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We report a mammalian-based promoter chromosomal array system developed for single-cell studies of transcription-factor function. Designed after the prolactin promoter-enhancer, it allows for the direct visualization of estrogen receptor α (ERα) and/or Pit-1 interactions at a physiologically regulated transcription locus. ERα- and ligand-dependent cofactor recruitment, large-scale chromatin modifications and transcriptional activity identified a distinct fingerprint of responses for each condition. Ligand-dependent transcription (more than threefold activation compared with vehicle, or complete repression by mRNA fluorescent in situ hybridization) at the array correlated with its state of condensation, which was assayed using a novel high throughput microscopy approach. In support of the nuclear receptor hit-and-run model, photobleaching studies provided direct evidence of very transient ER-array interactions, and revealed ligand-dependent changes in koff. ERα-truncation mutants indicated that helix-12 and interactions with co-regulators influenced both large-scale chromatin modeling and photobleaching recovery times. These data also showed that the ERα DNA-binding domain was insufficient for array targeting. Collectively, quantitative observations from this physiologically relevant biosensor suggest stochastic-based dynamics influence gene regulation at the promoter level.
Understanding transcription in the context of the nuclear environment has benefited from a combination of classic genetic systems and recent technological advances in molecular cell biology. A major goal is to document chromatin occupancy by transcription factors and other attendants at the molecular level, which can then be correlated with alterations in chromatin state and output from specific genes; however, cell-based inquiry of mammalian chromatin is limited. Nuclear receptors offer advantages in these pursuits because of their well-documented role in cellular responses to hormones.
Three experimental model systems in mammalian cells have been used to target transcription factors to integrated DNA. The spontaneous chromosomal integration of tandem mouse mammary tumor virus promoters in cell line 3617 (MMTV array) can be visualized using translational fusions of the green fluorescent protein (GFP) and the glucocorticoid receptor (GR) (McNally et al., 2000), the GR coactivator, GRIP-1 (Becker et al., 2002) and other transcription factors, including coactivators and a chromatin modifier (Muller et al., 2001). Chromosomally amplified lac-repressor-binding sites (lac arrays) have been used to visualize co-regulator interactions and chromatin changes in response to expression of a lac repressor, VP16 acidic activation domain and GFP chimera (Tumbar et al., 1999). An estrogen receptor α (ERα, herafter just called ER) chimera (GFP-lac-ER) was also used to document large-scale chromatin changes of the lac array in response to 17-β estradiol (E2) (Nye et al., 2002), and to record ligand-influenced, dynamic-recruitment of coactivators (Stenoien et al., 2001a). lac repeats linked to a repetitive Tet-responsive promoter were also recently used to observe chromatin changes in response to regulated activation of transcription (Janicki et al., 2004). In addition, live cell data using an HIV promoter array, and a synthetic NFκB binding site array, indicates rapid (seconds) oscillation of a gene regulator (NFκB) on a promoter (Bosisio et al., 2006). A general theme emerging from live single-cell studies of nuclear receptors and coactivators in individual mammalian cell nuclei is that their interactions (either at bulk chromatin or at specific DNA) are dynamic, with a half-time of residence in the order of seconds. The combined results of these model systems have greatly advanced our understanding of in vivo nuclear receptor (NR)-promoter, NR-co-regulator interactions and chromatin remodeling in general. However, each system individually lacks the ability to directly measure and correlate the multiple aspects of NR-mediated transcription, and has limited ability to assess antagonist-dependent transcriptional repression. The development of a single system with these multiplex capabilities would facilitate a greater understanding of the molecular mechanisms involved in transcription in vivo.
Our goal for the work described here was to develop a system that allows us to analyze the live cell dynamics of ER, including promoter interactions and associated large-scale changes in chromatin. For this purpose, we used an integrated array of ER synergy elements located in the enhancer of the prolactin gene, which also contains a binding site for the POU-class activator Pit-1. In addition to being induced in vivo by estrogen (Carrillo et al., 1987), the prolactin gene is also repressed by antiestrogens (Lieberman et al., 1983). Additionally, prolactin chromatin structure was differentially altered by agonists and antagonists (Cullen et al., 1993; Seyfred and Gorski, 1990). Our results indicate the establishment of a single-cell model to visualize quantitatively the hormonal regulation of ER action and provide evidence in support of the hypothesis that stochastic ER-promoter interactions are crucial for transcriptional regulation.
Large-scale ligand-mediated alterations in chromatin
Fig. 1 shows the DNA sequence of the prolactin promoter-enhancer region, and the Pit-1-ER synergy element that was multimerized in the construction of the plasmids used to establish our stable array-bearing reporter lines. To study large-scale chromatin changes, we treated GFP-ER-expressing PRL-HeLa cells with ligands or vehicle before imaging. We examined only cells with the lowest levels of fluorescence (typically the bottom 10% population, supplementary material Fig. S1C) to avoid overexpression artifacts. In this experiment, we quantitatively compared GFP-ER expression levels in PRL-HeLa cells with endogenous ER levels in MCF7 cells. The results indicated only a slightly greater level (<1.5-fold) of ER expression in PRL-HeLa cells relative to MCF7. In the experiments reported here, we ignored all transfected cells with levels of GFP-ER expression higher than that discussed above.
PRL arrays responded appropriately to antagonists by tight condensation (Fig. 2C,D), and to E2 by decondensation (Fig. 2B). E2-based decondensation contrasts with earlier results of multimerized lac-operator DNA using a lac-repressor-ER fusion (Nye et al., 2002). Most striking was the degree to which arrays condensed upon treatments with either the antiestrogens 4-hyrdroxytamoxifen (4HT; Fig. 2C) or ICI 182780 (ICI; Fig. 2D). These data revealed a remarkable plasticity in large-scale chromatin structure at the array, which appears similar to a smaller scale version of chromosome puffs. For an unbiased quantification of these responses, a high-throughput microscopy (HTM) approach was developed and applied to PRL-HeLa cells transiently expressing GFP-ER and treated with vehicle, E2 or 4HT (see Materials and Methods). Vehicle- and E2-treated cells had substantially larger size foci of fluorescence compared with 4HT and ICI, and E2 increased array size compared with vehicle (Fig. 2D). Time-lapse imaging demonstrated ligand-induced changes in array structure and illustrated real-time (minutes) decondensation (Fig. 3A) or condensation (Fig. 3B) upon exposure to agonist or antagonist, respectively.
Single-cell analyses of transcription by fluorescent in situ hybridization
For biological relevance, it was important to confirm transcriptional responses of the integrated PRL-based array to those previously documented for various ligand treatments in other systems. For this purpose, fluorescent in situ hybridization (FISH) was used to assay steady-state accumulation of reporter mRNA in response to ER expression and ligand (Fig. 4). Compared with non-transfected controls, there is a statistically significant higher level of array-associated reporter FISH signal in cells expressing wild-type ER in the absence of ligand. As expected from animal studies of pituitary responses to E2, the FISH signal localized at the PRL array increased at least 3.5-fold compared with vehicle controls within 30 minutes of exposure to E2. Interestingly, after 2 hours of ligand treatment, the level of induction was substantially higher than vehicle (more than twofold), but was reproducibly lower than the 30-minute treatment. Further demonstrating the hormonal responsiveness of the array, and correlating tightly to array size, 4HT and ICI completely repressed basal array FISH signal in the presence of ER. In the absence of ER, these antagonists had no effect on the array in terms of its size or constitutive levels of transcription (data not shown). The multiple, small FISH-signal foci in the 4HT-treated cells (arrows, Fig. 4B) are separate from the PRL array and were observed sporadically in cells under all treatment conditions. Importantly, the combination of array size and FISH data provided an early demonstration that ligand treatments lead to expected responses to agonist (decondensation of the array and increase in FISH signal) and antagonists (significant condensation of the array and undetectable FISH signal) (Figs 2 and 4, respectively). These data, which mimic the endocrinological response (e.g. agonist induction and antagonist repression) to hormones in the pituitary, provided confidence for the use of the multicopy, modified PRL promoter-enhancer in additional studies of the nuclear dynamics of ER and association of transcription co-regulators with the array.
Co-regulator and RNA polymerase II targeting, and modified histones
To further assess the biological significance of transcriptional responses of the PRL array, we performed immunolocalization studies using a series of antibodies directed against endogenous transcription factors, cofactors and modified histones. The p160 class coactivators (SRC-1 and SRC-3) accumulated over the array (versus nucleoplasmic levels) when GFP-ER without ligand or bound to E2 associated with the array (Fig. 5). In the absence of GFP-ER, neither of the p160 coactivators accumulated over the array (Fig. 5). As expected from previous lacER-p160 studies (Stenoien et al., 2001a), antagonist treatment resulted in a loss in accumulation of p160 coactivators at GFP-ER-occupied PRL arrays; interestingly, relative to SRC-3, reduction of SRC-1 was not always as robust with 4HT or ICI (Table 1, supplementary material Fig. S3). Similar assays were performed for the chromatin remodeling protein BRG1, which interacts with ER and is required by glucocorticoid receptor for chromatin remodeling (McKenna et al., 1999). BRG1 accumulated over the array with GFP-ER in vehicle-treated and E2-treated cells, but was not detected at arrays in antagonist-treated cells (Fig. 6, Table 1). Thus, each of these factors previously identified to be important for transcription activation appeared to have a unique signature of receptor- and ligand-dependent accumulation over the array.
In line with constitutive activity from the integrated transcription unit, the transcription elongation factors cyclin T1, CDK9 (Table 1; supplementary material Figs S4, 5) and the large subunit of RNA polymerase II (RNAPII) (Fig. 7) each showed array-associated targeting in non-transfected PRL-HeLa cells. By contrast, cells expressing GFP-ER and treated or not with E2 showed an increased accumulation of these factors over the array. Consistent with FISH results described above, exposure to 4HT or ICI eliminated the accumulation of cyclin T1 and CDK9 from GFP-ER targeted arrays. Similarly, 4HT resulted in loss of RNAPII accumulation at GFP-ER-targeted arrays and ICI treatment was not as effective in abolishing RNAPII array accumulation (Fig. 7).
We also characterized ligand-dependent histone modifications at the PRL array by immunofluorescence. Histone acetylation is associated with estrogen receptor activation (Kim et al., 2001), and histone methylation is associated with both transcriptional activation and repression (reviewed by Kouzarides, 2002). In cells not expressing exogenous estrogen receptor, acetylation of histone H3 at the array was observed (Fig. 8). This is indicative of a low level of constitutive transcription at the array (confirmed by colocalization with dsRed2 RNA FISH, data not shown). Surprisingly, GFP-ER-targeted arrays with or without ligands did not dramatically alter the levels of acetylated histone H3 array accumulation, even when highly condensed by 4HT or ICI (Fig. 8). Similar results were obtained for transcription-associated dimethyl-lysine 4 of histone H3 (Fig. 9). Repression-associated dimethyl-lysine 9 of H3 was not observed on condensed arrays at levels above those in the nucleoplasm, neither was acetylated histone H4 observed at the array under any ligand condition (data not shown).
In sum, these data on association of cofactors under varying conditions are consistent with the transcription responses observed by reporter FISH, and suggests that the integrated multicopy promoter-enhancer array responded to ER and its ligands in a biologically appropriate manner, albeit with characteristics specific for the prolactin promoter-enhancer.
Ligands affect ER mobility at prolactin array
To better confine real-time measurement of ER dynamics to a smaller nuclear volume containing a specific transcription locus, versus bulk nucleoplasm (Reid et al., 2003; Stenoien et al., 2001b), we examined live cell dynamics of GFP-ER at the PRL promoter array by fluorescence recovery after photobleaching (FRAP) methods.
Initially, we transiently transfected GFP-ER and performed a spot-FRAP analysis in both the nucleoplasm and at the array. As in our earlier studies, with minimal media (charcoal-stripped and dialyzed serum-containing), photobleaching the nucleoplasm demonstrated that ER mobility was extremely fast, with a half-maximal recovery (t1/2) of less than 1 second. The addition of E2 or 4HT resulted in significant reduction of ER mobility within the nucleoplasm (E2, 5 seconds; 4HT, 4 seconds) and, as expected, ICI immobilized ER (Table 3, data not shown). With improved normalization and analytical methods (see Materials and Methods), and in refinement of our previous work, we report here that 4HT led to a small but significant increase in mobility throughout the nucleus compared with E2.
FRAP was also used to specifically measure mobility over the PRL array (Fig. 10A,D; Table 2). GFP-ER recovery was markedly slower than that of nucleoplasmic receptor or Pit-1 (Fig. 10C). In the absence of ligand, t1/2 of GFP-ER was 12.19±1.92 seconds, n=30, which, interestingly, was not significantly different from that observed in the presence of E2 (11.64±3.41 seconds, n=30). 4HT treatments significantly increased mobility of array-associated GFP-ER (10.56±1.70 seconds, n=30), in concert with dramatic alterations in array size, the composition of recruited proteins and reductions of reporter mRNA shown above. As with nucleoplasmic receptors, ICI immobilized array-associated GFP-ER (Fig. 10A,D; Table 2).
Owing to concerns that the recovery dynamics observed at the de-condensed arrays could have been affected by the larger amount of bulk nucleoplasm interspersed with array chromatin, we reduced bleach regions to an area the size of the 4HT-induced condensed arrays. Because this meant that partial regions of larger arrays were photobleached, we refer to these as intra-array FRAP (FRAPintra-array). As shown in Table 2, FRAPintra-array data qualitatively agreed with the FRAP results above (absolute differences were due to different analysis method, see Methods and Materials), and demonstrated that differences in ER mobility were not the trivial result of differently sized arrays or bleach regions, or to the contribution of bulk nucleoplasmic dynamics in general.
FRAP and FRAPintra-array data provided recovery rates that represented both on- and off-rates of the photobleached proteins. To more directly assess GFP-ER loss from the array, we used inverse FRAP (iFRAP) (Becker et al., 2002; Dundr et al., 2002). In iFRAP, the entire nucleus, excluding the array, is bleached (Fig. 10B), and loss of fluorescence is directly related to the dissociation kinetics of GFP-ER from array chromatin. In absence of ligand, half-maximal loss (t1/2-loss) of GFP-ER fluorescence was 4.55±2.54 seconds, n=30) (Table 2). In contrast to FRAP or FRAPintra-array, addition of E2 resulted in a marked and significant (>50%, P<0.001) increase in t1/2-loss (6.49±1.79 seconds, n=30) (Fig. 10E, Table 2). iFRAP after treatment with 4HT, however, resulted in faster t1/2-loss than after treatment with E2 (4.11±0.85 seconds, n=30) (Fig, 10E, Table 2). Consistent with FRAP analyses, iFRAP also indicated that ICI immobilized GFP-ER in association with the array (data not shown).
In vivo mapping of ER domains required for array targeting
We next used PRL-HeLa cells to ask whether well-defined domains of ER contribute to the dynamics of GFP-ER interactions with array chromatin. ER domains associated with subnuclear localization, solubility and mobility have been previously reported (Stenoien et al., 2000; Stenoien et al., 2001a), with helix-12 of the ligand-binding domain (LBD) being implicated as a key intranuclear regulatory interface in living cells. To test this model with a biologically relevant, visible chromatin locus, we transiently introduced a GFP-ER-deletion series, including: (1) GFP-ER282 (AF1 with the DNA-binding domain, DBD), (2) GFP-ER534 (AF1, DBD and LBD up to, but excluding, helix-12) and, (3) GFP-ER554 (AF1, DBD and LBD including helix-12), Fig. 11A. In vitro and in vivo assays showed that GFP-ER554 retains agonist-inducible transcription function on an estrogen response element (ERE)-dependent reporter (Pakdel et al., 1993), whereas GFP-ER282 and GFP-ER534 are transcriptionally inactive (Tzukerman et al., 1994). In vitro, all deletion mutants bind DNA in an electrophoretic mobility shift assay (EMSA), and ER534 and ER554 bind ligand, whereas ER282 does not.
In stark contrast to its ability to bind DNA in EMSA, GFP-ER282 failed to demonstrate any detectable targeting to the PRL array (Fig. 11B). In the presence of E2, but not vehicle, GFP-ER534 associated with the PRL array, further supporting the ligand-binding potential of the truncated LBD. However, PRL arrays demarcated with GFP-ER534 remained condensed (Table 3) and showed no detectable recruitment of RNAPII (data not shown). Conversely, GFP-ER554 targeted the array indistinguishably from wild-type ER (Fig. 11B) and responded to 4HT and ICI, as evidenced by array condensation (Table 3).
Estrogen receptor domains contribute to its nucleoplasmic and PRL array dynamics
Our panel of mutated receptors was next analyzed for mobility in the nucleoplasm and at the PRL array. As we have shown previously (Stenoien et al., 2001b), GFP-ER282 was very mobile in the nucleoplasm and, as expected, unaffected by ligand (Fig. 11C, Table 5). Unliganded GFP-ER534 was also very mobile in the nucleoplasm and was significantly reduced by E2 (Fig. 11C, Table 4). GFP-ER554 demonstrated slower nucleoplasmic mobility relative to GFP-ER282 and GFP-ER534, consistent with a role for helix-12, and also exhibited reduced mobility upon exposure to E2 (Fig. 10C, Table 4). At the PRL array, GFP-ER554 demonstrated significantly faster recovery compared with GFP-ER in cells treated with vehicle (Fig. 11C, Table 4), similar to its faster reorganization into nucleoplasmic hyperspeckles after addition of E2 (Stenoien et al., 2001a). In cells treated with E2, GFP-ER534 demonstrated the fastest recovery times at the array, followed by GFP-ER and GFP-ER554 (Fig. 11C, Table 4).
We present here, for the first time, a direct and quantitative evaluation of spatiotemporal issues involved in transcription from a entirely mammalian promoter-enhancer array. The modified PRL promoter-enhancer array responds in a physiologically relevant manner to ER and ligands in terms of reporter-mRNA transcription. Its activity is linked to live cell dynamics of the receptor at the transcription locus and to readily measurable alterations in large-scale chromatin structure. Since ER is located predominantly in the nucleus, our system affords a new level of interrogation of transcription factor interactions with an integrated locus, both in the context of agonist-induced transcription and antagonist-induced repression. Further, this approach demonstrates that a transcriptional biosensor can be constructed from mammalian promoter components and establishes the feasibility of creating similar test systems with other types of DNA-binding factors that mediate transactions with chromatin. In addition to being an integrated reporter of chromatin structure-function relationships, arrays are highly amenable to analytical tools, including HTM as performed for this report, and should be a harbinger for systems-biology-level-approaches to transcription.
As we (Stenoien et al., 2000; Stenoien et al., 2001a; Stenoien et al., 2001b) and others (Htun et al., 1999) have shown, ligand exposure within minutes affects intranuclear targeting and dynamics of ER, with maximal reorganization peaking at about 30 minutes. Here, we more directly observed hormonally regulated transcriptional events at the integrated array, including remarkable ligand-dependent size and/or density plasticity of array chromatin marked by GFP-ER, which are tightly correlated to the ligand-regulated activation or repression of the array (Figs 1, 2). We are examining the potential of using HTM in live cell experiments to capture unbiased quantitative data on a large number of cells to assess array-size dynamics under variable experimental conditions similar those in Fig. 2. We currently interpret changes in array size as a reflection of alterations in chromatin state (closing and opening) that correlate directly with ER-mediated differences in transcriptional output and colocalization of RNAPII and its cofactors. The rapid loss of most of these factors from the array in response to antagonists is mechanistically consistent with marked changes in array size and undetectable levels of dsRED2skl mRNA by FISH.
Our ligand-based activation and repression studies document an interesting pattern of array-associated coactivators, chromatin modification enzymes, transcription elongation factors and RNAPII. Accumulation over the PRL array by cyclin T1, CDK9 and RNAPII is specific for transcription, be it constitutive or agonist enhanced. One unexpected observation was the incomplete disassociation of RNAPII from the PRL array chromatin in cells treated with the pure antagonist ICI. The interaction is antagonist-specific, because 4HT treatment reduced RNAPII association to nucleoplasmic levels. As neither cyclin T1 nor CDK9 is detected at arrays in ICI-treated cells, our data suggest that RNAPII is not activated (pre-initiation complex), is in an abortive cycle or is perhaps trapped with immobilized GFP-ER. The apparent association of the p160 coactivators, SRC-1 and SRC-3 is also not as straightforward. In our immunoassays of GFP-ER-expressing cells, colocalization with the array by SRC-3 appears to be strictly receptor dependent, and is regulated by agonist (increased SRC-3) and antagonist (dissociated SRC-3). SRC-1 colocalization, however, appears to be receptor-dependent (with or without agonist), and at least partially resistant to antagonist dissociation. It will be important to extend these immunoassays with experiments using fluorescently tagged SRC coactivators in photobleaching and protein-protein (FRET) studies to establish interactions following transcriptional stimulation or repression.
Significant changes in array size and/or density do not accompany major changes in several histone modifications that we have assayed. Previous histone modification studies have shown changes in acetylation of histone H3 or H4 after targeting repetitive, heterochromatic lac operator arrays directly with lac repressor-activator fusions (e.g. lacVP16, lacer) (see Nye et al., 2002; Tumbar et al., 1999). Also, a combined lac operator and tet operator array system regulated by tetOn also demonstrated histone modifications in cis (Janicki et al., 2004; Tsukamoto et al., 2000). With our studies, however, despite significant differences in array size (Figs 1, 2), acetylation and dimethylation of lysine 4 of histone H3 is not dramatically altered in immunofluorescence assays. Moreover, acetylation of histone H4 over the PRL array was never observed above nucleoplasmic levels. Finally, we examined the repression-associated methylated histone H3 (at lysine 9) and also did not see significant array signal above nucleoplasmic levels when the locus was repressed and highly condensed by 4HT or ICI (data not shown). One possibility is that minor changes in histone modification are not detectable by our assay, although such changes have been documented using biochemical approaches. Another possibility is that chromatin on the scale observed here may not be primarily mediated by changes in the acetylation and/or methylation of histones, but rather other modifications such as ADP-ribosylation (Tulin and Spradling, 2003), for which we are in the process of assaying. Also, the lack of changes may represent a form of memory in the histone code. Indeed, we are actively investigating the possibility of ligand-dependent delays in large-scale chromatin modification in hormone-switching experiments. It is also possible that, direct single-cell analyses give different results than cell-population-based biochemical (ChIP) studies that average results from millions of cells. In this vein, the ER molecular dynamics reported here, as with GR (McNally et al., 2000), differ substantially (range of seconds) from ChIP results (tens of minutes) (Metivier et al., 2003; Reid et al., 2003; Shang et al., 2000). Finally, it is possible that analyses beyond 2-hour time points can more readily show changes in histone modifications. Development of quantitative imaging approaches to better assess histone modifications at the PRL-array are required to help address this issue. Further, analyses of similarly created, hormonally responsive transcription biosensor lines containing other nuclear receptor-targeted arrays will also assist in better understanding these issues.
Accumulating evidence supports a non-ligand-dependent transcription function for ER (reviewed in Coleman and Smith, 2001). Thus, whereas it was not surprising to frequently observe non-liganded ER interacting with the arrays, it was interesting that unliganded receptor appeared to mediate significantly higher level of colocalizing reporter mRNA. Notably, there is a significant change in mobility of unliganded GFP-ER in the nucleoplasm versus over the PRL array (<1 second versus ∼12 seconds, respectively). Ligand-dependent activation data are consistent with an increase in the number of cells with GFP-ER targeted arrays after treatment with E2 (from ∼75% to >99%, supplementary material Fig. S2C).
Together, both FRAP and iFRAP data show that the fluorescently tagged nuclear receptor ER and the POU-class activator Pit-1 exhibit short resident times on a promoter-enhancer-based array, similar to the GFP-GR results reported using both the MMTV (McNally et al., 2000) and HIV array (Bosisio et al., 2006) models. An advantage in this system is the opportunity to document the molecular dynamics under conditions of both antagonists and agonist. In the case of ER and the PRL array, it was striking to find that ligand significantly influenced its time of residence. Whereas FRAP data from our lab (Stenoien et al., 2001b) and others (Reid et al., 2003) have shown that intra-nuclear mobility of ER is regulated by ligand, proteasome function or ATP, these data reflect the sum of multiple interactions in the nuclear volume that contains (spatially) few ER target genes. Nevertheless, we were surprised that E2 did not significantly change the `dwell time' over transcriptionally active array by standard FRAP. This result might represent a canceling out of putative, E2-mediated changes of (1) on- and off-rates of DNA-binding, (2) ER dimerization, (3) protein recruitment or, (4) proteasome-mediated turnover. Although we addressed potential trivial caveats of array size influencing mobility values using FRAPintra-array, we feel it is clearer to interpret ligand effects using the more focused koff data obtained from iFRAP. Our results from this approach show that the array dwell time is significantly increased after E2 treatment compared with vehicle or 4HT. This would be consistent with E2 promoting productive (additional) interactions at target promoters rather than increasing promoter binding. Differential GFP-ER array-dwell-time might represent assembly and disassembly of the focal and, probably very transient, nuclear receptor complexes, representing important mechanistic steps in regulating transcription and associated large-scale chromatin alterations. We interpret these live cell findings of activation- or repression-associated ER to be an important extension of the previously supported hit-and-run model of transcription activation by ligand-activated GR at the MMTV array (McNally et al., 2000). We also noticed significant agreement in the beaded morphology of activated and extended PRL loci (Fig. 1, supplementary material Fig. S1) with those bearing the MMTV array (Muller et al., 2001).
Our results using truncated receptors show that the GFP-ER282 (AF1 plus the DNA-binding domain) is not detectable at the PRL array, suggesting that the DBD is not sufficient for in vivo access to native ER-response elements in a chromosomal environment. Helix-12 appears to be required, presumably for fostering productive ER-protein interactions and array decondensation. We noticed that the rate of nuclear mobility of ER appears to be directly related to the number of functional domains present. The same correlation has been noticed for receptor residency time at the PRL array. The observations that GFP-ER534 interacts with the array only in the presence of E2, and the array remains condensed (and presumably inactive, i.e. no RNAPII recruitment) are consistent with previous in vitro and transient transfection data on this truncated receptor. This underscores the biological relevance of the PRL array-containing cell lines for future studies of ER-Pit-1 synergistic interactions.
Collectively, the data support a stochastic model of nuclear function (Misteli, 2001; Vermeulen and Houtsmuller, 2002) because fast (and regulated) ER exhibits altered exchange rates at the chromosomally integrated PRL array. As such, these results do not necessarily support the canonical view of pre-formed, stable holo-complexes recruited to promoters as the principal mechanism of transcription regulation but rather suggest that complex formation can be highly transient in living cells, and stochastically assembled (Peterson, 2003) influenced by probabilistic interactions. Our current efforts focus on documenting the highly dynamic nature of these interactions and quantitative multiplex read-outs of NR function, particularly through high-throughput screening technologies using high resolution and automated microscopy (Perlman et al., 2004).
Materials and Methods
Prolactin Pit-1 ID-ERE concatemer and reporter constructions
We generated cell lines bearing integrated rat prolactin (PRL) promoter-enhancer that contain no viral or bacterial components (e.g. entirely mammalian regulatory elements). Advantages of PRL control elements for this purpose are: (1) they are well characterized in terms of binding and transcriptional activation by Pit-1 and ER (Barron et al., 1989; Cao et al., 1987; Crenshaw et al., 1989; Day et al., 1990; Howard and Maurer, 1995; Ingraham et al., 1990; Mangalam et al., 1989; Nelson et al., 1988; Smith et al., 1995), (2) they are responsive to 17-β estradiol (E2) through transcriptional synergy by Pit-1 and ER (Day et al., 1990; Day and Maurer, 1989; Holloway et al., 1995; Nowakowski and Maurer, 1994; Seyfred and Gorski, 1990), (3) they were useful in some of the original work to characterize transcriptional downregulation by the antiestrogen 4-hyrdroxytamoxifen (4HT) (Gothard et al., 1996; Jordan et al., 1988; Lieberman et al., 1983; Seyfred and Gorski, 1990) and, (4) alterations of their chromatin structure in response to E2 (Cullen et al., 1993; Durrin and Gorski, 1985; Durrin et al., 1984; Gothard et al., 1996; Malayer and Gorski, 1995; Maurer, 1985; Seyfred and Gorski, 1990; Willis and Seyfred, 1996) and 4HT (Gothard et al., 1996; Liu and Bagchi, 2004; Seyfred and Gorski, 1990) are well studied.
The design of the Pit-1-ERE array began with a prolactin promoter enhancer construct (pDm66/Enh) originally described by Smith et al. (Smith et al., 1995), which consists of a transcriptional fusion of a short region (-66 to +1) of the prolactin (PRL) promoter containing the transcription start site and a high-affinity Pit-1-binding site (1P, Fig. 1A) driving expression of a luciferase (Luc) reporter. Linked upstream is the native distal enhancer of prolactin (-1807 to -1498) that contains four Pit-1 binding sites, 1D, 2D, 3D and 4D (Nelson et al., 1988), and five EREs (Day et al., 1990; Day and Maurer, 1989; Holloway et al., 1995; Nowakowski and Maurer, 1994; Seyfred and Gorski, 1990), PRL1-5 (Fig. 1A). This construct was stably integrated into the chromosomes of HeLa cells (HeLa-PRL-dm66-Enh, e.g. 1XPRL) and tested for Pit-1-ER synergy by transiently expressing one or both of these activators. As expected, expression of Pit-1 or ER alone results in increased levels of luciferase activity over background (seven- or fourfold, respectively, supplementary material Fig. S1B). Interestingly, ER activates this construct in the absence of Pit-1 and E2, which is consistent with GFP-ER interactions with the PRL-integrated array below. In the absence of hormone, exogenous expression of both Pit-1 and ER lead to an approximately twofold induction of luciferase activity compared with that promoted by Pit-1 alone, which increased to a fourfold induction in the presence of E2. These data are in agreement with previous experiments (Day et al., 1990; Day and Maurer, 1989; Holloway et al., 1995; Nowakowski and Maurer, 1994; Seyfred and Gorski, 1990), and indicate that this unit would serve well as an integrated reporter in HeLa cells carrying a visible array.
To construct the array-containing plasmids to establish the cell line used in this paper, we first added one unit of the ER-Pit-1 `synergy element' (Fig. 1B) onto the 5′-end of the native enhancer. This element contains one Pit-1-binding site and two EREs (1D, overlapping a full, imperfect site PRL1 and an inverted half site, PRL5, respectively). This complex-binding site (Fig. 1B) was previously shown to be cruial for the transcriptional synergy mediated by Pit-1 and ER (Nowakowski and Maurer, 1994). The relevant parts of plasmid p1X-PRLLuc, are shown schematically in Fig. 1C. In a separate cloning vector (pBS), reiterative cloning steps generated p2X, 4X, 8X, in which X refers to the number of element repeats in the array. A recombination event generated 13X and subsequent reiterative cloning generated 26X, 52X and 104X. These arrays were transferred into the PRL-Luc plasmids to test for functionality. Transient-transfection assays confirmed the qualitative functionality of three of these constructs (13X, 26X and 52X) with respect to transcription promoted by GFP-Pit-1 (supplementary material Fig. S1A). For integration into the chromosomes of HeLa cells, the Luc reporter was replaced with dsRED2-SKL fluorescent protein, which is similar to the CFP-SKL reporter protein used previously by Spector and co-workers (Tsukamoto et al., 2000). The SKL peptide represents the amino acids that target proteins to peroxisomes; in our hands, the signal of the dsRED2 reporter is much brighter than that of CFP, and allows an additional color to be used in labeling studies. Here, we describe results from analyses of one of our clonal HeLa lines (number 23) harboring the p52X-PRLdsRed2-Ser-Lys-Leu (SKL) plasmid, referred to throughout this manuscript as PRL-HeLa cells.
FISH and Southern analyses of PRL-HeLa cells
DNA fluorescent in situ hybridization (FISH) confirmed the integration of the transgene array (e.g. PRL array) that showed an intranuclear focus of signal (Fig. 2B). Colocalization of GFP-ER-Pit1 with DNA-FISH and RNA-FISH focus signals also confirmed the integration of the promoter array and demonstrated protein-DNA interaction and transcriptional activity, respectively (Fig. 2B). Quantitative Southern blotting indicated approximately 200-300 copies of p52X-PRL-dsRED2skl integrated into the HeLa genome (data not shown). These data indicate that the p52X-PRL-dsRed2-SKL-hygro vector integrated into the HeLa genome at a specific locus to form a promoter array that is transcriptionally active and visible through interaction with GFP-ER and/or GFP-Pit-1.
ER and Pit-1 interact with the PRL array separately
To determine whether GFP-ER or GFP-Pit-1 individually interact with the PRL array, single transfections of PRL-HeLa cells were performed. These experiments, originally carried out in OptiMEM (growth-factor-containing minimal essential medium) containing charcoal-stripped serum, showed both GFP-ER and GFP-Pit-1 can target the array individually. To properly assess ligand (ER) or growth factor (Pit-1) stimulation, we grew cells for a minimum of 48 hours in hormone-free stripped and dialyzed serum and DMEM (Dulbecco's modified Eagle's medium). Quantitative examination of the number of cells expressing Pit-1 alone (GFP-tagged or untagged) indicated inefficient targeting to the array under hormone-free conditions (data not shown). These data suggest an influence of cell signaling pathways on GFP-Pit-1 interactions with the PRL array, which is consistent with previously reported Pit-1 transcription assays (Xu et al., 1998).
In contrast to GFP-Pit-1, GFP-ER was capable of targeting the arrays in ∼75% of the cells in hormone-free medium. Interestingly, E2-treatment (2 hours at 10-8 M; a time and ligand concentration used throughout these studies) resulted in virtually all transfected cells demonstrating targeted arrays by GFP-ER (Fig. 2C). Consistent with our observations in the screens above, when CFP-Pit-1 was co-expressed with YFP-ER in PRL-HeLa cells cultured in hormone-free medium or in OptiMEM with serum, Pit-1 was found to target together with ER at the array (Fig. 2). Thus, in this system, under conditions of minimal global cell signaling, the data indicate that Pit-1 and ER can simultaneously interact with array chromatin. In the experiments reported below, we concentrated on ligand-dependent observations of ER at the array.
Antibody labeling was performed as described previously (Stenoien et al., 2000) using 4% formaldehyde fixation (30 minutes) and indirect labeling with Alexa Fluor-488, -546 or -633 (Molecular Probes) conjugated secondary antibodies. Since the majority of antibodies had previously been mapped to the nuclear compartment, we were able to perform localization experiments without confusing the well-defined dsRED2skl peroxisome signal with antibody label, even when using secondary antibodies with overlapping spectra. DAPI (1 μg/ml) was used to label DNA prior to mounting in Slow Fade (Molecular Probes). Affinity-purified rabbit antibodies against SRC-1 and SRC-3 were a kind gift of Jeimin Wong (Baylor College of Medicine, Houston, TX); cyclin T, cdk9, BRG-1, RNAPII large subunit, acetylated histone H3 and dimethylated histone H3 (K4 and K9) were obtained from Abcam or Upstate Biotechnology and all used at 1 μg/ml for 1 hour at room temperature or overnight at 4°C.
Fluorescent in situ hybridization (FISH)
The methods used here, including procedures for non-isotopic probe preparation and fluorescent in situ hybridization, have been published in detail elsewhere (Johnson et al., 1991; Tam et al., 2002). Briefly, coverslips with adherent cells were rinsed twice in PBS, dipped in cytoskeleton (CSK) buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES pH 6.8) (Fey et al., 1986), extracted on ice for 5 minutes in CSK buffer containing 0.5% Triton X-100 and 2 mM vanadyl-ribonucleoside complex (VRC; Gibco-BRL), rinsed in CSK-VRC buffer, fixed in 4% paraformaldehyde with PBS for 10 minutes, rinsed again in PBS and stored in 0.4% paraformaldehyde at 4°C until use.
Probes substituted with biotin-labeled deoxynucleotides were made by modifications of standard nick-translation procedures (Tam et al., 2002). For hybridization to DNA, cells were first denatured by incubation in 0.07 M NaOH, 70% EtOH for 5 minutes. This incubation denatures the DNA prior to hybridization and removes RNA. RNA hybridization refers to samples in which the cellular DNA was not heat-denatured, leaving it inaccessible for hybridization. Hybridization to RNA or DNA was carried out overnight at 37°C in rehydration buffer (2 mM VRC, 2 mg/ml BSA, 0.05 g/ml Dextran, and 2× SSC) containing 5 μg/ml probe and 50% formamide. After incubation, samples were rinsed in a dilution series of NaCl-Na-citrate (SSC) buffers, analysed for biotin using streptavidin-conjugated Alexa Fluor-594 (Molecular Probes, Eugene, OR) and rinsed in a dilution series of PBS. Where indicated, cells were counter-stained with 1 mg/ml DAPI (Sigma, St Louis, MO). Coverslips were then mounted on slides in Vectashield (Vector Laboratories, Burlingame, CA).
To quantify FISH signals, a 20 plane 128×128 (pixels), 0.2 μm Z-stack was collected (constant 100-millisecond exposure). A circular region containing the array was selected and a region of the same size was used to select the background nucleoplasm. Total array associated FISH signal was determined by quantifying the cumulative intensity of the array region after sum projection and background subtraction.
Fixed-cell- and time-lapse-imaging
XFP-fusions and immunofluorescently labeled cells were imaged using a DeltaVision Restoration Microscopy system (Applied Precision, Issaqua, WA) and applying a constrained iterative deconvolution process. Whole nuclear volumes were collected at 0.2 μm Z-steps, and images from select focal planes or 3D-projections were imported into Adobe PhotoShop. Histogram adjustments were made relevant to negative controls, which routinely included non-transfected cells and/or omission of primary antibodies. Live imaging was performed by collecting short Z-stacks (∼5-10 focal planes at 300-nm increments); neutral density for the green channel was set at 50% and the images were binned 2×2. Typical exposures were for <1 second, and time points from 3-10 minutes per stack. Projected images from each time point were used to create a QuickTime movie.
Live cell imaging
Cells were grown in 35-mm Delta T dishes (Bioptechs) and secured to a stage adapter for temperature control. HEPES-buffered media was gassed overnight in a 5% CO2 incubator, and circulated through the Delta T dish using a Bioptechs peristaltic pump and inflow-outflow tubing. The temperature was controlled to 37°C (± ∼0.1 degree); a Bioptechs objective-heating collar was also used (also 37°C). The Delta T dish was covered with a black plastic lid, with room for input-output tubing. For time-lapse imaging, a 63× objective (NA=1.4) was used.
Photobleaching parameters and calculations
For imaging, PRL-HeLa cells were plated and transfected in Delta T dishes. Live cell microscopy was performed using a Zeiss 510 confocal microscope using the 488-nm laser line of the argon laser at 75% of maximal power. In all experiments cells were maintained at 37°C and fresh media containing the appropriate ligand was cycled over the cells. All imaging was done with a 63× objective (NA=1.4) with pinhole set at either 4 Airy units (Au) (FRAP and FRAPintra-array) or 10 Au (i-FRAP). Scanning was bidirectional at the highest possible rate using a 3.5× zoom with laser power attenuated to 1% of bleach intensity. For all experiments, cells were selected for low levels of GFP-ER expression to limit experimental artifacts. For FRAP experiments, a single pre-bleach image was acquired followed by ten iterative bleach pulses of a total duration of 530 milliseconds using a circular bleach region of interest (ROI) of the diameter of either 32 pixels for large arrays or 18 pixels for small arrays. Single-section images were collected every 250 milliseconds for 60 seconds. Relative fluorescence signal in the bleached region was determined by two sequential normalization steps using the mean ROI (It) bleach signals and the mean nucleus signals (Nt): (1) (2) Pre-bleach values are not used in the second normalization step. Half-time of maximal recovery was determined by fitting a logarithmic equation to the recovery curve and determining the value of t when y=0.5 using the Excel curve-fitting function. Inverse-FRAP (i-FRAP) experiments were performed in a similar manner except that three iterative bleach pulses were used for the total duration of 600 milliseconds, with a bleach ROI containing the whole nucleus, excluding the array. Relative fluorescence and half-time of loss were also determined in a similar manner except pre-bleach values were retained.
For FRAPintra-array experiments, five pre-bleach images were collected followed by a single bleach pulse of 220 milliseconds using a circular bleach ROI of the diameter of 13 pixels. Single-section images were collected every 500 milliseconds for 75 seconds. The relative fluorescence signal in the bleached region was determined as above. The unbinding-rate constant and half-time of recovery was calculated by fitting a single-term exponential equation (1-e-kt) to the recovery curve (see Lele et al., 2004 for detailed methods).
Quantitative image analyses by HTM
PRL-HeLa cells transiently expressing GFP-ER were treated with 10 nM E2, 4HT, or ICI for two hours, fixed and DAPI-stained. Cells were imaged using the Cell Lab IC 100 Image Cytometer (Beckman Coulter) with a Nikon 40× Plan S Fluor 0.90 NA objective. Two channels were imaged: channel 0 (DAPI) was used to find the focus and nuclei and channel 1 was used to image GFP-ER. A proprietary algorithm (GPCR) developed at Beckman Coulter was used to identify and quantify the GFP-ER targeted PRL array. The parameters for the GPCR algorithm were: object scale = 30 and minimum peak-height = 10. Foci identified by the GPCR algorithm are masked. The area of the mask in pixels is the measure of PRL array size. Channel 1 was offset 2 microns from the DAPI focus for cells in all treatment conditions. This offset provided the greatest number of in focus arrays identified by the GPCR algorithm. After image acquisition and application of the GPCR algorithm the total cell populations for each treatment were progressively filtered (gated) using the same criteria. Nuclei clusters, mitotic cells and apoptotic cells were filtered from the total cell population using an intersection of DNA-content and DNA-cluster gates. In addition, low GFP-ER-expression gates and low aggregate-number-gates were generated and applied to produce the final cell population to be analyzed. From the final population of cells, the array size was determined using the GPCR mask. The images and masks were visually inspected for accuracy. Unpaired Student's t-tests assuming equal variance were performed to determine statistical significance (two-tailed, P<0.05).
The authors wish to thank Ann Nye-Carpenter, who provided early guidance in the establishment of the cell lines, and J. H. Price (Burnham Institute and Vala Sciences, San Diego, CA) for invaluable help with the automated image cytometry. We also thank B. W. O'Malley, J. A. Nickerson, J. Lawrence's laboratory, and the anonymous reviewers of the manuscript for many helpful suggestions. This work was supported from NIH grants DK55622 (M.A.M.), DOD W81XWH-04-1-0700 (Z.D.S.) and NASA NN A04CC96G (D.E.I.). All imaging was performed in the Department of Molecular and Cellular Biology Integrated Microscopy Core, supported by HD-07495 (Center for Reproductive Biology, B. W. O'Malley), NCI-CA64255 (Hormonal Regulation of Breast Cancer PPG, D. Medina) and departmental funds. C.H. and A.S. were supported in part by training grants in Reproductive Biology (DK12345) and Pharamoinformatics (GM12345), respectively.
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/119/19/4101/DC1
↵* These authors contributed equally to this work
- Accepted July 5, 2006.
- © The Company of Biologists Limited 2006


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Product Description
8 MP3 Series
Developing people skills is critical to achieving the quality and richness of life we desire. Our ability to demonstrate good people skills will often be the deciding factor on our levels of success in various areas of our lives. God is truly a "people-oriented" God and He wants us to experience His love through how we interact with each other.
In this 8-part series, "People Skills Matter," Dr. Mike Moore explores several major aspects of people skills such as kindness, flexibility, sensitivity, listening, humility, unselfishness, confrontation and conflict resolution.
"People Skills Matter" gives a comprehensive look at the various dimensions of people skills designed to help improve our communication, increase our influence and deepen our relationships -- as we are to be a shining light of God's love in the world.
*includes two (2) bonus Q&A MP3's.

subset: commoncrawl


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101 Funfetti Recipes
posted by Ashton July 27, 2017
About the author
Ashton Swank
Ashton is the owner and author of Something Swanky. Although first and foremost a wife and mother, she considers herself an online entrepreneur, freelance writer and photographer, and brand ambassador. Her focus is in food styling, food photography and recipe development.
posted in Becca, Recipe Collections
July 27, 2017


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Hello there and "The Emperor protects!"
I'm a BIG Fan of the Warhammer 40k Universe, at the Moment i try to build my first Tabletop Army (Imperial Guard). Also I love Computergames, and if the Game is also set in the Wh40k Universe, it's awsome. So it's not a Surprise that some of my favorite Videogames are Dawn of War + the 3 Addons, Dawn of War 2 + the one Addon & Firewarrior.
Also i like First-Person-Multiplayer-Shooter like the Battlefield-Series.
I'm REALLY annoyed about the Copy Protections of many Games, like Securom or that you must be online to play the SINGLEPLAYER!!! Settlers 7, Assassin's Creed 2, Command & Conquer 4, to name a few. I've installed Homeworld 2 with MY OWN CD and Securom tells my everytime when I want to start a Mod, that there's a Problem with Securom, I should insert the original Disc. And i cannot delete Securom, although Homeworld 2 is uninstalled. It makes my PC slower and I want to decide, which Programm is installed on my PC.
The Publishers don't give much at the Confidence of their Customers, this is why many Games are cracked. At the End, nobody will profit of the new Copy Protections, but it seems that the Publishers don't care.
No blogs were found matching the criteria specified. We suggest you try the blog list with no filter applied, to browse all available. Join now to share your own content, we welcome creators and consumers alike and look forward to your comments.
This group is open to anyone who loves the Stargate shows, movies and anything Stargate! Anyone caught making fun of Stargate (outside of certain boundaries...
For everyone that likes Warhammer 40k Tabletop and Dawn of War Games or anything to do with Warhammer 40K.
For all fans of Civilization-like games, the main Civilization series, Call to Power spinoffs, Alpha Centauri, and indies such as C-evo, FreeCiv and FreeCol...
That's my clan, so if you are a fan or you have warhammer Dawn of War, dark crusade or soulstorm join!!
Do you like Battlestar Galactica? Join the rest in the BSG Fan Group! That is the best way to tribute one of the best Sci-Fi series in the world.
No groups were found matching the criteria specified. We suggest you try the group list with no filter applied, to browse all available. Start a group and help us achieve our mission of showcasing the best content from all developers. Join now to share your own content, we welcome creators and consumers alike and look forward to your comments.
thanks you and have fun
NEW Battlestar Galactica Forum: Moddb.com
Thanks for watching SPACE 3102: ACE's Void
Nothing to thank for, Your Work speaks for itself (I hope you get the Meaning, it's a Word to Word-Translation)
Only registered members can share their thoughts. So come on! Join the community today (totally free - or sign in with your social account on the right) and join in the conversation.


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portrait of Mary Queen of France & Francis, Dauphin of France on their wedding day, April 24, 1558
portrait of Mary Queen of France & Francis, Dauphin of France on their wedding day, April 24, 1558
A new TV movie about Mary, Queen of Scots is being filmed.
|'Reign' – US TV movie about Mary Queen of Scots – is being filmed at Christ Church Cathedral.
|10 Apr 2013 : By Kevin Cronin
Currently shooting at Christ Church Cathedral in Dublin, 'Reign' charts the rise to power of Mary Queen of Scots in the 16th century when she arrives in France as a 15-year-old and is betrothed to Prince Francis against a backdrop of 'sexual intrigue'.
The TV movie is a US/Irish co-production between CBS Studios, World 2000 Entertainment and Octagon Films and is to be distributed by the CW Network.
Directed by directed by Brad Silberling, the project is described as a 'fantastical reimagining of the teenage Mary Queen of Scots' and pitched as 'Game of Thrones' meets Sofia Coppola's 'Marie Antoinette', according to industry reports.
Australian actress Adelaide Kane played Lolly on Neighbours and will next be seen in horror film 'The Devil's Rapture' and 'After the Fall' starring David Duchovny.
The young cast of 'Reign' includes Anna Popplewell (The Chronicles of Narnia); Caitlin Stasey (Tomorrow, When the War Began); Megan Follows (I am Number Four); and Toby Regbo (Harry Potter and the Deathly Hallows: Part 1).
As an historical figure, Mary Queen of Scots is also the subject of a big screen adaptation currently in development with Working Title Films for release in 2014 with Saoirse Ronan set to star and Michael Hirst (The Tudors, Elizabeth) writing the screenplay.


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iCloud Data Security
iCloud is a fantastic service for synchronizing photos, documents, notes and more between your Macs and IOS devices. But before your personal data can be pushed to your devices, the content has to be stored somewhere. In the case of iCloud, your data is stored on Apple's servers. So should you feel confident that your digital life is safe and secure in Apple's data center? Based on Apple's article on iCloud security, the answer seems to be a qualified "yes."
When dealing with information security, we talk about data in motion and data at rest. When data travels across the internet, it's in motion. When data is stored on a hard drive, it's at rest. Based on Apple's article, iCloud data is transmitted over the internet using SSL. This is a well-proven standard to encrypt data in motion. On Apple's servers, it seems that all data is stored encrypted, except for music, email, and notes. Unencrypted data would be much easier for a hacker or rogue Apple employee to steal. So think twice about storing your Swiss bank account numbers in notes if you use iCloud.
Apple hasn't publicly disclosed the encryption and authentication algorithms used with iCloud. Until this information becomes public, industries that require strong security and data privacy will likely pass on using iCloud.
3 Comments
Do you happen to know what key they use to encrypt the data in iCloud? I would imagine if you can access iCloud from any device you own, it must be based on your iCloud password, in which case the data is only as secure as the passphrase you use. Most people dont use very secure passwords. One problem I've always had with Apple's implementation of cryptography is their insistence on using the user's authentication password as the encryption password. This results in negative effects such as discouraging the use of cryptographically strong passwords, which users would have to enter each time they allow a process to escalate privileges. There should be a user's authentication password, which is separate from the encryption password.
But anyway I digress, I'm thinking of using iCloud, do you happen to have any specifics on how the encryption keys are generated that encrypt the data on iCloud? I'm thinking a good way to do that would be to generate a key on the user's local computer and encrypt all data with that prior to sending it to iCloud. Problem would be recovery of the data if your mac goes kaput or if you are on a new device trying to sync with iCloud. I've been thinking about using iCloud but wanted more info on the security protocols, if you have any insight would be helpful.
Actually that's even worse than I speculated…. if the data is not even encrypted on something derived from your computer/you but by apple and Apple has access to it, that's pretty lame.
Security through obscurity doesn't work. That's another thing apple seems to need to learn about security. Good systems have their mechanisms, algorithms, even source code if possible, open and freely inspectible, the secrecy of any of these things is never a reliable basis for security, and transparency allows inspection/discovery of systematic problems and thus their correction.
For online data storage/backup, I personally continue to favor JungleDisk as the data is encrypted on a user-provided password and even those hosting the data do not have access to the key.
Anyway thanks for the info.


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9:30 am to 12:30 pm
From humble beginnings in 1951 as a common market for coal and steel among six countries, the European Union today has 28 member states, an elected parliament, and a common currency used in 19 of those states. It is widely viewed as fostering postwar peace and prosperity in Western Europe and later in guiding the democratization of the post-communist states in Eastern Europe. Today, though, it faces a number of challenges: the refugee crisis, 'Brexit', and growing nationalism. In this seminar, we will examine the historical development of the EU to understand how it operates today and how that contributes to the crises facing Europe today.
Newberry Teachers' Consortium members may register for this seminar through their designated membership contact as space permits. Non-member educators may register for this seminar by purchasing an individual membership at the time of registration. Registration for all NTC seminars opens Wednesday, September 7, 2016. For more information about NTC membership, please contact Charlotte Ross, Teacher Programs Manager, at firstname.lastname@example.com.
The seminar will be followed by a catered lunch. Registrants should RSVP for lunch to assist Teacher Programs staff in reducing waste.
A link to the assigned pre-readings for this seminar will be distributed to participants via email.
If you believe you are registered for this seminar but have not received an email confirmation or reminder, please contact Teacher Programs staff.


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Infolines are information services for citizens of non-EU states traveling or residing in Poland.
It does not provide information on asylum-related matters.
Infoline in Poland:
22 490 20 44 - from Monday to Friday, 9:00 am to 5:00 pm (GMT +1)
Charges in accordance with your operator's price list
Please be advised that on July 31, 2018, the Migrant Info infoline ends its operations. We apologize for inconveniences.
Foreigners in need of voluntary return assistance are requested to contact IOM
by phone:
22 628 24 13
22 623 81 76
666 34 34 66
or email firstname.lastname@example.com.
Go back
The website is current as of 30/06/2018
The website is co-financed from the The Asylum, Migration and Integration Fund (AMIF) National Programme – 'Safe harbor' under the project "MIGRANT.INFO - support for immigrants and introduction of them in the Polish society" implemented by the International Organization for Migration in partnership with the Ministry of Internal Affairs and Administration and the Chief Labour Inspectorate. The sole responsibility for the content of this website lies with the authors. The European Commission is not liable for any use of the information provided on the site.


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Emus BMS Smartphone Connectivity Module is low energy adapter that allows to wirelessly monitor various battery parameters in real time on any Android and iOS device using Emus EVGUI app, or any PC running Windows operating system, using the usb dongle. The Smartphone Connectivity Module connects to the main controller using RS232 interface. A compatible EMUS EVGUI App is available on Google Play and App Store in Apps downloads section.
Please use part numbers below for ordering the products
|Part number
|Package Contents
|12-24V Smartphone connectivity module for Android, iOS, EVGUI
EMUS BMS Smartphone connectivity module – 1pcs
2018-12-07 15:25:07
G1 BMS system user manual which documents the whole BMS system components, connectivity, configuration, monitoring, troubleshooting and much more.Download Release Notes
2018-12-10 10:34:09Download Release Notes


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Have a Question?
Amish-Made Poly-wood Furniture
We carry superior Amish-made outdoor furniture in every style and size! From charming Mission-style outdoor poly wood dining sets to magnificent pine wood double-bench gliders, we have it all! Within our Amish Outdoor Furniture Collection you'll find patio dining and accent tables, patio bars and bar stools, outdoor benches, and chairs, outdoor gliders and rockers, picnic tables, settees, chaise lounges, and even porch swings! And 25 great colors as well!
Be sure to ask about our 10% discount and 6 month, interest free lay-away program.
Call us at: (636)295-9499
Current Inventory


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Published on May 13, 2017
Women's Race Champion Will Be Decided in Sacramento Tomorrow Where Men's Amgen Tour of California Kicks Off Weeklong Race
SACRAMENTO, Calif. (May 13, 2017) – Team Sunweb's Coryn Rivera (Tustin, Calif.) led a fast and furious group sprint over the finish line to claim Stage 3 of the Amgen Breakaway from Heart Disease™ Women's Race empowered with SRAM as it streamed into the Sacramento area today.
"The girls were perfect. There was some chaos on the side, but we just stuck to the plan, and we executed perfectly, and I couldn't have pulled it off without them," said Rivera.
With the final day of racing on tap in downtown Sacramento tomorrow, UnitedHealthcare Pro Cycling's Katie Hall (Saratoga, Calif.) retains the overall race lead with one second separating her from Boels-Dolmans' 2016 Olympic Games road race gold medalist Anna van der Breggen (NED). Her teammate and last year's race champion Megan Guarnier (San Mateo, Calif.) heads into tomorrow in third place overall, 29 seconds off the lead.
Hall, who likely locked up the Queen of the Mountain category win today, says of her one-second lead: "Better than no seconds!... It really is an honor to be wearing the yellow [Amgen Race Leader] jersey for the women in my home state." Of her team's chances tomorrow: "We do love the stage – we've been here a couple of times. We're excited to fight tomorrow."
24-year old Cuban cyclist Arlenis Sierra (Astana Women's Team) took over the Visit California Sprint Jersey as well as the TAG Heuer Best Young Rider Jersey with her second-place finish today. Wiggle High5 Pro Cycling's sprinter Giorgia Bronzini (ITA) rounded out the podium in third place for the stage.
Earlier in the day, a breakaway including Mexican champion Ingrid Drexel (Team TIBCO - Silicon Valley Bank), Rally Cycling's Erica Allar (Fogelsville, Penn.) and Hagens Berman | Supermint's Scotti Lechuga (High Point, NC) brought their lead over the peloton to one minute.
Visit Dallas DNA Pro Cycling's Mandy Heintz (San Antonio, Texas) rode aggressively throughout the day, including a stint in a solo breakaway after an attack with 13 miles to go that she nurtured to a 35-second lead. Her ride earned her the Breakaway from Cancer® / Breakaway from Heart DiseaseTMMost Courageous Rider Jersey
Presented by Lexus, Stage 3 brought the riders 73.3 miles from first-time Host City Elk Grove to Sacramento, where the race champion will be decided tomorrow after 20 blazing fast circuit laps around the State Capitol Building.
Stage 4 of the women's race runs 12:30-2:10 p.m. tomorrow in downtown Sacramento. Fans are in for a whole day of pro cycling action, as the men's week-long Amgen Tour of California race begins at the same location, rolling off at 12:25 a.m. and finishing the day at 3:30 p.m.
Stage 3 Podium
- Coryn Rivera (USA), Team Sunweb (NED)
- Arlenis Sierra (CUB), Astana Women's Team (KAZ)
- Giorgia Bronzini (ITA), Wiggle High5 Pro Cycling (GBR)
Stage 3 Jersey Winners
- Amgen Race Leader Jersey – Katie Hall (USA), UnitedHealthcare Pro Cycling Team (USA)
- Breakaway from Cancer® / Breakaway from Heart DiseaseTMMost Courageous Rider Jersey – Mandy Heintz (USA), Visit Dallas DNA Pro Cycling (USA)
- Lexus Queen of the Mountain (QOM) Jersey – Katie Hall (USA), UnitedHealthcare Pro Cycling Team (USA)
- Visit California Sprint Jersey – Arlenis Sierra (CUB), Astana Women's Team (KAZ)
- TAG Heuer Best Young Rider Jersey – Arlenis Sierra (CUB), Astana Women's Team (KAZ)
Breakaway from Heart Disease™
To leverage its sponsorship of the race, Amgen's newest initiative -- Breakaway from Heart Disease™ -- encourages Americans to be aware of their risk of heart disease through cholesterol and blood pressure screening at the Lifestyle Festival, courtesy of the American Heart Association. Amgen is proud to encourage Americans to take charge of their risk of heart disease by offering educational information at each Lifestyle Festival throughout the tour as well as providing activities to help everyone feel empowered to take action for good heart health. To learn more, visit www.breakawayfromheartdisease.com, or visit the Breakaway from Heart Disease™ booth at the Lifestyle Festival.
About the Amgen Tour of California
The Amgen Tour of California is a Tour de France-style cycling road race created and presented by AEG that challenges the world's top professional cycling teams to compete along a demanding course that traverses hundreds of miles of California's iconic highways, byways and coastlines each spring. The teams chosen to participate have included Olympic medalists, Tour de France contenders and World Champions, and awards important, world-ranking points to the top finishers. More information is available at amgentourofcalifornia.com.
The first-ever U.S. UCI WorldTour course will cover more than 583 miles through 12 Host Cities May 14-20, including Sacramento, Modesto, San Jose, Pismo Beach, Morro Bay, Santa Barbara, Santa Clarita, Ontario, Mt. Baldy, Big Bear Lake, Mountain High and Pasadena. The Amgen Breakaway from Heart DiseaseTM Women's Race empowered with SRAM May 11-14 will cover 256.6 miles through South Lake Tahoe, Elk Grove and Sacramento, concluding as the men's race gets underway.
About Amgen
Amgen is committed to unlocking the potential of biology for patients suffering from serious illnesses by discovering, developing, manufacturing and delivering innovative human therapeutics. This approach begins by using tools like advanced human genetics to unravel the complexities of disease and understand the fundamentals of human biology.
Amgen focuses on areas of high unmet medical need and leverages its expertise to strive for solutions that improve health outcomes and dramatically improve people's lives. A biotechnology pioneer since 1980, Amgen has grown to be one of the world's leading independent biotechnology companies, has reached millions of patients around the world and is developing a pipeline of medicines with breakaway potential.
For more information, visit www.amgen.com and follow us on www.twitter.com/amgen.
About AEG
Headquartered in Los Angeles, California, AEG is the world's leading sports venue and live entertainment company. With offices on five continents, AEG operates in the following business segments: AEG Facilities, which is affiliated with or owns, manages or consults with more than 120 preeminent arenas, stadiums, theaters, clubs and convention centers around the world including The O2 Arena, the Sprint Center and the Mercedes-Benz Arenas; AEG Presents, which is dedicated to all aspects of live contemporary music performances, including producing and promoting global and regional concert tours, music and special events and world-renowned festivals; AEG Real Estate, which develops world-class venues, as well as major sports and entertainment districts like STAPLES Center and L.A. LIVE; AEG Sports, which is the world's largest operator of sports franchises and high-profile sporting events; and AEG Global Partnerships, which oversees worldwide sales and servicing of sponsorships including naming rights, premium seating and other strategic partnerships. Through its worldwide network of venues, portfolio of powerful sports and music brands and its integrated entertainment districts, AEG entertains more than 100 million guests annually. More information about AEG can be found at www.aegworldwide.com.
# # #
Media Contacts:
Michael Roth, AEG
Josh Levitt, Canvas Blue


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As the boating season winds down, Formula owners want to spend every spare second on their boats, capturing some last minute fun and creating memories to warm winter-weary hearts when the snow begins to fall. So why not stretch your Formula day past the sunset and treat yourself to dinner on board with this simple yet delicious meal?
Per fish packet
Light the fire on the grill to preheat while you prep the fish. Place Talapia into individual foil packets. Top each filet with 1 Tablespoon butter, 1 Tablespoon of capers, and a few scallion slices. Squeeze the juice from a wedge of lemon (1/4 lemon) on top and add garlic pepper to taste. Seal the packets and let them cook on the grill 10 minutes or until fish flakes easily.
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Sunday School (for children aged from pre-kinder up to Grade 6) happens each week of school term (not in the holidays!) in the Upper Room. We are following the curriculum called 'On the Way'. Sunday School is a great time for children to learn of the claims of Jesus and how they can follow Him in the world.
All our Sunday School teachers and helpers have the necessary approvals and Working With Children's Checks.


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Cutlery Polisher
A tabletop cutlery polisher will do wonders for your cutlery, and frees up staff from spending hours manually polishing with a rag. Simply put your clean cutlery in one end of the machine, and it comes out the other side sanitary and shining. A cutlery polisher will be able to polish up to 8,000 pieces of cutlery per hour.
A cutlery polishing machine is designed with space-saving in mind, meaning they're ideal for compact café and restaurant kitchens. They combine high volume with the ability to reliably deliver hygienic and pristine cutlery, so that your customers get a consistently top-quality experience. Importantly, they save staff labour time, so a cutlery polisher will soon pay for itself.
We're pleased to supply tabletop cutlery polishing machines from Thomas Dörr and Sammic. It's also possible to lease a cutlery polisher from Dishwashers Direct.
TD 1500 - Tabletop cutlery polishing machine FROM THOMAS DÖRR
List Price: £2,080.00
Our Price: £1,899.00
Sammic 3001 Cutlery Polisher
List Price: £3,536.00
Our Price: £1,999.00
TD 2000 - Tabletop cutlery polishing machine FROM THOMAS DÖRR
List Price: £2,360.00
Our Price: £1,999.00
TD 3000 - Tabletop cutlery polishing machine FROM THOMAS DÖRR
List Price: £3,120.00
Our Price: £2,999.00
Sammic 5001 Cutlery Polisher
List Price: £5,771.00
Our Price: £3,149.00
Call for availability
Sammic 6001 Cutlery Polisher
List Price: £6,494.00
Our Price: £3,549.00
Call for availability


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Question # : 69376
Answer : 69376
Published on: Oct 8, 2016
بسم الله الرحمن الرحيم
(Fatwa: 1151/1140/M=12/1437)
If the said National Institute is located in the city or in the suburb of the city or in town or in big village where the conditions that make Jumuah salah valid are found then it is allowable to establish Jumuah salah with Khutba (in Arabic language) and jamat in college Hall.
Allah knows Best!
Darul Ifta,
Darul Uloom Deoband
Related Question


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A1 Garage Door Service & Repair In New River, AZ
Are you experiencing issues with your garage door? Trust only the #1 garage door specialist in Arizona — A1 Garage Door Service. Contact us now at 623-777-2833.
A1 Garage Door Service is the trusted service provider in installing, servicing and repairing all kinds and types of garage doors. We encourage you to check out the reviews of the people of Phoenix and Arizona on the kind of garage door service that we provide. Our customers have consistently given us A+ rating on BBB, 5-star ratings on Yelp and the Super Service Award on Angie's List to express their satisfaction on our service.
We service the garage door needs of everyone in New River, including nearby places and cities bordering it. When you call us, expect our technicians to arrive promptly and efficiently attend to your garage door problems.
We have the best team of expert professionals who know how to handle each and every type of garage door and its components. This is because all of our technicians go through a rigorous training process, covering every known garage door repair technique. We do not let them onto the field until we know that they are completely trained, and can perform a job to perfection. Aside from this, they are continually updated with all the latest knowledge, information and training in order to do their work efficiently.
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A1 Garage Door Service specializes in the installation, repair and maintenance of all commercial doors such as as security gates, roll-up garage doors, warehouse doors & more for local Tempe companies. Simply call us at 623-777-2833 .
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We have been talking about garage doors here ever since because it is our business. But we have not yet taken the time to look back at how the garage door industry started. We have taken it for granted that garages (and garage doors) are part of our houses but when...
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A-1 Garage door complete replacement
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mesosphere(redirected from mesospheric)
Also found in: Dictionary, Thesaurus.
mesosphere:see atmosphereatmosphere
[Gr.,=sphere of air], the mixture of gases surrounding a celestial body with sufficient gravity to maintain it. Although some details about the atmospheres of other planets and satellites are known, only the earth's atmosphere has been well studied, the science of
..... Click the link for more information. .
the atmospheric layer above the stratosphere, having an altitude from 50–55 to 80 km. It is characteristic of the mesosphere that the temperature decreases with altitude from approximately 0°C at the lower boundary of the layer to -90°C at the upper boundary.
The atmospheric shell between about 28-35 and 50-60 miles (45-55 and 80-95 kilometers), extending from the top of the stratosphere to the mesopause; characterized by a temperature that generally decreases with altitude.
1. the atmospheric layer lying between the stratosphere and the thermosphere, characterized by a rapid decrease in temperature with height
2. the solid part of the earth's mantle lying between the asthenosphere and the core


text:
Yes exactly yes! I'm not being negative or anything but… there is absolutely nothing to be negative about in this top class BBC comedy programme. There's more bikes, more of David casually thieving ideas and more of Will looking blank – it's W1A time…
Cycling Shorts ~ So Will (yeah, crap) had this idea – ok, cool, no worries, and Izzy has been driving it forward. Jack has interfered and made a pilot with unwitting Will's help and as if that wasn't hard enough for Izzy to get her head around, David Wilkes has had his paws on it yet again and shown Anna (yes exactly yes) the footage that Will has collated. It's doomed!
Anna on a bike (yes exactly) ~ In what I see as a bid to get another step closer to Ian – Anna, the most unlikely cyclist has a folding bike! How long before every member of the BBC staff is traveling by bike? It's definitely a strategic move from the has so little to say for herself, with hilarious consequences too.
How do you solve a problem like Ryan? ~ After his disastrous appearance on Match Of The Day, Ryan has been pulled back into the BBC to potentially join the line-up on Question Of Sport. It's Lucy's idea, Siobhan doesn't even know it's a programme, but Simon (and Tony I expect, after his mindfulness break) are buzzing with it. However, when the meeting happens and Ryan's new agent appears (Siobhan!) there's suddenly the suggestion of Top Gear 2 instead. Embraced by both Ryan and Siobhan as they take a selfie together!
Zoe & Chloe ~ The Charter renewal is in progress and Fiona Craig is in the building in charge and flanked by Zoe and Chloe. Zoe and Chloe are like the female version of Ben and Jerry. Genius!
Lucy's lament ~ Lucy is the token put upon member of the BBC team and it seems that leaving her role to take up a job in a Gardening Centre is still not entirely off her mind. Although Ian is wondering if she'll apply for the new role – and we all know that Lucy's likely to get lumbered with that job, even if she doesn't want it. From Better to Empowerment? Or will she be pulled by the call of the compost?
|Jack Patterson
|Jonathan Bailey
|Ryan Chelford
|Ben Batt
|Ian Fletcher
|Hugh Bonneville
|Faiza Rajah
|Priyanga Burford
|Tracey Pritchard
|Monica Dolan
|Jerry Guildencrantz
|Ivan Gonzalez
|Siobhan Sharpe
|Jessica Hynes
|David Wilkes
|Rufus Jones
|Izzy Gould
|Ophelia Lovibond
|Rose Matafeo
|Ben Rosenstern
|Max Olesker
|Anna Rampton
|Sarah Parish
|Fiona Craig
|Pooky Quesnel
|Clive Cook
|Derek Riddell
|Emma Sidi
|Will Humphries
|Hugh Skinner
|Lucy Freeman
|Nina Sosanya
|Simon Harwood
|Jason Watkins
|Neil Reid
|David Westhead
|David Tennant
|Paul Schlesinger
|John Morton
|John Morton
|Executive Producer
|Jon Plowman


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Elliott Smith emerged from the Pacific Northwest rock scene of the early 1990s, a performer soaked in both grunge's soul-baring angst and Beatles-infused pop. Smith's refined sense of arrangement and composition made him a singular figure among singer-songwriters and produced a stellar solo catalog until his presumed suicide in 2003.
The Texas native, born Stephen Paul Smith, spent his teenage years in Portland, OR. After graduating from Hampshire college in Amherst, Massachusetts, Smith moved back to Portland with university pal Neil Gust in 1992 and started the group Heatmiser. The band released two albums on Frontier Records — 1993's Dead Air and 1994's Cop and Speeder — and was signed to Virgin, which put out their final album, Mic City Sons in 1996 on subsidiary Caroline Records.
By that time, Smith was already two albums into a solo career. Roman Candle, a set of spare, acoustic songs, was released on Cavity Search Records in 1994. The songs were ostensibly home demos deemed not suitable for Heatmiser (Smith being one of the two principal songwriters in that band), evidenced by a quartet of songs being titled "No Name #1," "#2," etc. But songs like "Condor Ave." and "Last Call" showed an emotional potential that reviewers linked to Nick Drake and Bob Dylan.
Smith's second, eponymous LP was issued in 1995 on the Kill Rock Stars label and showed marked songwriting progression. The album was partially recorded and mixed at Heatmiser drummer Tony Lash's house and featured Gust's guitar on the song "Single File" as well as guest vocals from the Spinanes' Rebecca Gates on "St. Ides Heaven."
For the most part, Smith stuck to the instrumentation style of Roman Candle, focusing on the mix of his breathy, pained singing voice and his methodically melodic acoustic guitar playing. Lyrically, the album wallowed in depression, with hefty dashes of addiction and loneliness as seasoning. Smith admitted in interviews that his words drew from his own experience with drug and alcohol dependencies and suicidal episodes. Harrowing opener "Needle in the Hay" was later chosen by film director Wes Anderson to soundtrack a suicide-attempt scene in his 2001 movie The Royal Tenenbaums.
Tensions within Heatmiser, reportedly focused on Smith's preference for his burgeoning solo career (and his disparaging comments about the band's music in interviews) led to the group disbanding in 1996 before the release of Mic City Sons, which was produced by Tom Rothrock and Rob Schnapf.
Free to concentrate his musical inspirations wholly on his solo efforts, Smith set to creating his most fully realized album to date, 1997's Either/Or. The lion's share of songs on the LP were still sparsely arranged, but the songwriting had once again taken a step forward, Smith reaching for more melody in memorable choruses and adding wrinkles in mixing and production, such as the multi-tracked harmony vocals on uplifting closer "Say Yes" and the surprisingly evolved "Ballad of Big Nothing," which found Smith utilizing full-band instrumentation (he played and sung every note on the album himself) on an uptempo rock/pop song for the first time in three albums. Rothrock and Schnapf mixed the set, furthering a working relationship that would last until Smith's untimely death six years later.
The Either/Or track "Pictures of Me" best exemplifies Smith's progression toward becoming the studio-rat pop maven of his later years. The song starts out with representative finger picking and acoustic guitar strumming, but as Smith heads into the first bridge, a rough-hewn but catchy beat kicks in and propels the rest of the song. The obvious touchstone is latter-day Beatles at their rawest, with steady, head-shaking drums and the ham-it-up melodic bassline.
Either/Or was Smith's breakthrough album in that its success brought about two watershed points in his musical career: In 1998 he signed to DreamWorks records, his label for his next two LPs. His music also caught the ear of film director Gus Van Sant, who commissioned Smith to provide music for his 1997 film Good Will Hunting. Smith recorded an orchestral version of Either/Or's "Between the Bars" and also contributed a new original song, "Miss Misery" (two other Either/Or cuts, "Angeles" and "Say Yes," were also included on the soundtrack). Following the film's commercial and critical success, "Miss Misery" was nominated for an Academy Award for Best Song. Though it would ultimately lose out to the Titanic theme, the song brought Smith to a much wider audience when he performed it onstage at the Oscars in March 1998.
Smith recorded his DreamWorks debut, 1998's XO with help from LA studio veterans Jon Brion and Joey Waronker, and the album featured elaborate arrangements and lush instrumentation. Everything shines, from the candy-sweet staccato pop of "Baby Britain" to the hesitating maternal love letter "Waltz #2 (XO)" to a capella closer "I Didn't Understand," whose musical bed comprises a multitude of Smith's own harmonic vocals, echoing post Pet Sounds-era Brian Wilson.
XO hit No. 104 on The Billboard 200 album chart and Smith supported it with an extensive tour that included an appearance on Saturday Night Live in October of 1998. His backing band in this era often included former Heatmiser bandmate Sam Coomes, who contributed as a keyboardist and multi-instrumentalist as well as serving as one-half of sometime opening act Quasi. The next year, Smith moved permanently to Los Angeles and began work on his follow-up, Figure 8, which would be released on DreamWorks in 2000 and become the last album Smith put out in his lifetime.
The Figure 8 sessions found Smith (with Rothrock and Schnapf in tow) working in Los Angeles as well as in London at the Beatles' famed Abbey Road studios. As if the Beatles comparisons Smith drew in reviews weren't enough, he also contributed a cover of the Fab Four's "Because" to the American Beauty soundtrack in 1999.
Like its predecessor, Figure 8 is full of modern chamber pop, mixing in keyboards, strings and studio flourishes on melodically simple, yet structurally complex songs. "Somebody That I Used to Know" and "I Better Be Quiet Now" hearken back to the bare acoustic setting of Smith's first solo albums, while jangly opener "Son of Sam" and the rainy-day rocker "Junk Bond Trader" sound like long-lost radio singles (Figure 8 would reach No. 99 on the Billboard charts).
While the music on Figure 8 is some of the most upbeat in his career, the lyrics on Figure 8 still lean toward the melancholy. Even the chiming "LA," with its vaudevillian caricature of Smith's new hometown, portends toward self-destruction in its very chorus: "Living in the day / but last night I was about to throw it all away."
Recording the follow-up to Figure 8 proved more difficult than expected, as Smith's drug habits reportedly got in the way of separate sessions with Schnapf and Brion. He performed sporadically throughout 2001 and 2002, working on music for a new album by himself and with David McConnell of Goldenboy.
After playing only three shows during 2002, Smith returned to the stage in January 2003 with a trio of gigs each in Los Angeles and New York. He also played an east coast tour in early summer, appearing at the Field Day festival at Giants Stadium on June 7, 2003. Back in Los Angeles and working on a sizeable batch of songs under the working title From a Basement on the Hill, Smith released a two-sided vinyl single on Suicide Squeeze Records featuring two new songs, "Pretty (Ugly Before)" and "A Distorted Reality is Now a Necessity to be Free." He took time out of recording to make his last concert appearance September 19 at Redfest in Salt Lake City.
On October 21, 2003, Smith died from two stab wounds to the chest. The coroner's report called it "possible suicide versus homicide," leaving the mode of death officially unconfirmed. Smith had been arguing with his live-in girlfriend Jennifer Chiba and according to Chiba, she locked herself in the bathroom when she heard Smith scream, and emerged to find him with a knife in his chest. The report also noted Smith's reported longstanding narcotics habit (specifically, "heroin and crack") but that Chiba said he had been clean for a year.
Smith's estate hired Schnapf and longtime Smith associate Joanna Bolme to put together an album from the sessions the artist had been working on in the years since Figure 8. Anti- records released From a Basement on the Hill in October 2004. The album, which peaked at No. 19 on The Billboard 200, features 15 tracks whittled down from over 30 that Smith had left. It included different versions of both songs from the Suicide Squeeze single, and a handful of other notable compositions that rank with some of the artist's best work, especially the delicate, pastoral "Let's Get Lost," the XO-style nostalgia trip "A Passing Feeling" and the dark rocker "Don't Go Down."
In May 2007, Kill Rock Stars released New Moon, a two-disc compilation of songs Smith recorded while signed to the indie label, including demos, b-sides and a cover of Big Star's "Thirteen." The set debuted at No. 24 on The Billboard 200, and was warmly received by critics, with its glimpse into the depth of Smith's artistry at what was in retrospect considered by many to be his creative peak. "High Times," "Fear City" and "Either/Or," amongst others, are as compelling as much of the music on Smith's proper albums.
In December 2009, Kill Rock Stars announced that it had obtained the rights to re-release From a Basement on the Hill and Roman Candle, essentially consolidating the rights for all of Smith's independent albums. The press release on the label's website was accompanied by a free download of an unreleased Smith song, "Cecilia/Amanda."


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Black Helen is the working name of a project that I am involved with. More than just involved. I created the project.
*** updated April 2,, 2019
The name alludes to one of the most known...yet, UNKNOWN, Black women in history. the MOST BEAUTIFUL WOMAN IN THE WORLD. The "Face that Launched 1,000 Ships"..
Black Helen is about the Black woman called Helen of Troy!
This first blog entry is about the FILM (Black Helen).
Today is March 20, 2019. I have started to work on the script and outline of the film. Also, on the other back end, I am acquiring the equipment, writing the business and marketing plan and doing the virtual logistics and road map for the project.
Phase One:
Mar 20, 2019
Do you remember the book called "Black Athena: The Afroasiatic Roots of Classical Civilization (The Fabrication of Ancient Greece 1785-1985, Volume 1)"...by Martin Bernal?
The Black Athena book brought so much DRAMA in the mid eighties. Honestly, it seemed that book threatened to snatch the rug of the fraud called race and racism right from under the feet of the people that benefit and uphold that shameful "scientific racism.".. YANK! There was so much back and forth and desperation in academia to hold onto he false beliefs that the ancient Greeks were so called white people. Although, Black Athena came close, it FAILED to bring the REAL ancient history and identity of the Black people called Greeks.
I intend to bring that DISCUSSION and CONTROVERSY back to the minds of the people. It's been 30 years since that book was published and we have made HUGE advances on the knowledge and have discovered the more hidden and esoteric parts of ancient history (ancient Black history)!
Not ONLY was the Greek Goddess, Athena, a Black Woman (Athena was the Neith of the Egyptians). But the MAIN Greek God, called Zeus, was a Black man. Zeus is the Black Egyptian God/Man called Amen, Ammon, Ammon Zeus, Zeus Ptah, Jupiter, Jove, YHVH, Yahweh. Jehova, etc.
The ancient Greeks wrote that they got their knowledge and personifications of God from the ancient Egyptians. It has been VERY hard to get the modern world to ADMIT that the ancient Egyptians were Black people. We know they were. BUT...do you know the ancient GREEKS were Black people, too?
This film will be MONUMENTAL and instrumental in the END of racism. This changes EVERYTHING!
blackhelen.com T shirts are available here:
Mar 27, 2019
I am talking to a few musick producers than are going to provide the sound design for the film. no names yet because we are just meeting and discussing the possibilities.
The MOST BEAUTIFUL WOMAN IN THE WORLD...
That is what they called the Black woman known as Helen of Troy. We have started a line of beauty products in your honor. In her honor. The sales of these products will help fund the and promote the film. I have a Patreon that I am using for support as well. Only one person is patronizing this effort so far.
Join on to support...
Watch this video to get an idea about what was going on 30 years ago. We have learned a LOT since then...
This is an example of an actor reciting his lines for a part in the movie. Good luck, guy.
We also have a group on facebook. Here.


text:
Not all companies list their staff on their website, but many do and the site search technique is a wonderful way to find them.
The Google search syntax for this search is as follows:
The Google search syntax for this search is as follows:
site:URL (Job Title)
For example, if you are looking for profiles of people in the Executive Management at say Kforce Inc., the Google search script will be:
Clicking on the first result will take you to the company's webpage where their leadership is listed.
This particular command works in all the 3 major search engines - Google, Yahoo and Bing.