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Hepatocellular carcinoma (HCC) is sexually dimorphic in both rodents and humans, with significantly higher incidence in males, an effect that is dependent on sex hormones. The molecular mechanisms by which estrogens prevent and androgens promote liver cancer remain unclear. Here, we discover that sexually-dimorphic HCC is completely reversed in Foxa1- and Foxa2-deficient mice after diethylnitrosamine-induced hepatocarcinogenesis. Co-regulation of target genes by Foxa1/a2 and either the estrogen receptor (ERα) or the androgen receptor (AR) was increased during hepatocarcinogenesis in normal female or male mice, respectively, but was lost in Foxa1/2-deficient mice. Thus, both estrogen-dependent resistance to and androgen-mediated facilitation of HCC depend on Foxa1/2. Strikingly, single nucleotide polymorphisms at FOXA2 binding sites reduce binding of both FOXA2 and ERα to their targets in human liver, and correlate with HCC development in women. Thus, Foxa factors and their targets are central for the sexual dimorphism of HCC.
Sexual dimorphism is the biological inequality between females and males, favoring females in a variety of conditions including resistance to nutrient deprivation, prevention of premature aging, and resistance to diseases such as vascular and heart disease, brain disorders, and hepatocellular carcinoma (HCC) (Genazzani et al., 2007; Kalra et al., 2008; Stice et al., 2009). Sex hormones, i.e. estrogens in females and androgens in males, are the drivers of sexual dimorphism.
HCC is the fifth most common cancer and ranks third in annual mortality worldwide (Parkin et al., 2005). Women show significantly lower incidence of HCC than men (Parkin et al., 2005). Female rodents are also resistant to HCC compared to males during chemically-induced carcinogenesis (Kalra et al., 2008). Male mice treated with estrogen develop fewer liver tumors than control males, and ovariectomized females develop more liver tumors than normal females during chemically-induced carcinogenesis (Naugler et al., 2007; Shimizu et al., 1998; Tsutsui et al., 1992; Yamamoto et al., 1991). In addition, female mice deficient for the estrogen receptor alpha (ERα) lose their resistance to HCC (Naugler et al., 2007), while reduced incidence of HCC was observed in male mice lacking the androgen receptor (AR) (Ma et al., 2008; Wu et al., 2010). These data demonstrate that both protective effects of estrogens and deleterious effects of androgens contribute to the sexual dimorphism in HCC incidence. However, the molecular mechanisms of how this is achieved remain to be determined.
A recent report suggested that estrogens prevent HCC through inhibition of IL-6 expression in Kupffer cells, the resident macrophages in the liver, and that this in turn affects hepatocyte proliferation (Naugler et al., 2007). However, follow-up studies employing IL-6 antagonists or estrogen and its analogs to prevent HCC indicate that other mechanisms must also be involved in estrogen-mediated protection from liver cancer (Di Maio et al., 2006; Kalra et al., 2008; Lawrence et al., 2007). Comparatively little is known about the mechanism by which androgen signaling promotes HCC in males, although a recent study suggests that androgens enhance DNA damage and oxidative stress during hepatocarcinogenesis (Ma et al., 2008).
The vertebrate forkhead box A (Foxa) gene family of transcription factors consists of three members, Foxa1, Foxa2 and Foxa3, which are encoded by individual genes in mammals (Kaestner, 2010). Previous gene ablation studies of Foxa factors in mice have shown that Foxa1 and Foxa2 redundantly regulate liver development and metabolism, whereas the role of Foxa3 in the liver is limited (Bochkis et al., 2008; Friedman and Kaestner, 2006; Kaestner, 2005; Kaestner et al., 1998; Kaestner et al., 1999; Lee et al., 2005; Li et al., 2009; Shen et al., 2001; Sund et al., 2000). Though no liver forms in mice when both Foxa1 and Foxa2 are ablated in foregut endoderm following gastrulation (Lee et al., 2005), ablation of both Foxa1 and Foxa2 after liver specification does not affect hepatocyte development or proliferation (Li et al., 2009).
Genome-wide location analyses have revealed that FOXA1 and ERα or AR frequently bind to adjacent cis-regulatory elements in their target genes in human breast or prostate cancer cell lines, respectively, and that the recruitment of ERα or AR to their targets depends on FOXA1 (Carroll et al., 2005; Gao et al., 2003; Lupien et al., 2008; Yu et al., 2005). Hence, FOXA1 plays an essential role in estrogen and androgen signaling in breast and prostate epithelia.
We hypothesized that the effects of estrogens and androgens on HCC development are dependent upon Foxa factors. Using liver-specific gene ablation, we demonstrate that sexually-dimorphic HCC is completely dependent on Foxa1/2, and establish, using genome-wide analyses, how the winged helix transcription factors modulate the responses of the sex hormones in hepatic carcinogenesis.
To investigate the role of Foxa factors in hepatocarcinogenesis, we induced liver tumors in control and liver-specific Foxa1/2-deficient mice (mutant) of both genders. We employed the established N,N-diethylnitrosamine (DEN) followed by TCPOBOP two-step strategy to induce rapid HCC formation in mice. Consistent with previous reports (Kalra et al., 2008), male control mice developed severe HCC following carcinogen treatment, whereas female control mice were resistant to hepatocarcinogenesis, as evidenced by both direct visualization and NMR quantification of tumor nodules (Figure 1A and 1B). Strikingly, multiple and large tumors were found in female Foxa1/2-deficient livers after carcinogen treatment, while tumor growth in male mutants was reduced when compared to controls (Figure 1A and 1B). Thus, sexual dimorphic HCC is completely dependent on the Foxa factors in mice. Immunostaining for Cytokeratin 19 (CK19) and hepatic nuclear factor 4 alpha (HNF4alpha), markers of cholangiocytes and hepatocytes, respectively, confirmed that the tumors in Foxa1/2-deficient livers were HCC and not cholangiocarcinomas (Figure 1C), consistent with the genotoxic action of DEN on hepatocytes. In the two-step strategy for hepatocarcinogenesis, phenobarbital-like inducers are commonly used for `boosting' tumor growth following tumor initiation by DEN (Oliver and Roberts, 2002; Qatanani and Moore, 2005). These phenobarbital-like inducers are CAR (constitutive androstane receptor, or Nr1i3) and PXR (pregnane X receptor, or Nr1i2) agonists. We had included the phenobarbital-like inducer TCPOBOP in our protocol, leading to the question of whether Foxa1/2-dependent differential tumor growth was related to CAR/PXR signaling. However, neither CAR nor PXR expression levels were correlated with tumor load in our mice; in fact we saw the lowest levels of CAR/PXR in female mutant mice treated with carcinogen, which had the highest tumor load (Figure S1A).
To investigate the mechanisms of relative HCC resistance in female mice, we characterized gender-specific gene expression in mice with or without carcinogen treatment using microarray analysis. 6,612 genes exhibited gender-specific differential expression in control mice without carcinogen treatment (Figure 1D). Interestingly, only 2,019 genes were differentially expressed between female and male control mice after carcinogen treatment, among which 1,078 genes were expressed in a gender-specific fashion only after DEN treatment (Figure 1D). This latter group is comprised of the gender-specific and carcinogen-responsive genes, which may play a role in the resistance to carcinogenesis in female mice and/or the promotion of carcinogenesis in male mice.
To identify Foxa and ERα target genes in the liver, we determined genome-wide binding sites of Foxa1, Foxa2, and ERα in control female livers without carcinogen treatment by ChIP-Seq. We also analyzed target genes associated with these binding sites (Table S1). By intersecting these target genes with the 6,612 genes showing gender-specific expression identified above (Figure 1D), we found 52% and 54% (919 and 1,117 genes) of ERα-associated genes associated with Foxa1 and Foxa2 binding sites, respectively (Figure 2A), suggesting co-regulation of Foxa1/2 and ERα of the majority of genes modulating gender dimorphism in females.
Next, we asked whether regulation of gender-dimorphic gene expression was also dependent on the Foxa factors and ERα in livers of female mice exposed to carcinogen. As shown above, over 2,000 genes are expressed in a gender-specific fashion in the liver of mice exposed to DEN (Figure 1D). When we analyzed transcription factor binding in livers of female mice exposed to carcinogen (Table S1), we found co-occupancy of the Foxa factors with ERα greatly increased compared to the situation in livers of untreated females, with 77% and 92% of the ERα-bound genes also being targets of Foxa1 and Foxa2, respectively (Figure 2A), indicating enhanced co-regulation of Foxa1/2 and ERα in females during carcinogenesis. This finding suggests that the protection of females from hepatocarcinogenesis might result from co-regulation of protective target genes by both Foxa1/2 and ERα. Further analysis revealed that the Foxa/ERα dual target genes were almost equally distributed between those that were expressed in gender-specific fashion before and after carcinogen-treatment, which we define as `inborn protection' and `reactive protection', respectively (Figure 2C).
Next, we analyzed genome-wide co-localization of Foxa1/2 and ERα binding sites by calculating the distribution of ERα binding sites relative to the center of the Foxa1 or Foxa2 binding sites. Foxa1/2 and ERα binding sites were close to each other at the regulatory regions of dual target genes in control females (Figure 3A and 3B), suggesting co-regulation and/or co-operative binding Foxa1/2 and ERα. Remarkably, Foxa1/2 and ERα co-occupancy dramatically increased following carcinogen treatment in female livers, paralleling the increased number of binding sites of Foxa1, Foxa2, and ERα (Table S1, and Figure 3A and 3B), further supporting the notion that enhanced co-regulation of Foxa1/2 and ERα in response to carcinogen exposure plays a critical role in protecting female mice from HCC. More importantly, when we analyzed binding sites of ERα genome-wide in Foxa1/a2 mutant livers, we found ERα binding dramatically reduced (Table S1, and Figure 3A and 3B), providing genetic evidence that the recruitment of ERα to its binding sites depends to a very large extent on Foxa1/2.
FOXA1 has been found to directly interact with ERα in human breast cancer cell lines (Carroll et al., 2005; Gao et al., 2003). To investigate the mechanism by which Foxa1/2 and ERα modulated tumor progression in the liver, we employed immunoprecipitation with anti-Foxa1, anti-Foxa2, or anti-ERα antibodies followed by Western blot analysis. We found that both Foxa1 and Foxa2 are complexed with ERα in the female mouse liver with or without carcinogen administration (Figure 3C), further supporting the notion of co-regulation of target genes by Foxa1/2 and ERα. As expected, the interaction between Foxa1/2 and ERα was abolished in Foxa1/2 mutant livers, confirming the specificity of the immunoprecipitation assay (Figure 3C).
Genomic distribution analysis showed that Foxa1, Foxa2, and ERα binding sites were widespread in the genome, with preference at enhancer regions (intergenic and intron regions) (Table S1 and S2). In addition, the common targets of Foxa1/ERα were largely overlapping with those of Foxa2/ERα in the female liver (Figure 4A). In total, we found 609 Foxa/ERα common targets in genes for which gene expression changes in response to carcinogen exposure (Figure 4A).
Next, we analyzed the gene expression profile of these 609 Foxa/ERα dual target genes in female control and Foxa1/a2 mutant liver following exposure to carcinogen. Strikingly, we found that the change in gene expression for these Foxa/ERα dual targets following DEN treatment was largely (for ~77% of the genes in question) reversed between control and mutant female mice (Figure 4B). Thus, deficiency of Foxa1/2 caused differential expression of Foxa/ERα dual targets, indicating that the co-regulation of Foxa/ERα dual target genes depends on Foxa1/2.
To investigate the mechanism that caused enhanced co-regulation of Foxa and ERα, we analyzed the target occupancy of each factor (Foxa1, Foxa2, or ERα) before and after carcinogen treatment. We observed enhanced occupancy of Foxa1, Foxa2, and ERα in livers of female mice exposed to carcinogen (Figure S2A). To confirm this observation, we performed sequential ChIP-qPCR assays with anti-Foxa2 and anti-ERα antibodies for 20 Foxa2/ERα targets identified by ChIP-Seq analysis. 16 targets were confirmed for both Foxa2 and ERα with at least 2-fold enrichment in liver of female control mice not treated with carcinogen (Figure S3A). As expected, none of these genes were ERα-bound in male mice, although 13 of them were bound by Foxa2 (Figure S3C). All 20 targets showed significant binding for both Foxa2 and ERα in livers of female mice after carcinogen administration (Figure S3A). Linear regression analysis showed that carcinogen exposure significantly enhanced binding of Foxa2 and ERα to these common targets (Figure S3B). Interestingly, enhanced Foxa2 binding at these loci was also observed in male mice after carcinogen administration, although these sites lacked ERα binding as expected (Figure S3C). Strikingly, we found that ERα binding to all these targets was eliminated in Foxa1/2-deficient livers, regardless of exposure to carcinogen (Figure S3A), further demonstrating that ERα binding at these dual targets is completely dependent on the presence of Foxa factors. In conclusion, these findings suggest that Foxa1/2 and ERα cooperate to regulate gene expression in the female liver, and that Foxa1/2 is essential for estrogen signaling in the protection of female mice from chemically-induced hepatocarcinogenesis.
Detailed analysis of the Foxa/ERα dual targets suggested that these genes modulate multiple pathways in resistance to HCC, as indicated by their gene annotations (Figure 4C). One of these targets is the oncogene Myc. We found multiple Foxa2 and ERα binding sites in Myc promoter and enhancer regions, and confirmed Foxa2 and ERα co-occupancy at these sites by sequential ChIP (Figure S4A). In addition, we found that Myc expression was significantly higher in female mutant livers compared to controls after carcinogen treatment (Figure S4B). When we suppressed Myc expression in isolated hepatocytes from DEN-treated Foxa1/2 mutant mice using siRNA, hepatocyte proliferation was significantly reduced as shown by Ki67 expression (Figure S4C). These findings implicate Myc as an important target for repression by Foxa2/ERα in the livers of female mice. However, as mentioned above, multiple genes shown to be involved in cell cycle control and carcinogenesis are Foxa/ERα dual targets, and multiple co-binding sites of Foxa1/2 and ERα were also observed at regulatory regions of the `HCC-resistant genes' as defined above. These genes are involved in pathways related to cancer resistance, including xenobiotic metabolism and detoxification, DNA biosynthesis and replication, and cell cycling and proliferation (Figure 4C and Figure S6). Thus, Foxa1/2 and ERα co-regulate multiple pathways of hepatocarcinogenesis.
A prior study had proposed IL-6 as an essential mediator of gender-specific hepatocarcinogenesis (Naugler et al., 2007). We had found previously that gene ablation of Foxa1/2 after liver specification led to mildly elevated IL-6 expression and bile duct hyperplasia in the adult mouse liver (Li et al., 2009). Based on this model, we expected that Foxa1/2-deficient livers would develop more tumors than controls in both genders, because serum IL-6 levels were elevated similarly in male and female Foxa1/2 mutant mice (Figure S5). However, this was not the case, as female Foxa1/2 mutant mice developed dramatically more and larger tumors than male mutants (Figure 1A). Furthermore, while we observed higher serum IL-6 levels in male mice compared to female control mice after DEN administration, IL-6 levels in DEN-treated mutant mice did not correlate with carcinogenesis in males and females (Figure 1A and Figure S5). In addition, the tumor load did not correlate with the levels of IL-6 among all four groups (Figure 1A and Figure S5). Therefore, we conclude that IL-6 is not a major factor regulating sexual dimorphism of HCC.
Remarkably, Foxa1/2 deficiency significantly reduced tumor load in male mice after DEN administration (Figure 1A and 1B). To investigate the molecular basis of this phenomenon, we analyzed genome-wide binding sites and the corresponding genes for Foxa1, Foxa2, and AR in control male livers without carcinogen treatment by ChIP-Seq (Table S1). Genomic distribution analysis showed that Foxa1, Foxa2, and AR binding sites were widespread in the genome, with preference at enhancer regions (intergenic and intronic regions) (Table S1). The numbers of Foxa1, Foxa2, and AR binding sites were greatly increased in livers of male mice exposed to carcinogen as compared to controls (Table S1, Figure S2B). Next, we intersected Foxa1/2 and AR targets with the 6,612 genes showing gender-specific expression in control mice without carcinogen treatment (see Figure 1D). Among the intersect group, 75% or 72% (747 or 727 genes) of AR-associated genes were also bound be Foxa1 or Foxa2, respectively (Figure 2B), indicating potential co-regulation of Foxa1/2 and AR in controlling gender dimorphic gene expression in the male liver. We also analyzed genome-wide binding sites of Foxa1, Foxa2, and AR in livers of male mice exposed to DEN by ChIP-Seq (Table S1). Among the genes (2,019) differentially expressed in the livers of male and female mice following carcinogen treatment, Foxa1/2 and AR target co-occupancy was even higher than among the control group, with 86% and 86% of the AR-bound genes also being targets of Foxa1 and Foxa2, respectively (Figure 2B).
Next, we analyzed genome-wide co-localization of Foxa1/2 and AR binding sites from ChIP-Seq by calculating the distribution of AR binding sites relative to the center of the Foxa1 or Foxa2 binding sites. Foxa1/2 and AR binding sites were very close to each other at the regulatory regions of their common target genes (Figure 3D and 3E). Remarkably, Foxa1/2 and AR co-occupancy dramatically increased following carcinogen treatment in male livers (Figure 3D and 3E), further supporting the notion that enhanced co-regulation of Foxa1/2 and AR plays a critical role in promoting HCC in male mice. Strikingly, when we analyzed target binding by AR in Foxa1/2 mutant livers, we found AR binding to the common Foxa/AR targets almost completely abolished (Table S1, and Figure 2D and 2E), demonstrating that the recruitment of AR to its binding sites depends on Foxa1/2.
Both FOXA1 and FOXA2 have been found to directly interact with the AR in human prostate cancer cells (Gao et al., 2003; Yu et al., 2005). By immunoprecipitation assays, we found that both Foxa1 and Foxa2 were bound to AR in the male mouse liver, regardless of exposure to DEN (Figure 3F). As expected, the interaction between Foxa1/2 and AR was completely abolished in Foxa1/a2 mutant livers, confirming the specificity of our immunoprecipitation assay (Figure 3F).
In addition, the common Foxa1/AR targets were largely overlapping with those of Foxa2/AR in the male liver and were mostly found at putative enhancer regions (Figure 4D and Table S2). Just as was the case for the Foxa/ERa common targets, we found that the difference in gene expression of Foxa/AR dual targets between DEN-treated and -untreated control mice was reversed for most (~80%) genes in the livers of male Foxa1/a2 mutant male mice (Figure 4E). Functional annotation of Foxa/AR dual target genes revealed that Foxa1/2 and AR promote tumor growth through multiple pathways, including DNA replication, cell cycling, and cell growth and proliferation (Figure 4F). In conclusion, these findings demonstrate that Foxa1/2 and AR cooperate to regulate gene expression in the male liver, and that Foxa1/2 is essential for androgen signaling in promoting hepatocarcinogenesis in male mice.
Hepatocyte injury is considered the first step in hepatocarcinogenesis induced by DEN. Administration of estrogen to male mice treated with DEN prevents liver injury and carcinogenesis (Kalra et al., 2008), which we confirmed in our own study (Figure 5A). In addition, administration of the ERα-specific antagonist, fulverstrant (also called Faslodex), to female control mice led to loss of protection from DEN-mediated liver injury, confirming the importance of estrogen signaling in preventing HCC (Figure 5A). However, we found that estrogen enhanced liver injury in DEN-treated Foxa1/2-deficient male mice (Figure 5A).
The data on tumor load in Foxa1/2-mutant mice present a new puzzle (Figure 1A,B). If estrogen had only hepatoprotective effects, one would expect female Foxa1/2-mutant mice to develop exactly the same tumor load as male controls after exposure to DEN. However, as shown above, the tumor load in female mutants far exceeded that of male mice (Figure 1A,B). This suggests that in the absence of Foxa1/2, estrogens exert an additional tumor-promoting effect on hepatocytes. Estrogen itself has been found to act as a carcinogen due to the genotoxicity of its metabolites, such as 16 alpha-hydroxy estrone and 2- and 4-hydroxycatechol estrogens (Yager and Liehr, 1996). When we blocked estrogen signaling with fulverstrant, DEN-induced liver injury was significantly attenuated in female Foxa1/2 mutant mice (Figure 5A). However, this tumor-promoting effect of estrogen was only observed when Foxa1/2 was absent, indicating that the protective effect of estrogens is normally dominant over its tumor-promoting action. Estrogens have been employed clinically to treat many cancers with mixed results: in some cases, estrogens were found to prevent cancer progression; whereas in other cases, estrogens were found to promote tumor growth (Di Maio et al., 2006; Kalra et al., 2008; Lawrence et al., 2007). Our findings regarding the role of Foxa1/2 in estrogen carcinogenesis provide a possible explanation for these findings, in that estrogens prevent cancer progression when Foxa1/2 are present, but promote tumor growth in the absence of the Foxa factors.
In addition to the Foxa/ERα dual targets discussed above, there are also many genes solely bound by ERα in the female liver (Table S1). These are candidate genes to explain the cancer-promoting effects of estrogens in female mice lacking Foxa1/a2. Interestingly, most of these ERα-unique target genes showed opposite changes between female and male mutant mice after carcinogen treatment (Figure 5B). Pathway analysis revealed that most of them are involved in cell cycle, cancer development, and nucleotide/amino acid metabolism (Figure 5C). Thus, estrogen and its metabolites enhance hepatocarcinogenesis through both their genotoxicity and estrogen signaling when Foxa1 and Foxa2 are absent.
As shown above, we discovered gender-specific recruitment of ERα and AR to Foxa1/a2 binding sites in female and male liver, respectively. In addition, we did not observe significant differences of either Foxa1 or Foxa2 binding between males and females, or of their core consensus sequences (Figure S2C and S2D). Next, we compared the genomic locations of the 609 Foxa/ERα dual target genes that are likely mediators of carcinogenesis resistance in females with the 726 Foxa/AR dual target genes that are likely promoting carcinogenesis in male mice. Strikingly, we found that the vast majority of the targets (76% of Foxa/ERα and 64% of Foxa/AR) were overlapping between females and males (Figure 6A). For example, leukemia inhibitory factor receptor (Lifr) was bound by Foxa/ERα or Foxa/AR at the same location in female or male liver, respectively (Figure 3B and 3E). De novo motif analysis showed the presence of a strong consensus Foxa motif at these sites, as expected (Figure 6B). Next, we searched for estrogen response elements (ERE) and androgen response elements (ARE) in proximity (± 250 bp) to these Foxa binding sites. We found that the distribution of ERE/ARE pairs was random at these Foxa binding sites, with half of the ERE/ARE pairs clustered on either side of the Foxa core element, and the other half separated by the Foxa motif (Figure 6B). These data suggest that adjacent localization of Foxa binding sites with both steroid hormone receptor binding elements (ERE and ARE) enables gender-specific regulation of the same set of genes by each hormone in the liver.
Mutations in gene coding regions are the predominating cause of cancer. Where analyzed to date, hepatic FOXA1/2 levels did not differ significantly in HCC patients compared to controls (Chen et al., 2002; Lee et al., 2004; Lee et al., 2006), in concordance with our results regarding Foxa1 and Foxa2 expression in mice treated with DEN (Figure S1C) and previous reports in mice during hepatocarcinogenesis (Kalra et al., 2008). However, our findings suggest the possibility that mutations in cis-regulatory elements binding FOXA1 and/or FOXA2 could predispose humans to HCC. To investigate this possibility, we analyzed genome-wide FOXA2 binding in healthy human livers by ChIP-Seq and found 7,917 FOXA2 binding sites. Next, we retrieved single nucleotide polymorphisms (SNP) from the human GWAS database (NCBI, build 130). By intersecting these two datasets, we found 12,739 SNPs, associated with 3,357 genes, located within 5,303 FOXA2 binding regions (Figure 7A). We intersected this human gene list with the 609 dual Foxa/ERα targets (potential liver cancer-resistance genes) identified above in the mouse liver (Figure 4A). We obtained 228 potential `HCC-resistance genes' associated with 466 FOXA2 binding sites that contained 1,080 SNPs in the human orthologous loci (Figure 7A). Using the MATCH program to search the binding elements of transcription factors based on the positional weight matrices from the Transfac database (Kel et al., 2003), we further identified FOXA2 consensus binding elements in those 466 FOXA2 ChIP-Seq regions and found that 119 of them contained SNPs within the FOXA2 core binding element (TRTTT, R=G or A) (Figure 7A).
In order to investigate the impact of FOXA2 SNPs on estrogen signaling, we determined FOXA2 and ERα binding at regions of ± 200 bp surrounding these FOXA2 core binding elements by ChIP-qPCR in samples from four healthy and four HCC livers from women. Strikingly, among the 113 FOXA2 binding sites tested, 18 sites showed significant reduction in binding to both FOXA2 and ERα in some or all HCC livers compared to controls (Figure S6). To identify SNPs at these FOXA2 binding sites, we sequenced these 18 loci in the HCC samples and found multiple point mutations, deletions, and insertions that were either heterozygous or homozygous (Figures S6 and Figure 7B). In most cases, SNPs at FOXA2 binding sites were associated with impaired FOXA2 and ERα binding (Figures S6). In particular, the BTG1 (B-cell translocation gene 1), FGL1 (fibrinogen-like 1), and ABCC4 (ATP-binding cassette, sub-family C (CFTR/MRP), member 4) loci had mutations at one or two alleles causing loss of binding of both FOXA2 and ERα in all four HCC patients, while three out of four PPM1L (protein phosphatase, Mg2+/Mn2+ dependent, 1L) loci contained mutations at one or both alleles (Figure S6B) and all loci with mutations also showed impaired binding of both FOXA2 and ERα (Figure S6B). BTG1 is an anti-proliferative factor (Rouault et al., 1992), FGL1 is an acute phase reactant for inflammatory response (Liu and Ukomadu, 2008), ABCC4 mediates the membrane transport of various molecules in multi-drug resistance (Russel et al., 2008; Vlaming et al., 2006), and PPM1L is a tumor suppressor (Thean et al., 2010). To confirm this observation, we analyzed additional seven normal and seven HCC livers from women. When analyzing all 22 samples, we found that both FOXA2 and ERα binding were highly correlated with the sequence of the FOXA2 binding elements (Figure S6C), and that FOXA2/ERα dual occupancy was significantly different between normal and HCC livers (Figure 7C). Impaired binding of both FOXA2 and ERα occurred predominantly in female HCC livers (Figure 7C), which we postulated would lead to altered gene expression of BTG1, FGL1, ABCC4 and PPM1L, which in turn might contribute to the development of HCC. Indeed, when we performed immunostaining of these four proteins for liver sections from female HCC patients and normal controls, we found that PPM1L expression was reduced and FGL1, BTG1 and ABCC4 expression was increased in HCC livers compared to normal controls (Figure 7D). These data suggest that impaired regulation by FOXA2 and ERα contributed to altered expression of these dual target genes in HCC and supports the notion that SNPs at FOXA2 binding sites could contribute to HCC risk in women.
Hepatocellular carcinoma is an often lethal malignancy with limited treatment options. Here, we have uncovered a central role for the winged helix transcription factors Foxa1 and Foxa2 in controlling estrogen and androgen signaling through recruitment of ERα and AR to their relevant targets in the liver, thereby explaining the sexual dimorphism of liver cancer in mammals. Not only are the Foxa factors required for the sex hormone receptors to bind to many of their targets, importantly, tumor growth is also strongly dependent on Foxa1/2. Thus, tumor load is dramatically increased in the livers of female Foxa1/2 mutant mice, and decreased in the livers of male Foxa1/2 mutants exposed to hepatocarcinogens. Our current view of how the Foxa1/2 factors control multiple aspects of hepatocarcinogenesis is summarized in the schema provided in Supplementary Figure S7.
The Foxa factors play a dominant role in determining the gender specificity of HCC development. Interestingly, without carcinogen treatment, Foxa1/2 mutant mice maintain much of the sexual dimorphic gene expression profile that is present in control livers (data not shown). This suggests that the loss of gender specificity in Foxa1/2-deficient mice occurs with the onset of carcinogen exposure. Given the fact that multiple physiological and pathophysiological processes are sexually dimorphic in the organs expressing Foxa proteins, we speculate that other stresses, like dietary deprivation and aging, might also invoke gender-specific responses from Foxa factors, which we aim to address in the future.
A previous study had attributed the disparity in liver cancer between the sexes to differences in IL-6 production by Kupffer cells in response to chemical carcinogens (Naugler et al., 2007). In fact, DEN-induced tumor incidence was reduced in both male and female IL-6 null mice. However, while we confirmed higher IL-6 plasma levels in wild type male mice treated with DEN compared to female controls, IL-6 levels did not correlate with tumor load in Foxa1/2-deficient mice (Figure 1A and Figure S5). In addition, because Foxa1/2 were not ablated in Kupffer cells but in hepatocytes in our model, the effect of the sex hormones on tumor susceptibility shown here is exclusively due to direct action of the Foxa factors on the parenchymal cells in the liver.
Estrogen promotes and estrogen antagonists (such as tamoxifen) prevent the growth of breast cancer cells, at least in estrogen receptor-positive tumors (Hollingsworth et al., 1998; Zumoff, 1998). Androgen promotes and androgen deprivation prevents the growth of prostate cancer cells (Paulson, 1984; Smolev et al., 1977). As discussed above, in both cancers, binding of the sex hormone receptor occurs near FOXA1 binding sites. However, in liver cancer, a different scenario has been recognized for years, in that estrogen prevents cancer development, opposite to the situation in the mammary gland. As we have shown here, both the effects of androgens and estrogens are Foxa1/2-dependent in the liver. Thus, a puzzle emerges: how can it be that estrogen signaling, dependent on Foxa factors in both the mammary gland and the liver, is tumor-promoting in the former, and tumor-preventing in the latter? This paradox suggests the existence of tissue-specific targets or tissue-specific co-regulation of the Foxa/ERα axis in the two tissues, an issue that will be of great interest for future investigation.
Thus far, no mutations in the FOXA1 or FOXA2 gene have been linked to human HCC. This might not be too surprising given the redundant function of the two proteins, which suggests that mutations in one or the other might not be sufficient to affect tumor initiation or progression. However, strikingly, we found multiple examples of single nucleotide polymorphisms in FOXA binding sites that affect FOXA and ERα occupancy, which were significantly more frequent in HCC samples than in normal livers from women. Not only does this finding provide supporting evidence that the co-regulation of target genes by FOXA and ERα extends from mice to humans, but it also suggests that SNPs in FOXA binding sites could contribute to the risk of hepatocarcinogenesis in women. Future large-scale studies investigating HCC risk in women with respect to these SNPs appear warranted.
We previously found that glucocorticoid receptor (GR)-mediated transcriptional regulation also depends on Foxa1/2 (Li et al., 2009; Zhang et al., 2005). Full activation of certain genes in the liver in response to a prolonged fast, and engagement of their cis-regulatory elements by GR requires Foxa2 (Zhang et al., 2005). Likewise, repression of the IL-6 promoter by GR is dependent on the Foxa factors in hepatocytes (Li et al., 2009). Why are Foxa factors required for steroid hormone signaling? Our current study suggests that Foxa factors serve as a scaffold for steroid hormone receptors to regulate gene transcription in the liver on a genome-wide scale, and extends to at least three nuclear hormone receptors. Our finding that two sets of sex hormone cis-regulatory elements, AREs and EREs, are found close to the same Foxa binding site indicates that Foxa-dependent estrogen and androgen signaling regulates the same set of genes in the liver (Figure S7). More importantly, our studies showed that ERα and AR had opposite effects on this gene set between females and males (Figure 4 and Figure S7), which provides a molecular explanation for previous findings that estrogen and androgen were able to reverse HCC incidence in the opposite genders (Naugler et al., 2007; Shimizu et al., 1998; Tsutsui et al., 1992; Yamamoto et al., 1991). In conclusion, we have identified a set of regulatory units in which juxtaposition of Foxa binding sites next to both EREs and AREs allows for the mediation of the gender-specific effects of the sex hormones in the liver.
The derivation of Foxa1loxP/loxP;Foxa2loxP/loxP (control) and Foxa1loxP/loxP;Foxa2loxP/loxP;AlfpCre (mutant) mice has been reported previously (Gao et al., 2008; Li et al., 2009; Sund et al., 2000). A two-step strategy was employed for hepatocarcinogenesis for 5–6 mice in each group. 32 microarray analyses were performed in livers from control and mutant mice of both genders with or without carcinogen treatment (four mice in each group for eight groups) for gene expression profiling. 17 ChIP-Seq experiments were performed and analyzed, for Foxa1, Foxa2, ERα, AR in control and Foxa1/a2 mutant livers of both genders with or without carcinogen treatment, and FOXA2 in normal human liver. Detailed experimental procedures are provided in supplemental information.
We thank Drs. Jeff Albrecht, Linda Greenbaum, and Snorri Thorgeirsson for valuable comments on the manuscript. We gratefully acknowledge Alan Fox, Olga Smirnova, Karrie Brondell, Amber Riblett, James LaRossa and Reina Aoki for their excellent technical support. We also thank Dr. Erik Knudsen for sharing his hepatocarcinogenesis protocol. This study was supported by the NIDDK (P01-DK049210 to KHK). Z. Li was supported by NSREC and JDRF postdoctoral fellowship awards. We thank the University of Pennsylvania Diabetes and Endocrinology Center (DERC) for the use of the Functional Genomics Core (P30-DK19525).
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Supplemental Information Supplemental Information includes seven supplemental figures, two supplemental tables and extended experimental procedures.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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By Maria Puente, USA TODAY
There will be no fairy-tale coach to the church door for Kate Middleton when she marries Prince William of Wales on April 29. Palace officials announced Wednesday that she would travel to her nuptials by car.
It's a break with tradition: Recent royal brides have arrived at their weddings in the 130-year-old Glass Coach. Still, the couple will leave Westminster Abbey as newlyweds in a royal carriage, although it's not clear yet which one of the scores of royal coaches will be brought out for the occasion.
"It's symbolic," says royals biographer Kitty Kelley, pointing to the attention paid to Middleton's non-royal, non-aristocratic, middle-class origins. "If she got into a royal coach going to the abbey, everyone would go crazy, but if she's married to a future king, when she gets into it, then it will be all right."
According to a press release and tweets from Clarence House (the office of William's father, Prince Charles), Middleton will travel to the abbey by car along a route that will include The Mall, Horse Guards Parade and government offices in Whitehall and Parliament Square.
It's the traditional route; in fact, it was the route William and his family walked to the abbey behind the funeral cortege of his mother, Diana, Princess of Wales, in 1997. Huge crowds lined the route then, and similar numbers can be expected for the wedding of Diana's eldest son, 28, and his fiancée, who is soon to turn 29.
The wedding will begin at 11 a.m. The Archbishop of Canterbury, the spiritual leader of the Church of England and the man who crowns British monarchs, will marry them. The Dean of Westminster will conduct the service, and the Bishop of London will deliver the sermon.
Afterward, the couple will return by carriage along the same route to Buckingham Palace at the end of The Mall, where the groom's grandmother, Queen Elizabeth II, will host a reception for guests drawn from the couple's official and private lives. Prince Charles will then host a private dinner at the palace for close friends and family, followed by a gala with dancing.
These, too, break with tradition: At previous royal weddings, the queen would have hosted a breakfast for the family on the day of the wedding and a ball a few nights before, Joe Little, managing editor of Majesty magazine, told the Associated Press. But a reception would allow more people to attend, and it follows a new custom set at Prince Charles' second wedding, in 2005, when the queen hosted a reception for 800 at Windsor Castle.
Clarence House did not explain why Middleton would not take a coach to the wedding, as did Lady Diana Spencer when she married Prince Charles in 1981 and Sarah Ferguson when she married Charles' brother Andrew, the Duke of York, in 1986. The queen herself rode in the Glass Coach when she married as a princess in 1947. Commentators suggested it might be part of the couple's effort to avoid the appearance of excess in a time of painful austerity in Britain.
There may also be security concerns: Though the Glass Coach isn't actually made of glass (it has large glass windows), it's not bulletproof, either. Last month, a royal car carrying Prince Charles and his wife, Camilla, was attacked by rioters protesting increases in university tuition, resulting in dramatic front-page photos around the world of the frightened couple and recriminations about the lack of security.
"It's both," Kelley says. "There have been so many security incidents in the U.K., they really can't afford to take a chance. And given the economy, they can't afford to put on glitzy show. They'll put on something befitting and dignified and royal, but it won't be over-the-top with 500 bridesmaids and 55 footmen and trumpets and glass coaches."
Then again, the decision to forgo a coach may be a dress issue. Diana's silk taffeta dress had such a long train (25 feet) that it filled the Glass Coach and appeared deeply wrinkled when she alighted pulling it behind her.
Details about the wedding have trickled out since the couple announced their engagement in November, after nearly nine years of courting. It's still unknown who will design Middleton's dress, how many and who will be invited (the abbey can hold up to 2,000), who will perform the music at the service, and whether a proposed concert in nearby Hyde Park will be organized to celebrate the wedding on the big day, which has already been declared a national holiday in Britain.
We've updated the Conversation Guidelines. Changes include a brief review of the moderation process and an explanation on how to use the "Report Abuse" button. Read more.

subset: commoncrawl


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Prices, promotions, styles, and availability may vary. Our local stores do not honor online pricing. Prices and availability of products and services are subject to change without notice. Errors will be corrected where discovered, and Lowe's reserves the right to revoke any stated offer and to correct any errors, inaccuracies or omissions including after an order has been submitted.
3M Heavy Duty Stripping Pad
Item # 44639 Model # 10112NA
Product Information
- For flat surfaces
- Use with chemical paint strippers
- Removes paint, varnishes and other finishes without harming wood
- Replacement pads for 3M™ Heavy Duty Stripping Tool
|Length (Inches)


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1911 Triangle fire remembered as spur to unions, safety laws
NEW YORK It was a warm spring Saturday when dozens of immigrant women and girls leapt to their deaths, some with their clothes on fire, some holding hands, as horrified onlookers watched the Triangle Shirtwaist factory in Manhattan burn.
The March 25, 1911, fire that killed 146 workers became a touchstone for the organized labor movement, spurred fire safety laws and shed light on the lives of young immigrant workers.
The 100th anniversary comes as public workers are fighting efforts to limit their collective bargaining rights. Labor leaders and others say we need only look to the Triangle fire to see why unions are necessary.
This is a story that needs to be told and retold, said Cecilia Rubino, writer-director of From the Fire, an oratorio inspired by the Triangle fire. We dont have that many moments in our history where you see so clearly the gears of history shift.
To mark the centennial, hundreds of theatrical performances, museum exhibits, lectures, poetry readings, rallies and panel discussions are taking place nationwide. Two documentaries have aired on TV; PBS Triangle Fire premiered Feb. 28 and HBOs Triangle: Remembering the Fire on Monday.
Descendants of victims and survivors of the fire will gather Friday for a procession to the site in Manhattans Greenwich Village. The building now houses New York University classrooms and labs.
Suzanne Pred Bass, a Manhattan psychotherapist and theater producer, is the great-niece of Katie Weiner, who survived the Triangle fire, and Rose Weiner, who did not.
Bass ticked off the reasons why people remain fascinated by the Triangle fire after 100 years.
Its the youth of these women, she said. Its the tragedy, its the changes it spawned and its the immigrant experience.
The fire started at end of the workday and raced from the eighth floor to the ninth and 10th. As hundreds of workers mainly Jewish and Italian immigrant women and girls, the youngest 14 tried to escape, they found a crucial door apparently locked.
They were panic-stricken, said Eileen Nevitt, whose grandmother, Annie Sprinsock, survived. It was hellacious, and they ran for their lives the best they could.
Firefighters rushed to the scene but their ladders reached only to the sixth floor. The fire was under control in 18 minutes too late.
At the trial later that year of Triangle owners Max Blanck and Isaac Harris on manslaughter charges, survivors testified that their escape had been blocked by a locked door on the ninth floor. Some said the door was kept locked to prevent theft.
Katie Weiner said she felt for the door, which she could not see in the smoke, and turned the knob.
I pushed it toward myself and I couldnt open it and then I pushed it inward and it wouldnt go and I then cried out, The door is locked! she testified.
Meanwhile, the elevator shuttled up and down carrying as many workers as could cram into it. Weiner joined the crush for the last elevator but was pushed back. She testified that she grabbed the elevator cable and threw herself in, landing on girls heads. She was the last person out of the burning building.
The jury heard from 155 witnesses before returning a verdict of not guilty.
I believed that the door was locked at the time of the fire, one juror said. But we couldnt find them guilty unless we believed they knew the door was locked.
Workers advocates continued to blame Blanck and Harris, who had resisted a union drive in 1909.
Blancks granddaughter, Susan Harris, said she is saddened when people demonize her grandfather, who died before she was born
Its really important for them, I think, to have a villain, she said.
Blanck and Harris were on the 10th floor when the fire started and were able to escape to the roof. But several of Susan Harris relatives died in the fire, including Jacob, Essie and Morris Bernstein, members of Blancks wifes family who worked at Triangle.
Harris lives in Los Angeles but is spending March in New York to take part in Triangle commemorations. An artwork she created to honor the fire victims made of antique shirtwaists and handkerchiefs will be displayed at the New York City Fire Museum for a month.
One witness to the Triangle workers death plunges was Frances Perkins, who later became the first female Cabinet member when President Franklin Roosevelt appointed her secretary of labor. Perkins was having tea nearby and heard the commotion. She ran to the scene as the first body hit the ground.
That fire is the event that changed her life and that really changed the course of American history, said Kirsten Downey, author of a book about Perkins, The Woman Behind the New Deal.
Perkins was appointed to the Factory Investigating Commission, convened in response to the Triangle fire, and the panel held hearings all over New York state before drafting 20 laws aimed at improving workplace safety. Some of the new laws required fire drills, set occupancy limits in buildings and required exit signs to be clearly posted.
Policies that were enacted because of that fire permeate American workplaces now, Downey said.
Days after the Triangle fire, 100,000 mourners marched in a funeral procession through the streets of New York, while another 250,000 lined the route. Their grief built support for the right of garment workers to unionize.
It created a strong garment workers union, said Bruce Raynor, president of Workers United, the 21st-century heir to the International Ladies Garment Workers Union. It helped to really start the modern labor movement.
He said the Triangle fire commemoration resonates strongly today, given the labor struggles across the country and in Wisconsin, where a law passed this month limits public workers collective bargaining rights.
One hundred years later, 150,000 people are protesting in Madison, Wis., over the same issue, he said: the right of working people to organize.
Copyright 2011 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.
Posted | Updated


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Last year, one out of 13 people lost money due to internet fraud and identity theft, says a recent report. However, you can insure your password and data by following these easy-to-follow tips.
Always keep the cyber thieves guessing. Never use personal information to create a username, login or password. It could be the name of your pets, relatives, nicknames, dates of birth, etc.Identity theft experts have become savvy at ferreting out these details. Hence, it is crucial to choose usernames and passwords that have nothing to do with your personal history.
Avoid using the same login and password across multiple sites and/or cards and accounts. If a thief gains access to one, it will be like a house of cards, allowing them to quickly wreak havoc across your entire financial portfolio.
Studies have consistently shown that a large fraction of all user-chosen passwords are readily guessed automatically. Shorter passwords are more susceptible to commercially available password recovery tools. Such software is capable of testing 200,000 passwords per second. To improve the cipher strength of your password, longer passwords are better. Include a minimum of 8 characters - using both upper and lower case letters and a mix of letters, numerals and symbols. Do not use words found in the English dictionary. Put yourself into a thief's shoes - don't even think about using an overly simplified password such as '12345678,' '222222,' 'abcdefg.'
Avoid sequential passwords or using passwords derived from the use of adjacent letters on your keyboard; this will not make your password secure. Also, avoid using only look-alike substitutions of numbers or symbols. Criminals and other malicious users who know enough to try and crack your password will not be fooled by common look-alike replacements, such as replacing an 'i' with a '1' or an 'a' with [email protected][email protected][email protected]
Traditional Naga system of learning encompassed the understanding of cultural practices, history as passed down orally, agricultural science as demonstrated through practices in th


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Praised for her "bountiful gifts and passionate immersion into the music she touches" (The Plain Dealer), Chinese pianist Fei-Fei Dong is a winner of the Concert Artists Guild Competition and a top finalist at the 14th Van Cliburn International Piano Competition. She continues to build a reputation for her poetic interpretations, charming audiences with her "passion, piquancy and tenderness" and "winning stage presence" (Dallas Morning News).
Her burgeoning career includes a number of prominent concerto engagements in the 2016-17 season, including performances with the Anchorage Symphony, Spokane Symphony, DuPage Symphony (IL) and a special concert with the Pacific Symphony, featuring Fei-Fei playing both Beethoven Concerto No. 1 and No. 4 with conductor Carl St, Clair at Soka Performing Arts Center in Aliso Viejo, CA. Featured recitals this season include the SUNY Purchase Performing Arts Center and the Howland Chamber Music Circle Piano Festival (both NY), Macon Concert Association (GA), and Atlantic Music Center (FL). This follows a busy summer 2016 schedule internationally, including recitals at Spain's Auditorio Nacional de Madrid, and in Shenzhen, China and Nantucket, MA, as well as a concerto with Germany's Norddeutsche Philharmonie Rostock and chamber music at the Music at Menlo Festival.
Fei-Fei's 2015 summer festival highlights include Bravo! Vail Valley, the Highlands Chamber Music and Lake George Music Festivals, and a recital for Chicago's Dame Myra Hess Concert Series. She was showcased prominently in the documentary film, Virtuosity, about the 2013 Cliburn Competition, which premiered on PBS in August 2015, and she has also been featured numerous times on New York's WQXR radio.
Career concerto highlights in the US include the Fort Worth Symphony, Kansas City Symphony, Aspen Music Festival Orchestra, Hong Kong Philharmonic Orchestra, Corpus Christi Symphony, Austin Symphony, Youngstown Symphony, Fort Collins Symphony, Denver Philharmonic, and the Juilliard Orchestra, and in China with the Shanxi and Shenzhen Symphony Orchestras. She has worked with such prominent conductors as Leonard Slatkin, Michael Stern, Jeffrey Kahane, Randall Craig Fleisher, and John Giordano.
Fei-Fei has performed in recital at Alice Tully Hall as the winner of Juilliard's 33rd Annual William Petschek Recital Award. Other notable recent recitals in the US include her Weill Recital Hall debut at Carnegie Hall, Gilmore Rising Stars (Kalamazoo, MI) and The Cliburn's spring 2015 Chopin Festival, and in Europe at Warsaw Philharmonic Concert Hall and the Louvre.
She is a member of the Aletheia Piano Trio, which debuted at the Kennedy Center in February 2014 as part of its Conservatory Project, and performs across the US. Deeply committed to sharing her joy for music and connecting with communities, Fei-Fei also engages students and community audiences through frequent school and outreach concerts and master classes.
Born in Shenzhen, China, Fei-Fei began piano lessons at the age of 5. She moved to New York to study at The Juilliard School, where she earned her Bachelor and Master of Music degrees under the guidance of Yoheved Kaplinsky.
Debussy: Danse
Chopin: Rondo in E-Flat Major, Op. 16
Clementi: Sonata in F-Sharp Minor, Op. 25 (Excerpt)
Lowell Liebernmann: Gargoyles, Op. 29
Schumann: Novelletten, Op. 21 - No. 8 in F-Sharp minor
Scarlatti: Sonata in F minor, K. 466
Mozart: Piano Sonata No. 18 in D Major, K. 576, Allegro
Liszt: Piano Sonata in B minor, S. 178 (Excerpt)
Volodos: Concert Paraphrase on Mozart's Turkish March
- Quotes
"She has tremendous technical facility, a laudable ability to bring out a work's narrative arc, and a great deal of coloristic resource… you got the distinct impression that this is an artist who is honest, responds with spontaneity to the music as it's happening, and who is unafraid to take risks to get her musical thoughts across."
–Palm Beach Arts Paper
"In her mid-20s, she is one of the most engaging and promising rising stars in the musical firmament."
–Times Herald-Record
"In this performance, Dong extracted every last ounce of energy where needed and gentle nuance when required. The audience held its breath in rapt attention to this amazing musical experience."
–Palm Beach Daily News
"The evening's highlight, an excellent performance of the popular Edvard Grieg Piano Concerto in A Minor, Op. 16, featured the young and remarkably talented Fei-Fei Dong as soloist."
"…performed by Dong with steely precision and poised expression. She showed great rhythmic control and played her solo passages masterfully…"
–Cleveland Classical
"Fei-Fei Dong is already marketable as an accomplished pianist, well trained, with generally good musical instincts and a winning stage presence."
–Dallas Morning News
"She displayed a natural musicality and beauty of tone; I enjoyed her fluid technique and her presence at the piano."
–Cincinnati Enquirer
"The 22-year-old Chinese pianist played with exceptional control of dynamics and articulation, and she gauged the second movement's conflict with the strings to striking effect. Given Dong's bountiful gifts and passionate immersion into the music she touches, it should be intriguing to hear how she approaches the Beethoven fourth – and other works – in a decade or so."
–The Plain Dealer
"Fei-Fei Dong teamed up with the Brentano in a Brahms Piano Quintet that made most of the right gestures, if not always in the right degrees. There was passion, piquancy and tenderness, and every one of the five musicians lent eloquent nuances."
–Dallas Morning News
"Dong's unforgettable Beethoven Fourth stretched the dimensions of the music's time and space with risk-taking rapture… It was illuminating, poetic Beethoven…"
-Huffington Post
- Reviews
- Orchestral Repertoire
Beethoven: Concerto No.1, Op.15
Beethoven: Concerto No.2, Op.19
Beethoven: Concerto No.3, Op.37
Beethoven: Concerto No.4, Op.58
Brahms: Concerto No.1, Op.15
Mozart: Concerto No.20 in D minor, K. 466
Mozart: Concerto no.23 in A Major, K. 488
Mozart: Concerto No.21 in C Major, K. 467
Chopin: Concerto No.1 in E minor, Op.11
Chopin: Concerto No.2 in F minor, Op.21
Gershwin: Rhapsody In Blue
Grieg: Concerto in A minor, Op.16
Liszt: Concerto No.1 in E-flat Major, S. 124
Mendelssohn: Concerto No.1 in G minor, Op.25
Schumann: Concerto in A minor, Op.54
Tchaikovsky: Concerto in B-flat minor, Op.23
Shostakovich: Concerto No.1 in C minor, Op.35
Rachmaninoff: Concerto No.2 in C minor, Op.18
Rachmaninoff: Concerto No.3 in D minor, Op.30
Rachmaninoff: Rhapsody on a Theme by Paganini, Op.43
Yellow River Concerto


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Sept. 25, 2015, Fairfax, Va.—Americans for Limited Government President Rick Manning today issued the following statement in response to the pending retirement of House Speaker Rep. John Boehner (R-Ohio):
"Speaker Boehner has led the House through some tumultuous times. While we have not always agreed with his tactics, we all shared a common desire to restore fiscal sanity in Washington, D.C. and his legacy will be the three-year leveling of outlays that have slowed the growth of the national debt.
"The transition provides a unique opportunity to reestablish the separation of powers between the branches of government. Rep. Ken Buck has been at the forefront of the push to the defend the House's Article I power of the purse, a vision shared by all House Republicans led by Majority Leader Kevin McCarthy.
"The budget discussions ahead provide the perfect opportunity for House Republicans to set the spending priorities for 12 of the remaining 15 months of the Obama Administration. The shift in power provides a golden opportunity for Congress to put its imprimatur on policies that stop Obama's executive branch regulatory overreach."
To view online: http://getliberty.org/boehner-retirement-presents-golden-budget-opportunity/
Americans for Limited Government is a non-partisan, nationwide network committed to advancing free market reforms, private property rights and core American liberties. For more information on ALG please visit our website at www.GetLiberty.org.


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Nite Bedside Table
Written by Katie on May 28, 2008. Permalink
This rather clever bedside table is wall mounted so it'll take up less room. This would also be perfect for the entryway or for a central phone/gadget resting place.
Made in Sweden.
Material: white Plexi.
Height 380, depth 250, width 330 mm.
Purchase Information:Price: $279.00
Availability: Buy Nite Bedside Table here


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Data Science Engineer (Search) SoundCloud is the world's leading audio platform, allowing everyone to share and discover unique content anywhere, anytime, on the web and on mobile. We reach hundreds of millions of listeners globally every month, many of which are listening on mobile. We are now looking for an experienced data scientist to join our search team. The search team builds and maintains systems that empower our users to actively and easily discover SoundCloud's huge and unique catalog of music and audio. You will lead efforts to improve the search experience with respect to ranking algorithms, user assistance and user interface in order to enable listeners to find the most relevant and engaging content and discover hidden gems. You are experienced in information retrieval or a related field (such…
Link to Full Article: Search


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It's not a regular occurrence that you discover you need a replacement Window Motor, Power to fix your vehicle. You bought your Cavalier because its dependability and long-lasting quality appealed to your senses; so why would you be okay with anything but the best when it comes to the highest quality auto parts? Your Chevrolet dealership isn't the only place to locate trusted parts to repair your Cavalier. While it's not hard to find replacement parts for Chevrolet vehicles, these days the necessity for nothing but the absolute best are required to maintain your car or truck.
In order to maintain a basic level of convenience, your windows have to roll up and down. If you have power windows, it would be your power window motor that does the job of moving the glass up and down. Your Chevrolet Cavalier power window motor either turns a gear on the window regulator that moves its arms or it spins a pulley on the regulator that pulls a cable one way or another. It's hard to imagine your vehicle manufactured without a power window motor, but easy to see how much you've come to rely on it when you can no longer use it. It's not fun to be forced to drive your Chevrolet Cavalier with the windows down in the snow because your power window motor doesn't work.
Bring your Cavalier back to life with the highest quality repair parts from the experts at Car Parts Discount. Don't let a Window Motor, Power repair bring about the demise of your car, truck, or SUV, and don't gamble with its condition by using anything but the best repair parts. At Car Parts Discount, we have real customer support agents available with enough expertise to help you purchase the appropriate Chevrolet Cavalier part for your needs. All the Chevrolet Cavalier Window Motor, Power parts on this page will fit vehicles made in 2005, 2004, 2003, 2002, 2001, 2000, 1999, 1998, 1997, 1996, 1995, 1994, 1993, 1992, 1991, 1990, 1989, 1988, 1987, 1986, 1985, 1984, 1983, 1982. Choose your year below to refine your search.


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Tips & highlights for the younger animal lovers
Zoo Berlin offers the younger animal lovers plenty of highlights to explore the exciting animal kingdom and even to experience it up close. You'll find tips of what simply can't be missed with your children here.
Petting zoo
Loving strokes are welcome here! Pygmy goats, sheep, donkeys and ponies love the caring hands of children. Our goats especially like the special food from the automatic feeders.
Adventure playground
Pirates beware: Climbing opportunities, slides and a maze await young sailors, adventurers and buccaneers! Junior pirates with a head for heights can breathe in true seaman air at a height of six metres on the figurehead. And should the weather be bad, then the cabins are the place to romp around.
Watch the hippos underwater
See how the eyes of even the smallest of children light up: Visitors can watch our hippos on an underwater stroll through the large panoramic window – and be only a few centimetres from the pachyderms.
On night safari: Among the animals of the night
We've turned day into night in the nocturnal house. In doing so, our visitors are able to see what's normally hidden away: Snooping aardvarks, hopping bush babies and the sweet slow lorises with their round button eyes.


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|Reviews for Second Guessing
| Aravis'sSilverQuill chapter 1 . 11/29/2007
I love YOU! Ok that might be going a bit over the top but DAMN I love your little one shots so much. I love George! He's grown up so much in this fiction, I can imagine him being just like this!
You are FANTASTIC! Please don't stop, I think the Angelina Johnson fans are starting to creep back onto the site finally. We were all a bit drained from reading so many "morning fred" fics that I thnk we all decided to give it a rest for a while. Then you came back!
*cries in delight*
I hope I get to read more from you soon! This is an instant fave*
I love
I love
ASQ Xx
| CrzyAngelchic chapter 1 . 11/29/2007
Yum. Short, sweet, and loveable. :)
| Alice-in-french chapter 1 . 11/29/2007
Very good! This was a very sweet story, I very munch enjoyed it.
| Malvolia chapter 1 . 11/29/2007
AW, George. What a sweetie. I love how there for her he is in this fic, and how he knew her mind better than she knew it herself. I also liked the bit about how women like that romantic twaddle. So true...but we like it sounding real, like this does, and not over-the-top melodramatic, like most other fics do. (There were no tears welling up in the endless pools of her deep chocolate brown orbs, for example.)
Brilliant, as expected. :)


text: SBR Forum Members Discuss eSports Betting By SBR Friday, September 30, 2016 Share with: SBR Forum members are discussing the Counter Srike Global Offensive ESL tournament which began today in New York. Optic Gaming is a +260 underdog vs. SK and team Liquid +222 vs. Navi. eSports is a niche market that has slowly gained in popularity among gamblers, though not all sportsbooks offer eSports betting markets. Sportsbook Review has a rating guide filter which allows bettors to browse all online sportsbooks offering eSports. 63 sports betting sites offer eSports wagering markets; this number is expected to grow in 2017, though perhaps not at the pace once projected by industry insiders. eSports betting opens a can of worms with respect to market manipulation among lower ranked leagues and suspicious betting patterns and match-fixing; think low-level tennis multiplied by 100. The fact remains that wherever there is market interest, betting sites will offer lines, challenges to the lines manager departments aside. Sportsbook Review welcomes feedback from eSports bettors.


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We spent four nights at the Levni and couldn't have asked for a better spot for our first trip to Istanbul. It is within walking distance of all the major attractions but also feels slightly removed from the hustle and bustle. The staff were also very friendly and helpful. We arrived at about 130am from the airport and were greated by two very lovely and enthusiastic gentleman. We were taken immediately to our room where a small bottle of wine and fruit plate were waiting. Our wifi didn't work so they spent time with me and my iPad to fix the issue. Breakfast was also nice and the pool area very relaxing. I would recommend this hotel. There was construction while we were there for the new metro, which may increase foot traffic when it is finished but that will make it even more convenient. Also there was new mosque being finished in front of the hotel. We noted speakers, so you may want to request a room at the back to avoid being woken up for prayer in the morning, once it is finished.
- Official Description (provided by the hotel):
- Levni Hotel & Spa Old City is a special category (5 star category) hotel located in Sirkeci Istanbul within 5 min. walking distance to many museums and palaces - Topkapi Palace, Santa Sophia, Blue Mosque, Basilica cistern. All rooms are decorated in modern style. 1 suite and 30 deluxe rooms , 5 Excecutive rooms with jacuzzi some with a very nice Bosporus Sea view. SPA center is avaiable with an indoor pool, sauna, Turkish bath and gym. there are 3 therapy rooms including one for couple massages. Rich Open buffet in the Gourme Restaurant, which has a capacity of 100 pax also terrace cafe is avaiable. There are 2 meeting rooms with 100 pax and 30 pax capacity connected to each other with a lounge and one exclusive VIP room with capacity upto 12 pax. our business center is avaiable with free internet and printing services nex to meeting rooms. ... more less
- Reservation Options:
- TripAdvisor is proud to partner with Cheap Tickets, Expedia, Booking.com, Hotels.com, Travelocity, TripOnline SA, Hotwire, getaroom.com, HotelQuickly and HRS so you can book your Levni Hotel & Spa reservations with confidence. We help millions of travelers each month to find the perfect hotel for both vacation and business trips, always with the best discounts and special offers.
- Also Known As:
- Levni Hotel And Spa
- Levni Hotel Istanbul


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The 80th Annual Reunion of the Strohl Family will be held Sunday, Aug. 5, at Palmerton Memorial Park.
Those attending are asked to bring a covered dish and join everyone for the 1 p.m. luncheon. Sodas and hot dogs will be available for purchase.
A brief business meeting will be held immediately after lunch.
Cakes should be brought for the cakewalk and a small prize for bingo. Family members should bring old family pictures and other Strohl mementos for discussion.
The 70th annual Marsh family reunion will be held on Aug. 12 at Indian Trail Park, Pavilion 1. The meal will be held at 12:30 p.m., and a business meeting will convene at 1:30.
Prizes will be awarded to the youngest and oldest males and females, traveling the farthest, most recently and longest married couples, and the largest family present.
Cake donations for the cake walk and donations for bingo prizes are appreciated. Drawings for door prizes will be held.
Family members with last names that begin A through M are asked to bring a dessert; N through Z, salad.
Panther Valley High School Class of 1974 will have a 40-year class reunion planning meeting at 6:30 p.m. Wednesday, July 25 at the Sports Zoo in Lansford.
The Panther Valley Class of 1992 will be holding a 20th reunion on Saturday, Aug. 4, from 3 to 8 p.m., at the Fire Company Roadside Rest in Nesquehoning. Notifications have been sent out via Facebook and through word of mouth.
If any classmate has not been reached, please contact Paul McArdle at (570) 645-8707 or firstname.lastname@example.com or Yvonne Stoffey at firstname.lastname@example.com or (570) 778-6017.
PV Classes of '90, '91, '93, and '94
The Panther Valley High School Class of 1987 hosted its 25-year reunion on June 9 at the Lakeside Ballroom in Barnesville. Classmates attending included, front row from left, Chris Aungst and Mary Kelly Reese Aungst. Second row Amy Toniatti Blasko, Suzanne Sommers Milkowich, Brian Ouly, Christine Fritzinger Chinchar, Tammy Smith Faenza, Cindy Macalush and Mary McLaughlin.
Anyone interested in helping plan a "get together" of the Lehighton Area High School class of 1954 is invited to meet at the Community Grove pavilion at 6 p.m. today (Tuesday).
Panther Valley High School Class of 1974 will hold a 40-year class reunion planning meeting at 6:30 p.m. Wednesday, July 25 at the Sports Zoo in Lansford.
Members of the Tamaqua High School graduating class of 1943 held their annual reunion recently at the La Dolce Casa Restaurant in Tamaqua. In attendance were, front row from left, Pearl Leiby, Pauline Fenstermacher, Pauline Edling, Josephine Havrilko, Ruth Blaschak, Alice Truskey and Jean Derr. Middle row, Jim Valentine, Harry Fetterman, Theresa Calabrese, Sarah Miller, Patricia Kennedy and Dick Jones. Third row, Harold Boyer, Paul Lewis, Marlin Reinhart, Daniel Gangloff, Ted Bogosh III and Tom Edwards.
The Lehighton Area High School Class of 1982 is holding its 30-year reunion on Friday, July 13 at the Lehighton Orioles from 6-11 p.m.
It will be a pig roast/luau-style event, and there is a charge. There will be live entertainment.
If you have not received an invitation or for more information, please contact Robin Fatzinger at email@example.com or (610) 718-9091.
The Lehighton Area High School Class of 1982 is holding its 30-year reunion on Friday, July 13 at the Lehighton Orioles from 6-11 p.m. It will be a pig roast/luau-style event, and there is a charge. There will be live entertainment.


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