO. I'm curious though, have any web administrators responded to your email template saying that they ARE in fact victims and that the link removal requests aren't genuine?
Either way just because it hasn't happened yet doesn't mean it won't and I think this is a common sense standard for people to adopt. Great work


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2011 November 07
This was our first visit to this public dog park and we were well pleased! Incredibly nice dogs there and very nice people as well. This visit I took some overview photos to give you some idea of what the park is like, and a few dog shots...mostly of these two little cuties wrestling. The park is on Lake Shore Drive where it meets up with Walter Rd. Entrance is double-gated and there is running water, picnic tables and shade.Read More


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Barkatullah University Madhya Pradesh has declared the result of bba 1st year .Those candidate how are interested bba result this link will help you . If you are a bba 1st year student and doing bba from
Barkatullah University, Bhopal (Madhya Pradesh)
BBA 1st Year Examination Results
Click Here to Check BBA First Year Result
BBA from Barkatullah University Madhya Pradesh
Bachelor of Business Administration or BBA is a 3-year professional undergraduate course in Business Management, the minimum eligibility for which is class 12th from a recognized educational board with minimum score of 50%.The course is divided into six semesters and comprises variants like BMS, BBM and BBS.
Some of the major highlights of the course have been mentioned below.
|Course Level
|3 years
|Examination type
|10+2 from a recognized educational board
|Admission Process
|Entrance exam based
|Course Fee
|INR 1 to 2.5 lacs
|Average Starting Salary
|INR 1.5 to 2 lacs
|Top Recruiting Companies
|EY, HP, ICICI Bank, Microsoft, Mc Kinsey and Company, IBM, Sony, Deloitte, Nokia and others such
|Job Positions
|Financial Analyst, Business Development Executive, Project Manager (IT), Data Analyst, Human Resources Manager, Operations team leader, SAP Consultant etc.
About Barkatullah University Madhya Pradesh
Barkatullah University, formerly known as Bhopal University, was established in 1970 in the capital city of Madhya Pradesh and rechristened in 1988 as Barkatullah Vishwavidyalaya in the living memory of the great freedom fighter Prof. Barkatullah who belonged to Bhopal. The University has been accredited 'B' grade by NAAC in 2015.
University campus covering an area of approx. 400 acres of land is located along Hoshangabad highway at a distance of about 3 km from Habibganj Railway Station. University covers almost full spectrum of higher education, offering courses in its affiliated Colleges and University Teaching Departments in the faculties of Arts, Social Science, Science, Life Science, Engineering, Law, Commerce, Education, Physical Education, Technology, Home Science and Management. Territorial jurisdiction of the university extends to eight districts namely Bhopal, Sehore, Vidisha, Raisen, Hoshangabad, Harda, Betul and Rajgarh.
Similar Posts:
- Bubhopal Result
- MPBSE (MP Board) Exam Class 10th,12th Results 2018
- Veer Bahadur Singh Purvanchal University Result of Regular/Private Students (Session 2017-18)
- Rajasthan University Admission
- Deen Dayal Updahya Gorkhapur University Result for M.Sc. (Home Science) Extension Education II Sem
- Mp Bhoj University Result
- TMBU Admission 2018. Application Form, Eligibility, Dates
- JNVU Result 2016 Name Wise. BA 2nd Year Exam Result 2017


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Found this article on Crosswalk.com.
1. I wish someone had told me just to be myself. "I am a people-pleaser by nature, so for me, not being prepared to handle being a pastor's wife with my personality was a heavy burden to carry early in our ministry."
2. I wish someone had prepared me to deal with criticism of my husband and me. "It was hard to deal with negative experiences, conflicts, or criticisms, especially in relation to my husband and our area of ministry. So I would harbor feelings of resentment when it came to ministry and my man."
3. I wish someone had reminded me that my husband is human. "I wish someone had told me that my husband could not be God for me. I was disillusioned at first to find out that he indeed is just a man."
4. I wish someone had told me that others were watching us (the glass house syndrome). "Even though they are watching us, we don't need to be controlled by what they expect of us."
5. I wish someone had told me there are some really mean people in the church. "I was really surprised. I had to learn not to pay too much attention to them or they would get me down."
6. I wish someone had told me how much my husband needs me to build him up. "I need to be his cheerleader. Dealing with critics in the church is difficult. He needs to hear that I respect him now more than ever."
7. I wish someone had told me that my schedule will never be normal again. "Your husband will be very busy. Expect that. But come alongside him in the areas of time management and organization."
Here's the link for the full article: http://www.crosswalk.com/faith/women/7-things-pastors-wives-wish-they-had-been-told.html


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Rick Ross Is So Rich That He Doesn't Even Need Lights In 'Gold Roses' Video
His chains, teeth, and ceiling of gold flowers light the way for him
His chains, teeth, and ceiling of gold flowers light the way for him
He gets fitted for a suit for two whole minutes
It was God's plan to break out the excavator
'Port Of Miami 2' is the sequel to his 2006 debut, 'Port Of Miami'
Cue the money counter sound!
Swizz Beatz brings the ad-libs to the party
The 'Love, Simon' duet gets a romantic remix
They're playing the game by their own rules and winning
Leonardo da Rozay
Industry friends have taken to social media to show their support
He tells MTV News why Rick Ross and J. Cole's shout-outs are so important
The rapper gives his trademark parables an intriguing political makeover
'Let's unify. Let's talk business. Let's hold hands. Let's get this money.'
His new album draws crystal clear lines in the sand
It'll have you seeing dollar signs
'Trap Trap Trap' features Young Thug and Wale
Gaby Wilson sits down with Rick Ross to discuss his forthcoming ninth studio album 'Rather You Than Me'
Hear Ross's 'I Think She Like Me' and Larsson's 'So Good'
Barack Obama's relationship with rap music will be sorely missed
Time to banish 2016 to the annals of history
They're 'bout to 'Buy Back the Block'
Molly Lambert on the Atlanta kingpin's chilly, grown-up new album
Rick Ross signs Isa Muhammad, who rapped for him on the street, to Maybach Music Group.


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Nebuliser Omron Compessor Nebuliser NE-C801
Supplier: NZ Health Delivery
Omron NEC-801 Compressor Nebuliser
The Omron NEC-801 is the smallest, lightest, most convenient tabletop nebuliser on the market. Omron's advanced design and engineering allows it to administer medication as effectively as table top models three times its size. The Virtual Valve Technology offers an affordable and efficient alternative for patients experiencing asthma, COPD or other respiratory conditions.
Product Features
- Compact and easy to use
- Virtual Valve Technology (V.V.T)
- Safe and easy to clean
- Small partilcle size of 3um MMAD
- LightWeight 270g
- Quiet
- Includes carry case with accessory compartment
- AC adapter


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UNITY of Greater New Orleans, which leads community campaigns to end homelessness, is hiring:
1) Fiscal Grants Manager who will review and process sub recpient reimbursement requests, monitor fiscal compliance, manage project budgets, and provide technical assistance to improve fiscal controls of partner agencies. Must have excellent math and communication skills, attention to detail, and ability to learn grant management software systems. Preferred candidates will have experience in cost or fund accounting, government grants, and non-profit accounting. Email resume and cover letter explaining interest by October 23 to email@example.com.
Job Description CoC Fiscal Grants Manager Oct2019
2) Contract Manager to work with partner agencies to help coordinate the work of partner agencies, develop system improvements, improve outcomes, and ensure compliance with HUD and other funding sources. Required: Demonstrated commitment to social justice, excellent communication skills, ability to use Excel. Experience serving homeless, low-income, or special needs populations preferred. Email resume and cover letter explaining interest by October 15 to email@example.com.
Job Description for CoC Grants Manager Sept2019
3) Housing Specialist to assist homeless people with mental & physical disabilities to remain stable in housing. Work w/clients to calculate rents, complete annual re-certifications, & housing inspections. Required: Demonstrated commitment to social justice, experience providing housing services to low-income people or people w/disabilities, excellent detail in paperwork, basic math skills.
4) SSI Specialist to assist homeless people with mental and physical disabilities to qualify for Supplemental Security Income (SSI). Work w/clients to obtain documentation of disabling conditions & drive clients to appts. Required: Demonstrated commitment to social justice, experience working with people with mental or physical disabilities, excellent writing & analytical skills. Apply by sending resume, with cover letter explaining which job you are applying for, interest & qualifications, to email@example.com.
5) Development/Communications Coordinator: Assisting with fundraising, grant-writing, website updates, social media, blogging, newsletter-writing & production. Required: Well-organized, motivated individual with excellent skills in writing & layout design; attention to detail. APPLY by sending resume, with cover letter explaining qualifications for position & commitment to social justice, to Frances Misenheimer at email@example.com. For more information, see www.unitygno.org.
Volunteer Opportunities
If you are looking for other ways to get involved with our work, please contact us at (504)483-9300 for volunteer opportunities.


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Work neighbor. Customer. Friend. Those are just a few terms INB Peoria's Market Manager Trip James uses to refer to Dr. Khurram Rashid, owner of Rashid Putnam Plastic Surgery. He recently added "workout buddy" to the list when Khurram challenged Trip to run the Chicago Half Marathon. Trip says, "I played rugby and football growing up, but I didn't run long distances. Actually, if you'd have told my 18-year-old self that I'd be running for distance one day . . . my coaches and I would have said, 'Never.'"
But run he did on Sept. 24, finishing his first half marathon just 2:25 minutes behind the experienced runner, Khurram. In return for Trip taking the running challenge – and completing it on a very hot, late September day — Khurram is spending some time in the weight room, something he has not done before.
Trip says, "We challenged each other because we simply wanted to help make each other better. He wanted to learn weight training, and I was open to learning about long-distance running."
They put their plan in action, using any spare time to train, working around the doctor's surgery schedule. 5:30 a.m. on Sunday. 6:15 p.m. on a weeknight.
Trip says it was his time as a wilderness guide that got him through the training and marathon itself. "We'd have to go out a 4 a.m., paddle for hours at a time, and carry a canoe through the woods. You had no choice but to keep going." Trip also ran a 10K in Peoria prior to the half marathon with Dr. Rashid. He learned from that experience. "You need headphones and music. I need a knee brace . . . you learn from your mistakes."
To mark the half marathon, Trip's wife, Colleen James, and Khurram's wife, Dr. Mary Elizabeth Rashid, an orthopedic surgeon with OSF Healthcare, were on hand. Colleen to cheer them on and document the occasion. Dr. Mary Elizabeth ran the half marathon as well.
You are leaving INB's website. INB is not responsible for the content of
the linked site. Please be advised that you will no longer be subject to, or under the protection of, the privacy
and security policies of our website. We encourage you to read and evaluate the privacy and security policies of
the linked site, which may be different than ours. Please confirm that you would like to continue.


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Season 4
Episode 1: American Alien (Season Premiere)
Episode 5: "Parasite Lost"
Plot Synopsis
Colonel Haley makes a surprising decision regarding Supergirl. Kara writes s series of articles highlighting aliens in National City in the hopes that humans will stop being so fearful of them. Unfortunately, the article puts some of the aliens in harm's way.
Full Recap:
Kara, Alex, J'onn, James and Lena are at a bunch on the roof of Kara's building. Alex, J'onn and Kara catch up as James and Lena discuss the increase of nationalist groups are stepping out in support of Guardian. Brainy interrupts after having drunk too many mojitos and ends up briefly passing out. Later Nia arrives, and Kara introduces her to J'onn and Alex. Brainy sobers up and finds the group and notices Nia at the party. Nia asks why he never found her after the incident at the Pizzeria and he spouts off all her information but says he never contacted her because she said just to find her. Alex tells the group about Colonel Haley being assigned to the D.E.O. and that she is nervous to meet her. Kara mentions that she has an interview with Amadei Deios, an alien healer and recent recluse.
Children of Liberty Hideout:
Agent Jensen is doubled over in pain from the parasite Liberty inserted into him. Lockwood tells Jensen to follow him, and the two approach an alien who generates fire chained up as a prisoner. Liberty instructs Jensen to touch him and when he does the parasite inside Jensen absorbs the alien's powers, transfers them to him, but also killing the alien.
The D.E.O.:
Alex meets with Colonel Haley who commends her for the work she has done and encourages Alex to keep up the good work.
Apartment of Amadei Deios:
Kara and Nia arrive for their interview and witness Amadei heal a young alien boy. During the interview, Amadei says that he can only heal aliens but expresses his wants of being able to heal humans as well.
James is watching a video of Ben Lockwood on the computer when Lena walks in and recognizes Lockwood from their earlier encounter when he went to beg Lena to continue to buy steel from his father (Episode 3).
The D.E.O.:
Brainy locates Jensen at the National City Promenade as Alex assembles a strike team to capture Jensen.
National City Promenade:
A street performing alien shapeshifter is entertaining the crowd as Jensen shows up. As he is about to touch the entertainer Supergirl lands and engages Jensen wants to steal. Jensen is able to touch Kara and hold her at bay long enough to absorb the shapeshifters powers. As Alex and the D.E.O. agents converge on the scene Jensen shifts into a little girl and is able to escape.
The D.E.O.:
Alex briefs Colonel Haley on the events of the situation that just occurred at the National City Promenade. Alex orders Kara stand down from the fight as a precautionary measure.
Kara and Nia are reading through the comments section from Kara's interview with Amadei interview. At first they find ones that support and praising the healer, but they also find negative comments from people, several trolling comments which are death threats directed against all aliens.
Alien Bar:
J'onn is leading Fiona's support group meeting as some of the attendees mention that they have stopped using the image inducers. At the end of the meeting Amadei's helper, Cassian, arrives and asks J'onn for help. He and
When they arrive at Amadei's apartment, J'onn discovers that the healing metahuman in ailing health and his amulet has been stolen. Kara arrives after hearing the news and J'onn informs her that Amadei is dying.
Cassian's grants J'onn the permission to read Amadei's mind and sees an image of the last person he thought about or saw. J'onn calls Brainy at the D.E.O. and morphs into the person in Amadei's mind and has him run a visual scan looking for the person. It turns out that the woman is named Elizabeth Hawkings, and Kara and J'onn fly to Hawkings' home.
Hawking's Residence:
Kara knocks on the door and talks to the girl while J'onn sneaks into the house to search for the missing amulet. As J'onn moves through the house he is spotted by Hawking's mom, who screams. Elizabeth runs in the room and J'onn and Kara tell the two women that Elizabeth was the last one to see Amadei before he became ill. Elizabeth reveals that she's never met Amadei and her mother says that he is Elizbeth's deadbeat father.
Children of Liberty Hideout:
Jensen is still in pain and tells Agent of Liberty that he is having trouble maintaining the powers he absorbs. Lockwood tells him not to worry that they are in the process of acquiring something to help him and make him invincible.
The D.E.O.:
Haley, Alex, and Brainy are working on the way to deal with the Parasite-Jensen. They plan to use a version of the method that that was used last time to overload Parasite.
National City Media Luncheon:
James and Lena arrive at National City Media where the two are approached by Ben Lockwood. Lockwood praises James' work as Guardian but James turns around and informs Lockwood that he doesn't agree with Lockwood's anti-alien stance. Even after hearing this, Lockwood continues trying to convince James that the vast majority of Americans are anti-alien and that CatCo and The Guardian would be great additions to the movement.
Kara's Apartment:
Kara and J'onn are talking about the revelations they learned about Amadei Deios. Kara laments that she wrote a glowing piece about Amadei earlier, disgusting about his actions against his daughter. J'onn isn't so sure the Hawkins' story is entirely accurate, so the pair does some digging, and finds out that Amadei and his wife more than the one night stand she let on about. Alex arrives with pizza and tells the two that working with Haley is not as bad as she thought it was goin to be.
Hawking's Residence:
J'onn and Kara return to the Hawkins residence, where Amadei's wife admits that he broke her heart, which caused her to intentionally keep Elizabeth away from her. Mrs. Hawkins reveals that she's the one that cut out Amadei's amulet and sold it to the Children of Liberty she met on the dark web. She also says that her daughter found the letters from her dad that her mom had hidden and left.
They soon realize that Amadei's amulet is most likely going to be used to help the Parasite powered Agent Jensen maintain his powers and drain more aliens.
Amadei's Apartment:
Brainy figures out that Jensen is probably going to attack the attendees of the vigil for the dying Amadei. Alex and the D.E.O. agents converge on the spot as Jensen starts killing aliens. The plan to overload Parasite with a nuclear blast requires that they enclose a several block radius in a magnetic dome that Brainy built. Haley is running the mission from a van with Brainy as the D.E.O. arrive on the scene and immediately begin fighting Jensen. Alex shoots Jensen with the concentrated blast but they need to increase the power du to the amulet Jensen is wearing. Brainy informs them that there will be mass casualties, but Haley orders Alex to shoot Jensen. Alex spots Kara and tells her to help move civilians out of the dome. As is grabbing people, she is confronted by Jensen.
National City Media Luncheon:
The mayor announces that a mass attack is happening in midtown and Lockwood speaks up, and suggests that James should go help out as Guardian.
Amadei's Apartment:
Jensen finds himself in a standoff with Kara and Alex. Alex tries convincing Jensen to stop what he is doing and appeals to the person she knows Jensen was as an agent. Haley is ordersed Alex to kill Jensen, but Alex ignores the order as the rogue DEO agent removes Amadei's amulet, and falls to the ground.
Up in the apartment, J'onn reinserts Amadei's amulet, and the alien is almost instantly healed. He's introduced to his daughter as the two make amends. Amadei also thanks J'onn and Kara for helping him to finally heal a human
The D.E.O.
Colonel Haley congratulates Alex on her work earlier in the day and reveals that Jensen is still alive, being kept at the desert facility, but braindead. She then goes on to comment to Alex that she felt the J'onn was an imposter and never supported him running the D.E.O. but is glad it back in human hands. She even reveals that she helped launch the campaign to remove J'onn from power. Alex tells her that J'onn is not only her mentor but also her family. Haley says that Alex had better watch herself and makes Alex salute her before she leaves.
Alien Bar:
J'onn is having a drink and the bartender mentions that another patron has inquried about him looking into a case for her.
James and Lena are discussing the day's events, as James tells Lena he needs to get to the bottom of the anti-alien nationalist movement and to do that he will nee to infiltrate their
group. Lena warns him that is dangerous but James says that he knows Lena will help him.
Kara tells Nia about a pitch a weekly column which would feature a different National City alien each week, allowing people to get to know them and see that they are just the same as humans.
Children of Liberty Hideout:
Lockwood is brooding over Jensen's failure when he receives a phone call from James, who says he has thought about what Lockwood told him at the reception and wants to meet up and discuss the matter.
— — — — — — —
Tosses of the "S" Shield
Episode 5 Rating: 3 out of 5 "S-shields"
This was a decent episode which continued to lay the foundation in predictable ways. The overall theme and current socio-political parallels continues to be a bit heavy-handed and forced.
The most predictable revelations were James deciding to infiltrate the Children of Liberty group and Haley being anti-alien, which the show probably has to do to move the seasons plot along.
I still am hoping that we eventually see the CW version of Director Bones becoming involved with the D.E.O.
Colonel Lauren Haley
Created by: Dan Mishkin & Don Heck
First Appearance: Wonder Woman #325 (May 1985)
Death: Wonder Woman #329 (February 1986)
Little is known about Lauren Haley's early life other than she came from a military family, and as a young woman, she joined US Air Force. Haley distinguished herself for being an extremely proficient and trustworthy officer, climbing the ranks to become a Lieutenant, and earning the attention of her superior officers. She was regarded as one of the best officers of her year and was sent to Washington D.C. to work for General Phillip Darnell, as her first assignment for the Secret Intelligence.
Lt. Lauren Haley is a capable and professional military officer, a woman with a strong sense of duty and a genuine will to serve her country and her people, ambitious enough to be always willing to do her best in any situation and in every mission. She's a well-trained soldier, an expert pilot and a skilled martial artist, perfectly able of defending herself in nearly every situation. As pragmatist and level-headed as she is, though, Haley might be too unprepared to face situations that go far beyond what she's been taught in the academy.
Other Thoughts:
J'onn seems to be returning to his comic book roots as a detective.
- In the comics, when J'onn realized he was stranded on Earth he became a police detective in the fictional town of Middletown.
- At the end of the episode he puts a fedora on his head much like his early DCU counterpart
Brainy's dome looks similar to the dome that Tellos /Brainiac trapped the cities during the Convergence event.
Haley orders Alex to salute her which doesn't seem to be proper protocol as the D.E.O. is not part of the military.
I feel that like in her origin (see Who's Who above) Haley is being used without her knowledge and a scapegoat which will play out at the end of the season leading to changes in the operation of the D.E.O.
Next Episode: Call To Action
Everyone gathers for Thanksgiving but Kara is feeling down after her televised debate with Ben Lockwood about the anti-alien sentiment coursing through National City. Meanwhile, James and Lena argue about the best way to handle the Children of Liberty.


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Toby Alderweireld looks set to become the second Atlético defender to leave this week after Atlético agreed a €16 million fee with Tottenham Hotspur.
The Belgian centre-back spent the whole of last season on the south coast of England with Southampton, and was thought to be close to a permanent move.
The Saints even had the option to buy Toby for €10 million at the end of the agreement, but Atleti seem to have instead taken up the option of paying a €2 million penalty fee to cancel the clause, and sell to the highest bidder.
The Tottenham chairman Daniel Levy is reported to have flown to Madrid this weekend to tie up the move. The deal is reported to be worth €16 million plus a potential €2 million in bonuses.
Alderweireld only joined Atlético in the summer of 2013 for €7 million, so the move represents a tidy profit for Atleti.
Chelsea were also interested in Toby, but for once, one of our players has decided to not move to Stamford Bridge. The Belgian has decided to join his compatriots Jan Vertonghen, Moussa Dembele and Nacer Chadli at White Hart Lane.
Following Miranda out of the Calderón, Toby's departure opens up the door to sign another central defender, with Atleti heavily linked with Chancel Mbemba of Anderlecht.


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'It's something that I think about every day:' Greeley woman reflects on raising a baby with a congenital heart defect
February 24, 2019
The day Calista Faith Libretti, at the time 2 months and 1 day old, was released from the hospital, her family rushed straight to her older brother's graduation from University High School in Greeley.
Calista, now almost 11 months, had spent the first eight weeks of her life recovering from open heart surgery. She was born with a congenital heart defect called hypoplastic left heart syndrome, which means half her heart does not work. Because of this, doctors had to operate on her when she was just 2 days old.
Calista Faith Libretti
Age: 11 months
Number of siblings: Three
Parents: Maria & Mike Libretti
Congenital birth defect: HLHS
Open heart surgeries to date: 2
Her mother, Maria Libretti, found out about her daughter's heart issues when she was 20 weeks pregnant. Calista is her fourth child and her three previous children had been born with no health issues.
"A lot of parents told me during this process it's an up and down roller coaster. And it was," Maria said. "One minute she would be doing great, everything would be going fine and then the next minute her blood pressure would drop."
After Calista was born, her parents hoped she would have at least a week before she would have her first surgery. Instead, it was only two days.
"We missed the newborn stage," Maria said. "We didn't get to cuddle her as much as we wanted to. I didn't get to do a lot of skin-to-skin right away. I didn't get to breastfeed. Because of her complications, week to week it was so up and down. I feel like we missed a lot of that."
Only a few months after Calista was released from the hospital, she went back for another surgery. During this one, doctors transplanted some of her own stem cells into her heart, which had been saved when she was born. The procedure is supposed to strengthen her heart.
Calista is the first baby in Colorado to receive this type of operation. She was home two weeks after her second surgery.
According to the Center for Disease Control and Prevention, 960 babies are born with Calista's heart defect every year. About 40,000 births per year are affected by some sort of congenital heart defect.
Maria works as a nurse at the North Colorado Medical Center, and she described her medical knowledge as both a blessing and a curse.
"I think me as a nurse, I see the reality of it," Maria said. "Working in the emergency room, I see how fragile life is and how quick it can go."
However, Maria said employers have been very supportive at both the hospital she works at and for her husband Mike, who works for the city, allowing them the time off they need to take care of their baby.
One of the biggest issues for Calista, according to her mother, is feeding. Because of her heart issues, she gets tired very quickly and only eats so much with her mouth. She has a tube inserted in her stomach to give her nutrition directly.
Calista will have another open heart surgery when she is 2 or 3. After that, she is still not cured and future operations will depend on how strong her heart becomes. If it is not enough, she may need a heart transplant one day.
"It's something that I think about every day," Maria said. "I don't know where we'll be next year, where we'll be in two years."
For her parents, the day to day with Calista can be a challenge because of the amount of care she needs, and the constant uncertainty about her health. The flu and cold season presents a risk that means the family spends a lot of time at home.
"A common cold for us could be life-threatening for her," Maria said. "Her oxygen sits between 75 and 85. Ours is 90 and above. When she's sick, her oxygen goes down even lower. I don't think people realize how serious it is."
However, Calista still goes to the store and attends church with her family every Sunday. Many parts of her life are that of a normal, loving baby and for that her mother says she is grateful, especially after months of fear and difficulties.
"Now I just think when we're home together, she's giving me kisses on the cheek and stuff like that," Maria said. "It's all worth it."
Calista turns 1 on March 24, which her mother said will be a big landmark as 20 to 30 percent of children born with critical heart defects do not live past 12 months. The life spans for those living with HLHS are significantly shorter, with many not living past 40. This uncertainty is something her family must live with.
"We just enjoy every day with her that we can because we don't know what the future will bring," Maria said. "As scary as it is to look at it, you have to."
But for now, Calista remains feisty, full of personality and smiles. Her parents do all that they can and believe in her strength.
"Her middle name is 'Faith' because we have faith in her," Maria said.


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Sleepy Hollow screencaps
250 years after the bloody battle near the quiet town of Sleepy Hollow, events are gaining new strength. Ichabod crane is killed after he beheaded his terrible enemy. Soon they both rise from the dead, the headless horseman rises from the bottom of the river, which was with Crane. For many years they rested dead dreams, but now it is their time. At this time, Ichabod will again destroy his worst enemy and save the inhabitants of the city from the dark forces. Hellraiser rider has already begun his revenge on the inhabitants of the city, every night he goes on his horse for new victims. The police can not do anything in this case, the forces are not equal. To Crane joins one girl who wished to help with Affairs on these awful events. They have to find out why the villain woke up from a dead sleep and what he wants.
Televison show Sleepy Hollow seasons available for download - 1,2,3,4,54 seasons
Sleepy Hollow Season 113 EpisodesDownload
Sleepy Hollow Season 218 EpisodesDownload
Sleepy Hollow Season 318 EpisodesDownload
Sleepy Hollow Season 413 EpisodesDownload


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Service & Parts Specials and Deals at Delehanty Ford
Make the drive to Delehanty Ford to Flushing, near Flint, for all your service and repair needs. The well-equipped team of technicians at our Ford dealership are ready to deal with piratically any kind of auto repair or maintenance concern, including brake work, transmission or engine repair, tire installation, basic tune-ups, and even the basic oil change. We know that you have high expectations, and we enjoy the challenge of meeting and exacting those standards each and every time. Our car dealership offers service and parts specials for your Ford cars, trucks, crossovers, and SUVs so that maintaining its quality is absolutely hassle-free.


text: Easy Rider. One of the best movies ever. I saw it soon after it came out. Had quite an effect on me. The start is brilliant. Throw your watch away, what does time matter, then ride off into an adventure on the open road. 2 guys on 2 bikes and that great feeling of freedom. What bikes, too. Special indeed. I bet I was dribbling, as a 14 year old in the movie theatre. A fantastic collection of sounds too. Steppenwolf nailed it. Very sad to hear the creator, Peter Fonda has died. He wrote the story, starred in and directed Easy Rider. He also had the bikes built to his design. The Captain America bike is worth over a million dollars now and inspired countless bike builders. It still looks cool and radical. {} Ride On Peter. Thanks for the wonderful movie.


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Rose Lily and Freesia Coffin Spray
£160.00 – £300.00 Or from £53.33 each when you split the cost between 3 using Chippin
A truly special scented spray for the top of the coffin which has been expertly designed in a classic style using fragrant lilies and freesias alongside complimentary roses and other matching blooms. Shown here in yellow and white.
Also available in white and green, lilac purple and white, soft pink and white, shades of pink and purple, autumn shades, or mixed colours. Please let us know if there are any favourite flowers you would like included.
Please note we always select the best blooms for your tribute which means that the selection may vary from the picture due to seasonal availability and variety strength.
A complimentary card featuring Sarah's original botanical drawings, with your personal message, will be delivered with your order.
We are of course happy to design a piece for you using other colours and flowers, please telephone 01926 424826 or call in to discuss your ideas.


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- Advanced use of conditional functions and logic
- Dynamic panel
- Use of variables
- Creation and management of widgets library
- collaborative functions
This training is the follow-up of Level 1 Axure : The fundamentals. We see in a more advanced way how to create rich interactions. Always based on practical exercises, participants explore the true potential of Axure !


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SHE OCEAN YOGA (SOY) is collaborating with selected marine protection organisations worldwide.
Coral Guardian is our Partner for our most recent Project #doyogasavecorals. The aim: to build the first SHE OCEAN YOGA coral reef in Indonesia in autumn 2019. Coral Guardian´s local team of divers and scientists has mastered coral transplantation, a technique used to rebuild coral reefs by implanting strong coral particles to artificially provided underwater steel consturctions. The corals are breeded in a coral nursery to help coral reefs grow strong and healthy, and become more resilient against warmer water temperatures.
To be part of the solution SHE OCEAN YOGA is organizing charity flows >> co-hosted by local yoga studios | health institutions | wellness hotels here in Vienna >> enabling YOU to become an OCEAN WARRIOR to help #colortheocean!
Our workshops are usually themed for one of our latest marine protection projects. We, Carmen and Ela the founders of SOY, are inviting you to join the next charity flow.
WHEN: August 30th 2019 | 7-9PM
WHERE: YOGAlounge | Seidlgasse 30 1030 Vienna
AIM: to build the first SOY coral reef in Indonesia in Autumn 2019
LEVEL: No matter if it is the first time you want to try yoga or if you practice daily, this yoga session is designed for beginners and advanced ♡
What are we going to do during the charity flow?
Make some time and create space for two yummy hours of:
♡pranayama to gain strength and focus through guided breathing
♡gentle and at times strong movement during our creative flow designed for you to reconnect to the elemental force of water;
♡and learn new meditation as well as body scanning techniques.
When you will walk out of this yoga class you will most propably feel a sensation of ease and lightness. Why not walk as light as a snowflake - or a sunbeam, whichever you prefer. One thing is certain: yoga is awesome and so is growing healthy corals with your asanas!
We are looking forward to welcome you soon!
We will be happy to keep you tuned about the progress here on our website and on Instagram. If you have any Questions do contact us!


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Our Services
Ketamine Can Help Treat The Following Conditions
"Really excellent staff! This is the first place I've ever been able to feel like I am a part of my care."
"Dr. Jugan and her staff have given me the proverbial new lease on life. The treatment here has given me back the ability to feel joy again."
"Kind, caring, compassionate. Saved me a lot of pain!"
"This is the most significant change ever. I feel so much better. It's a true miracle – OMG!
I'M SO RELIEVED AFTER ONE TREATMENT! You are giving my life back to me! Thank you for studying this field
and taking care of people like me. I'm so happy to have HOPE! I just wanted you to know, thank you for helping me, and don't forget it!"
"Dr. Carol and her staff are compassionate and empathetic. Beautiful, serene office setting. Individual care by a wonderful staff."
"Dr. Carol Jugan and her staff are incredibly caring. I just completed my first round of treatments; they went above and beyond to ensure my comfort, and I felt safe in their care."


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It was the Big Uh-Oh. An inch of lemon poppy seed sludge was left in the bottom of the KitchenAid mixer bowl when I poured out my cake batter. Could the gap between the bottom of the paddle and the bottom of the bowl be out of adjustment?
It was easy to find out and all it took was one thin dime.
It's called the "dime test." Place a dime in the bottom of your steel KitchenAid mixer bowl and attach the paddle. Raise the bowl to its working position, or if you have a tilt head model just tilt the head down. Turn the mixer on at speed 1. If the paddle pushes the dime along all around the bottom of the bowl, there's not enough clearance. If it doesn't move the dime at all there's too much.
But if the beater moves the dime just a quarter to a half inch on each revolution it's jusssssst right. If one of the first two you can lower or raise the beater using the adjustment screw in the neck of the mixer.
My mixer didn't move the dime at all. So I turned the adjustment screw. And it still didn't move the dime at all. So I turned it all the way. Still no joy. The bowl needed to come up a tiny fraction of an inch more, but there was no adjustment left. Now what? My brilliant solution is pictured below.
Just one washer on each of the mounting pegs shimmed up the bowl enough to get a perfect dime test.
1. This method only works with the KitchenAid steel bowl, not the glass or ceramic models.
2. Use the paddle attachment only, not the whisk or hook, for this test.
3. Turn the adjustment screw in the neck of the mixer only. Don't mess with any other screws.
Naturally, I called Nancy Leson immediately to tell her what a genius I was. Nance diplomatically reserved comment on that, but immediately set to testing her standing army of stand mixers.
I was so pleased with my hack that I immediately mixed up a batch of that Rosemary Shortbread we talked about a few episodes back. Hope I remembered to take that dime out first.
"A nickel ain't worth a dime anymore." –Yogi Berra


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New Delhi: Prime Minister Narendra Modi on Monday wished former president Pranab Mukherjee on his birthday and said he prayed for his long and healthy life.
Mukherjee turned 82 on Monday. "Spoke to Pranab Da and wished him on his birthday. His impeccable service endears him to every Indian. I pray for Pranab Da's long and healthy life," the prime minister tweeted.
Mukherjee was born this day in 1935 in Mirati, a village in the Bengal Presidency of British India (now in Birbhum district, West Bengal).


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Vintner's House
Get away and enjoy private time with your family and friends in the Southern Styrian vineyards. You can now stay at the Vintner's House, our exclusive holiday residence directly on the premises of the winery.
As the first rays of sunlight fall on your face, a breakfast basket full of all kinds of delicious specialties is waiting for you outside the door. In no time at all, a table is set by the pool and you are enjoying the amazing view, all against thephenomenal backdrop of the vineyards. Nowhere else does a croissant taste this good. While the adults enjoy a glass of Muskateller sparkling wine, the kids dive into the pool. What more could your heart desire?
Room for up to eight people
Two families, a group of friends—six to eight people feel completely at home in our SKOFF ORIGINAL Vintner's House. Three double rooms, two single rooms, two baths with shower and/or bathtub, 3 WCs offer plenty of privacy. Everyday amenities are also available: washing machine, dryer and air conditioning. Park your car comfortably and safely in the garage.
Overview of the Vintner's House:
Total living space 350 m²
3 double rooms
2 single rooms
2 bathrooms with shower and/or bathtub
3 WCs
Kitchen with stove and oven, microwave, refrigerator, coffee machine
Terrace and green space with adjacent vineyard
Swimming pool (12m x 6m)
Well-stocked wine refrigerator(for a charge)
Flat screen TV
Tiled stove
WLAN throughout the house
Washing machine and dryer
Air conditioning
Additional space for sunbathing near the vineyard
Let's meet at the tiled stove
On cooler days, everybody meets at the cosy tiled stove for a pleasant evening watching the flat screen TV. Would you like a good glass of wine? No problem! The wine refrigerator in the vintner's house is well stocked with treasures from the SKOFF ORIGINAL wine cellar. If you get hungry, simply walk over to SKOFF ORIGINAL Hospitality, and there is no need to warm up the stove.
Culinary artists welcome
Do you prefer to stir the pot yourself? Passionate cooks can live it up in the large, fully equipped kitchen of the Vintner's House. Eat in the living room or on the terrace, whichever you prefer. No worries about having enough room here—the Vintner's House is spacious!
Exploring Gamlitzer Eckberg
If the weather cooperates, everyone will be drawn outdoors: The area surrounding the picturesque Eckberg lures you into taking a variety of hikes and exploring. If you want to leave your car behind, you can get around easily in our guest taxis. Gamlitz and its Motorikpark exercise zone—afavourite with children—is only a few minutes away by car. In town, you can find everything you need for daily life: supermarkets, a bank and a variety of boutiques and shops.
Price: EUR 385 per night
Price for 6 nights or more: EUR 355 per night
Minimum stay: 2 nights
Large breakfast basket: EUR 12 per person/day
Alternatively, you can eat breakfast at SKOFF ORIGINAL Hospitality.
(50 m away)
Final cleaning: EUR 70, one-time fee
No pets allowed.


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BurgaBox Review: The BEST Burger You Will Ever Make + Piles of Sides
BurgaBox Review: Ready to make the best burger ever? Take our BurgaBox challenge to see if you can feast on the entire box of yummy goodness. The first ever burger box subscription easily rates five stars and then some. The burgers are piled high with meat, cheese, and toppings. The sides are mouthwatering – convenient, no prep, heat & serve style. A food coma waiting to happen, well worth every bite.
BurgaBox Video
The Full BurgaBox Experience – Unboxing & Cooking BurgaBox
What Is BurgaBox?
From the founders of The Boston Burger Company, the BurgaBox is a burger subscription box that sends you all of the ingredients you need to make a huge feast including your piled high burger, heavenly delicious mac & cheese, the perfect potato wedges, baked beans, and coleslaw. You can choose the BurgaBox of the Month Club to get a monthly themed BurgaBox or select a one-time purchase of a feast kit and extra sides.
BurgaBox Cost: starting at $59
BurgaBox Coupon $10 off
Click here for $10 Off your BurgaBox of the month subscription using our exclusive BurgaBox promo code: BESTSUB10
What's In BurgaBox?
BurgaBox of the Month Club Subscription Details
Your monthly themed BurgaBox ships on the 3rd Wednesday of every month. BurgaBox will announce the theme of the box on the 1st of the month, so you can decide to enjoy, pause, cancel, or renew your subscription.
BurgaBox Review February 2017 – The Muffaletta Burger
I have immensely enjoyed every part of the cooking and eating experience of BurgaBox. You get two gigantic burgers piled high with toppings for a burger-lover's paradise. I've always been terrible at making burgers at home, but these burgers were over the top delicious. You can't fail with the ingredients they give you for the most amazing burger you will ever make.
These burgers are rightfully famous too – The Boston Burger Company won Rachael Ray's battle of the best burger and Guy Fieri stopped by and did an episode of Diners, Drive-Ins, and Dives. So not only are you getting competition winning burgers, but you're able to get them at home. You don't have to travel to Boston just to try these famous burgers!
The Burgers
We received the February BurgaBox themed after the famous Muffuletta sandwich, first created in 1906 at Central Grocery in New Orleans, LA. I had the pleasure of eating a Muffuletta sandwich from Central Grocery when I visited NOLA in 2015, so I can honestly compare the quality and taste. I enjoyed the Muffaletta Burger far more than the Muffuletta sandwich from Central Grocery.
February's BurgaBox, The Muffaletta Burger, was over the top delicious. The olive tapenade was so good, I piled on as much as the bun could hold. BurgaBox's creation of the Muffaletta Burger was outstanding.
The BurgaBox Experience
The burgers were really easy to prepare. All you have to do is season the meat, cook, and top with the other meats and cheese. Heat up the bun, chop the lettuce and tomato, then layer it on. The sides come already prepared. Just heat them up and put them on your plate.
BurgaBox Mac & Cheese Review
If you're a huge fan of mac & cheese you may be saying enough about the burger, what about the mac? The BurgaBox Mac & Cheese for February was Jambalaya Mac n Cheese and it was phenomenal. It is the best macaroni and cheese I have ever eaten at home. It had some sausage in it, macaroni, cheese and whatever else made it taste amazing.
BurgaBox Potato Wedges Review
I don't know how they do it, but the potato wedges come in a really thin coating of awesomeness. Add a little oil to them and bake. They come out perfect.
February's BurgaBox came with Bayou Fries – potato wedges, cajun seasoning, and remoulade.
BurgaBox Baked Beans & Coleslaw Review
Baked beans and coleslaw all taste about the same to me. That being said, they were pretty good. BurgaBox's baked beans are ready to heat and serve. They coleslaw is ready to put on your plate.
BurgaBox Nutrition Facts?
My BurgaBox review has shown how delicious this food is going to be, but one thing we noticed is that BurgaBox leaves out the nutrition facts. Not showing the nutritional information may be misleading, but you can bet on the box being high calorie. My husband and I only needed to share one burger and it was still way more than we could eat. The two burgers plus sides are easily a 4 person meal!
Subscribing to this box once a month, we say don't worry about the nutrition facts and just go for it!
BurgaBox Review
Ready to have an amazing better-than-restaurant quality burger experience at home? Sign-up here for your BurgaBox experience today. Use BurgaBox promo code: BESTSUB10 for our exclusive offer of $10 off your BurgaBox of The Month Subscription. It's a genius gift idea too.
BurgaBox is so sure you'll love it, they offer a 100% money back guarantee. We'd love to know what you think after your BurgaBox experience. Sign-up here.
Share your subscription feedback with others by leaving a comment or review. Help us grow by sharing, liking, or saving this to Pinterest.


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Beyond Marketing
BizReport : : November 27, 2019
Expert: How 2020 will impact digital campaigns
With Thanksgiving upon us, the countdown is now on for January, updated campaign and marketing strategies, and whatever else the new year brings. Just what might merchants and brands find with the change of the calendar? We asked a digital expert how blockchain might impact marketers in 2020.
Kristina: As 2020 approaches, how should brands and marketers be updating their campaign strategies?
Gowthaman 'G'Man' Ragothaman, CEO, Aqilliz: There are two key trends that will influence strategies.
The fragmentation of platforms to reach consumers will continue to rise on the back of new and emerging OTT. A fresh look at the "subscription vs subsidy" model of advertising funding the platforms versus the opportunity to directly reward the consumer for communication is definitely needed.
The second trend is the increasing awareness of safe, secure and consent-driven use of consumer data and the related increase in both the volume and velocity of data. Marketers will need to take a deep and hard look at their data strategy and how they can get a unified view of their consumer at one end and on the other, identify what can be done with data in a more agile way that influences outcomes.
Kristina: We're hearing more and more about the difference in personalization and individualization - what are your thoughts on this?
G'Man: Personalisation is an approach to use a particular consumer's data to give that consumer something back in return as a reward, one that enhances and/or makes the consumers experience better. In my personal view, individualisation takes this one step further where the data is used to prescribe something from a position of authority, where a specific need or want is asked by the consumer. Personalisation can be made serendipitous, whereas individualisation is addressing a particular need, upfront.
Kristina: How important is it for brands to stay true not just to their brand's image but to the wants/needs of their core shoppers?
G'Man: This is extremely important. A brand has a particular personality and stands for certain values that differentiate itself from other competing brands. The needs and wants of the core customers can only be enhanced if there is some consistency in the brand's positioning, otherwise, the brand can quickly degrade itself to a commodity.
Kristina: How can data help brands better engage as we enter 2020?
G'Man: Right now, companies have access to untold amounts of data and overall, the marketing industry, in particular, is still grappling with what can be done with this data. The ideal approach is to test a hypothesis with the data, verify it, and fine-tune it. Secondly, we also need to "cut the junk" from the cacophony of information and make some "music" out of it. We need to create "events" out of the data, that can then be meaningfully attributed to brands sales or growth. These "events" can then help in exchanging value with consumers, in return for the sharing of their information.
Kristina: We started hearing more about blockchain this year - what is happening with blockchain currently?
G'Man: "Is blockchain a solution looking for a problem?" -- you may have heard this oft-quoted question a number of times over the past year. It is sufficient to say that across many industries, we have moved on from the position of identifying specific problems, issues or opportunities that need a solution, to implementing the technology and solving the problems at hand. In this process, we have also identified problems that cannot be solved by blockchain. As we reach the end of 2019, I think there has been a tremendous focus on the areas that blockchain or the wider distributed ledger technology can play a meaningful role, driving both efficiencies and effectiveness.
Kristina: How will this impact 2020 and beyond?
G'Man: 2020 is the year that we will significantly graduate from identifying use cases of blockchain, to providing proof of concepts and delivering business benefits. At Aqilliz, we have identified three core areas where blockchain can help marketers - building trust, providing transparency, and delivering convenience. Blockchain has the potential to become a very valuable facilitator to brands, platforms and the consumers -- this is Aqilliz's core proposition.
Kristina: How might blockchain help brands and marketers improve their transparency?
G'Man: Transparency is a very powerful word. Blockchain is a team sport and cannot improve transparency on its own. It needs willing partners to come on board who have a genuine interest in making this industry a better place. When partners are willing to collaborate, the improvements in campaign metrics are significant. Collaboration helps in managing the campaign in a much more agile way that helps not just brands, but equally the platform and its consumers. In a simple way, there are too many "point solutions" to solve many problems in the AdTech ecosystem today - from audience profiling and discovering the right inventory, to measuring whether an impression is fully viewed or not, and whether the impression appeared at the right place, right time or in the right context -- the technological interventions are endless. These point solutions are now being used as a "post facto" report but unfortunately the system is not able to assemble all the information at the same time in order to reconcile and authenticate that the impression that is truly delivered in the correct way -- blockchain helps in doing this whereby transparency is applied for delivery; not just for reporting.
Tags: advertising, advertising tips, advertising trends, Aqilliz, ecommerce, mobile marketing
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Product Description
Evident Crystal on Talisman: Rose Quartz and Rose Quartz Beads
Orgonite Materials: (Crystals) Clear Quartz, Rose Quartz, Smoky Quartz, Black Tourmaline, Magnetite, Black Mica; (Metals) Copper, Iron, Zinc, Aluminum, Graphene; Resin
ROSE QUARTZ is the pure stone of love – for oneself, one's partner, children, family, friends, community, the Earth, the Universe and the Divine. Meditating with Rose Quartz brings an envelope of love around oneself and activates the heart chakra. Healing the heart of its wounds and reawakening its trust are among Rose Quartz's gifts. Its soothing vibrations are a balm to the emotions, and they calm and cleanse the entire auric field. It engenders the release of tension and stress, the dissolution of anger and resentment, the dispelling of fear and suspicion and the rebirth of hope and faith. It links one's personal heart to the heart of the Earth and the Heart of the Universe. Its love vibrations can penetrate to the cellular level, reprogramming the cells for joy and longevity rather than despair and death. Rose Quartz can stimulate the crown, third eye and throat chakras, bringing them into harmony and unity with t he heart. Even the lower chakras respond favorably to the abundance of love energy which flows from the heart under the influence of Rose Quartz.
-Chakras: Hearth (4th)
-Element: Water
-Physical: supports the heart in healing from trauma and/or disease
-Emotional: aids in releasing past wounds, teaches trust and hope
-Spiritual: encourages spiritualilty of love, links one with the Great Mother "Feminine Energies"
-Keywords: love, gentleness, emotional healing, release of stress, uniting with the Divine
All crystal descriptions are from,"The Pocket Book Of Stones: Who They Are & What They Teach" by Robert Simmons. (Revised & Expanded Edition)
PAKU QI Talismans
Paku Qi Talismans are unique crystal art pieces that function as energy healing tools, to support one's bio electromagnetic system. They may be of assistance during meditations, in crowded environments, day to day activities, and just for extra support.
PQT helps with boosting, balancing-optimizing, stabilizing-fortifying one's biofield. They radiate uplifting and healing scalar frequencies that works with the aura (bio-electromagnetic energy): which are subtle energy fields that permeate the living body. PQT enhances energetic flow throughout meridians, giving the wearer more vitality, and may assist in clearing one's energy blockages.
Each Paku Qi Talisman is created with the intentions based upon:
- Crystal/Gemstone Healing
- Color Therapy
- Reiki Energy
- Shamanic Practices
- Biofield Boost System TM
Harmoniks Incorporated presents our own "homegrown" BIOFIELD BOOST SYSTEM TM (scalar technology)
Our Biofield Boost System TM is a collection of frequencies that have imbued (programmed) for over 100 hours into our crystals and "Orgonite" material using a frequency generator. It affects the bio electromagnetic field of the human body creating uplifting synergistic vibrations.
Combinations of crystals are: Combinations of metals are:
-Clear Quartz -Zinc
-Smoky Quartz -Copper
-Rose Quartz -Iron
-Black Tourmaline -Aluminum
-Magnetite -Graphene
-Biotite (aka Black Mica)
PACKAGING includes:
(1) hand-carved 4X6 wooden box
(1) small selenite stick
(1) 5X8 velvet bag
(1) Paku Qi Flyer
-Handle with care
-Crystals are fragile
-Do not get wet due to adhesive
-Crystals maybe cleaned with clean dry/wet rag
-Occasional cleansing of the crystals is recommended different ways.
For example:
-Smudging (sage, cedar, sweetgrass, lavender, copal, myrrh, frankincense,etc.)
-Crystals (selenite, citrine,etc.)
-Energy clearing techniques (visualization, intention, Reiki, etc.)
- Crystals
- Metals
- Resin
- Hologram
- Leather
- Metal Jump Rings
- Glitter
- Length: 2 inches = 5.08 centimeters
- Width: 2 inches = 5.08 centimeters
- Height: 1 inch = 2.54 centimeters
- Necklace Length = 18 inches
Contact Us
email: email@example.com


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From a redesigned website to a new mobile app and from a planogram booklet for store managers to helpful "SmartTips" for customers, the SmartStop brand was completely revamped to have a consistent visual system, portraying a friendly and approachable look.
The mobile app allowed customers to easily "tag" their belongings within their storage space using QR codes for easy tracking.
The planogram booklet provided instructions to store manager for hanging the new signage, along with helpful SmartTips they can share with customers.
Envelope for paperwork when customers rent a storage space.


text: We are the well established firm of market highly dedicated to provide high quality assortments of Fresh Mango to our esteemed clients. It contains a number of health benefits that helps in stimulating your body functions. It improve digestion and strengthens eye health. It is also good for hairs and skin. Our Fresh Mango is incredibly tasty and keeps body perfectly nourished.


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The Potato Eaters
Jump to navigation Jump to search
The Potato Eaters is a painting made by Vincent van Gogh in 1885. It was one of his first paintings he has made. He painted it at age 32 in 1885. Potato Eaters is a painting of peasants that lived in his town.[1]
References[change | change source]
- ↑ "Vincent van Gogh: Visitor Submissions". www.vggallery.com.


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Jessica Swinford received her law degree in 2013 from the University of Arkansas, Little Rock. She also received her Master's Degree in Social Work from the University of Illinois, Urbana-Champaign in 2009.
Christopher Bocci & Associates
Christopher Bocci received his law degree from the University of Oregon in 1977. He began solo legal practice in 1979, focusing on criminal law, personal injury, and domestic relations. He is a member of the following: Oregon State Bar and Federal Bar (U.S. District Court), Oregon Trial Lawyers Association, National Association of Criminal Defense Lawyers and Oregon Criminal Defense Lawyers Association.
Our attorneys understand that when you need to hire an attorney, you want someone who is dependable, honest, efficient, and will promptly respond to your concerns and keep you informed about your case. We understand the importance of your case and will give it the treatment that it deserves.