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The helix-loop-helix protein, E47, is essential for both B- and T-lineage development. Here we demonstrate that in vitro E47 and Notch signaling act in concert to promote T cell development from fetal hematopoieitic progenitors and to restrain development into the natural killer and myeloid cell lineages. The expression of an ensemble of genes associated with Notch signaling is activated by E47, and additionally, Notch signaling and E47 act in parallel pathways to induce a T lineage–specific program of gene expression. Enforced expression of the intracellular domain of Notch rescues the developmental arrest at the T cell commitment stage in E2A-deficient fetal thymocytes. Finally, we demonstrate that regulation of Hes1 expression by Notch signaling and E47 is strikingly similar to that observed during Drosophila melanogaster sensory development. Based on these observations, we propose that in developing fetal thymocytes E47 acts to induce the expression of an ensemble of genes involved in Notch signaling, and that subsequently E47 acts in parallel with Notch signaling to promote T-lineage maturation.
Hematopoietic stem cells (HSCs) continuously generate progenitor cells of distinct lineages, including T, B, NK, dendritic, myeloid, and erythroid cells. Long-term HSCs mature into short-term repopulating cells. The short-term repopulating cell compartment contains multipotent progenitor cells, of which a fraction has the ability to differentiate into common lymphoid progenitor cells (CLPs) (1–3). Many different bone marrow populations have been proposed to seed the adult thymus, including HSCs, multipotent progenitor cells, early lymphoid progenitors, and CLPs (1, 2, 4). Thymus-colonizing progenitors in the fetal ages, however, were shown to develop from different origins (5–7).
Once the progenitors enter the thymus and interact with Notch ligands expressed on the thymic epithelium, T cell development is initiated. The earliest T cell precursor cells, known as early T-lineage precursors, exhibit high expression of c-kit and lack the expression of the coreceptors CD4 and CD8 (8–10). Early T-lineage precursors are present in the CD44+CD25− (DN1) subset, which maintain the ability to differentiate to diverse cell types, including B, NK, and dendritic lineages, but have minimal myeloid potential. Progenitor cells become committed to the T cell lineage at the CD44+CD25+ (DN2) and the CD44−CD25+ (DN3) cell stages.
The E2A gene encodes for two isoforms, E12 and E47, generated through alternative splicing of exons encoding the DNA binding and dimerization domain (11). The E2A proteins are classified as class I HLH proteins also named E proteins (12). They include E12, E47, HEB and E2-2, and the Drosophila homologue, daughterless. daughterless is most closely related to E12 and E47. In B lineage cells, the predominant E-box–binding complex is comprised of E47 homodimers, whereas in thymocytes, E47 readily forms heterodimers with HEB (12, 13).
Thymocyte development is perturbed in adult E2A-deficient mice, albeit partially, at the DN1 stage (14). Enforced expression of Id3 in human T-lineage precursor cells and expression of a dominant-negative form of HEB arrest thymocyte development also at the DN stage (15–17). Several target genes, including pTα and RAG1/2, have been shown to be regulated by E2A and HEB in developing T-lineage cells (18, 19).
T-lineage commitment requires Notch1-mediated signaling (20–26). Four mammalian Notch receptors have been identified, designated as Notch1–4. Notch receptors are activated upon interacting with different ligands, named Delta-like 1 (DL1), Delta-like 3, Delta-like 4, Jagged1, and Jagged2. Binding of the ligand to the Notch receptor activates proteolytic cleavage, resulting in the release of the intracellular portion of the receptor (ICN) from the cellular membrane. ICN is transported to the nucleus, where it interacts with CSL (CBF1, RBP-Jκ, suppressor of Hairless Su(H)) to activate down-stream target gene expression. Among its targets are Hes1 and pTα (22, 24). In the thymus, T cell development in Notch1-deficient mice is blocked at the DN1 stage (27). In contrast, enforced expression of ICN promotes T cell development in the bone marrow, whereas B cell development is perturbed (28). Conditional inactivation of the CSL gene and ectopic expression of antagonists of Notch signaling, including Deltex1, Nrarp, and Lunatic Fringe, inhibit T cell maturation at a similar stage (29–32). Consistent with being a key target for Notch1-mediated signaling, thymocyte maturation in Hes1-deficient mice is blocked at an early stage (33). How Hes1 contributes to T-lineage developmental progression is still poorly understood.
The critical roles for E proteins and Notch signaling at the early stage have raised the question of how these two ancient regulatory modules interact to control thymocyte commitment. Initial studies have suggested that Notch signaling acts to modulate the transactivation activity of E47 (28, 34). Further studies have indicated that enforced Notch signaling together with mitogen-activated protein (MAP) kinase signaling readily promotes the degradation of E47, suggesting a linear relationship (35, 36). Collectively, these studies suggest that E proteins and Notch signaling act in a linear pathway.
Here we show that in vitro E47 and Notch signaling act in concert to promote thymocyte development from fetal hematopoieitic progenitors. Furthermore, E47 and Notch signaling act in synergy in fetal progenitor cells to restrain NK and myeloid development. E47 also induces the expression of a subset of genes involved in Notch-mediated signaling, including Notch3, Hes1, Hes5, Grg6, and, albeit modestly, Notch1, and E47 and Notch signaling act synergistically to induce the expression of pTα and CD7. An early block of T cell development in E2A-deficient fetal thymocytes is rescued, in part, by the overexpression of ICN. Based on these and previous observations, we suggest that in the fetal thymus E47 acts to induce the expression of an ensemble of genes involved in Notch signaling and additionally that it acts in concert with Notch signaling to induce a T lineage–specific program of gene expression.
Global analysis of gene expression profiles in E2A-deficient hematopoietic progenitor cells in response to E47 activity
Previously, we described E2A-deficient hematopoietic progenitor cells (HPCs) that exhibit T, NK, dendritic, myeloid, and erythroid lineage potential (37). To identify E47 target genes in hematopoietic progenitor cells, E2A-deficient HPCs were transduced with retrovirus carrying an E47–estrogen receptor (E47ER) fusion protein. Control cells were transduced with virus carrying the bHLH region but lacking the NH2-terminal transactivation domains. Both retroviral constructs also contained the coding sequence of hCD25 to allow rapid isolation of transduced cells (Fig. 1 A). 2 d after infection, the cells were cultured in the presence of S17 stromal cells and incubated with 4-hydroxytamoxifen (4-OHT) for a period of 6 h to activate the E47ER fusion protein. hCD25-positive cells were purified using magnetic beads, and RNA was isolated (Fig. 1 B). The resulting RNAs were converted into cDNA that in turn was used to synthesize biotinylated cRNA, which subsequently was hybridized to mouse oligonucleotide arrays. Fluorescence intensities for each probe were normalized to the signal from oligonucleotides representing a group of housekeeping genes. The normalized hybridization intensities from each of the samples were then compared with that of RNA derived from control infected E2A-deficient HPCs. We identified those oligonucleotides that had ratios of either >4.0 (representing candidates for positive regulation by E47) or <0.5 (representing genes potentially repressed by E47). We focused our analysis on a selected group of genes involved in cell survival, proliferation, and developmental progression. Among this focused group of genes, there were 53 whose expression was increased by a factor of at least fourfold (Fig. 1 C). Among these were, interestingly, a set of genes that are known to play essential roles in T cell development and cell growth, including MHC class II, IL-7Rα, syk, pTα, Hes1, LAT, Id2, Grg6, Bcl2A1b, CD7, and cyclin D3 (Fig. 1 C). 21 genes repressed by a factor of twofold or more include Bcl11b, RORα, and Gfi1b (Fig. 1 C).
To validate the data obtained from the microarray analysis, mRNA was isolated from the transduced cells and analyzed by real-time PCR (Fig. 2 A). As predicted from the data obtained from the microarray analysis, pTα, IL-7Rα, and LAT transcript levels were significantly elevated in cells expressing E47 (Fig. 2 B). To determine whether E47 directly activates the expression of these target genes, cells were induced with 4-OHT in the presence of cycloheximide (CHX). Each of the targets, including pTα, IL-7Rα, and LAT, were activated in the presence of CHX, indicating that the induction of transcription by E47 was direct (Fig. 2 C). These data indicate that enforced E47 expression in hematopoietic progenitor cells both activates and inhibits the expression of genes associated with a T lineage–specific program of gene expression.
E47 activates the expression of an ensemble of genes involved in Notch-mediated signaling
Unexpectedly, the bHLH protein, Hes1, was among the target genes induced by the expression of E47 (Fig. 1). To explore whether additional components of the Notch signaling module are regulated by E47, RNA was analyzed by real-time PCR for the expression of Hes1, Hes5, Notch1, Notch2, Notch3, NumbL, Grg3, Grg6, and Deltex1 (Fig. 2 D). Hes1 expression was readily induced by E47 activity, as measured by real-time PCR, consistent with the data obtained from the microarray analysis (Fig. 1). Deltex1, Hes5, and Notch3 were also activated by E47. Furthermore, Notch1 expression levels were modestly elevated by E47 (Fig. 2 D). Transcript levels of Groucho-related corepressors, Grg3 and Grg6, which act together with the Hes gene products to inhibit down-stream target gene expression, were also modulated by E47-mediated transactivation (Fig. 2 D). The expression of genes involved in Notch-mediated signaling was increased by the enforced expression of E47, even in the presence of CHX, indicating direct regulation by E47 (Fig. 2 E). These data indicate that enforced expression of E47 in E2A-deficient HPCs rapidly leads to the induction or elevation of components of the Notch signaling pathway.
To exclude the possibility that the E47ER fusion protein aberrantly activates target gene expression, we enforced the expression of E47 in the absence of an ER fusion component. We found that also in the absence of the estrogen domain E47 induces the expression of Hes1, Notch3, and Notch1 in E2A-deficient HPCs (Fig. 2 F).
Because E2A-deficient cells are grown on S17 cells, we considered the possibility that Jagged-mediated signaling would contribute to the induction of, for example, Hes1 expression by E47. To exclude this possibility, E2A-deficient cells transduced with E47ER were cultured in the presence of γ-secretase inhibitor IX. Hes-1, Notch1, and Notch3 mRNA levels were significantly induced by E47 in the presence of the γ-secretase inhibitor IX, indicating that the induction of the expression of these genes was not mediated by Notch signaling (Fig. 2 G).
To further define the involvement of E47 in the regulation of these genes, we examined the expression of these genes in fetal E2A+/+, E2A+/−, and E2A−/− thymocytes derived from day 15 embryos. RNA was isolated and analyzed by real-time PCR using the appropriate primers. pTα, IL-7Rα, and LAT transcripts were reduced in E2A-deficient thymocytes (Fig. 2 H). Hes1, Hes5, Notch1, and Notch3 transcript levels also were significantly lower in E2A-deficient thymi when compared with wild-type thymi (Fig. 2 I). E2A heterozygous thymocytes also showed a decrease in LAT, pTα, Hes1, and Hes5 mRNA levels as well, indicating that the regulation of these genes by E47 is dosage sensitive (Fig. 2, H and I). Collectively, these data indicate that in fetal thymocytes E2A proteins contribute to the regulation of a subset of genes involved in Notch-mediated signaling.
E2A directly regulates Hes1 gene expression
The observations described indicate that, surprisingly, Hes1 gene expression is activated by E47 in hematopoietic progenitor cells. Because Hes1 activation by E47 occurred in the presence of CHX, we conclude that E47 directly regulates Hes1 gene expression. The activation of Hes1 by E47 is interesting because it is reminiscent of the regulation of the Drosophila homologue of Hes1, enhancer of split, in Drosophila sensory organ development (38). Enhancer of split gene expression is regulated by daughterless, a basic helix-loop-helix (bHLH) protein strikingly similar to E47 (39). To identify the regulatory elements in the murine Hes1 locus that are responsive to E47 binding, we compared the DNA sequence of the human, murine, and chicken Hes1 loci. Four domains, named CR1-CR4, were identified that were highly conserved (Fig. 3 A). One of these conserved elements, CR3, contained two E47 high-affinity binding sites that are located 10 bp apart, in both the murine and human sequences, respectively (Fig. 3 A). To determine whether the CR3 element has the ability to activate gene expression, we inserted it upstream of a minimal promoter linked to the luciferase coding region. The reporter construct, as well as a construct lacking the CR3 element, was transfected into E2A-deficient HPCs in the presence of a vector carrying the E47 gene placed under control of the β-actin promoter. The reporter construct was activated by a factor of 2.5-fold compared with the reporter lacking the CR3 region (Fig. 3 C). To determine whether the increase in luciferase activity was dependent on the E47 binding sites, mutations were generated either in one of the sites (named E-box(1) and E-box(2)) or in both E-box sites (Fig. 3 B). Mutation of each of the binding sites substantially lowered luciferase activity (Fig. 3 C). These observations indicate that a highly conserved E47 responsive regulatory element is present in the Hes1 locus.
Hes1 is normally expressed at the double-negative (DN) cell stage but upon β-selection its levels decline significantly (33, 40, 41). To determine whether E47 binds to the CR3 region in DN and double-positive (DP) cells, we performed chromatin immunoprecipitation (ChIP) assay. Thymocytes from either wild-type mice or mice harboring a knock- in mutation in the E2A locus generating a E2A–GFP fusion proteins were sorted, and DN and DP populations were isolated, fixed with formaldehyde, and then disrupted by sonication (42). The cross-linked DNA was immunoprecipitated using a GFP-specific antibody. Immunoprecipitated DNA was amplified using Hes CR3 primers and examined by DNA gel electrophoresis. E47 binding to the Hes1 CR3 element was not significantly enriched in DP thymoctes derived from mice expressing the E2A–GFP fusion protein compared with wild-type thymocytes (Fig. 3 D). On the other hand, the CR3 element was substantially enriched in DN cells in E2AGFP mice (Fig. 3, D and E). Because Hes1 transcript levels are high in DN thymocytes but virtually absent in DP thymocytes, the binding activity of E47 to the Hes1 regulatory element correlates with Hes1 gene expression. These observations, in conjunction with the induction of Hes1 expression by E47 in the presence of CHX (Fig. 2 E), suggest that E47 directly regulates Hes1 gene expression by binding to a highly conserved regulatory element. These data imply that the molecular mechanism underlying enhancer of split gene expression has remained conserved in mammalian hematopoietic progenitor cells to regulate Hes1 transcription.
Notch-mediated signaling and E47 activate a common set of target genes
Previous data have established that Notch signaling activates the expression of Hes1 and Hes5 (21–24). Our data indicate that E47 directly activates Hes1, Hes5, and Grg6 expression. Collectively, the data raise the possibility that Notch signaling and E-protein activity act in parallel to induce target gene expression. To identify targets activated by Notch-mediated signaling alone and in conjunction with E47 expression, E2A-deficient HPCs were transduced with retrovirus carrying the E47 bHLH coding sequence. Transduced cells were cultured in the presence of stromal cells expressing the Notch ligand DL1 (OP9-DL1) or stromal cells expressing GFP (OP9-GFP) for a 6-h period (Fig. 4 A). The hCD25-positive cells were subsequently purified using magnetic beads, and RNA was isolated and analyzed for differences in down-stream target gene expression using microarrays. Interestingly, culturing E2A-deficient HPCs in the presence of the Notch ligand, DL1, showed elevated insulin and olfactory receptor transcript levels significantly within a 6-h period (Fig. 4 A). Additionally, GATA-3 and Hes1 mRNA levels were increased, consistent with them being targets for Notch-mediated signaling (24, 43). Notch signaling also activated the expression of CD7, MHC class II, EHOX, FGF21, and IRF7. We note that this set of target genes was also activated by the enforced expression of E47 alone.
To identify target genes activated by both E47 and Notch signaling, E47ER-transduced cells were incubated for 6 h in the presence of 4-OHT and OP9-DL1. RNA was isolated from the transduced cells and examined by microarray analysis (Fig. 4 B). As expected, E2A-deficient cells exposed to both Notch signaling and E47 expression induced the expression of genes including Grg6, LAT, pTα, unc93 homologue, NFκBIε, PirA1, Bmf-pending, EHOX, Notch4, CD7, and Hes1, which also responded to either E47 and/or Notch-mediated signaling alone (Fig. 4 B). Notch signaling and E47 also acted to repress Mmp16, Wnt7b, Egr3, RORα, and Ndr1 expression (Fig. 1 and Fig. 4 B).
To examine whether Notch signaling and E47 act in synergy to induce the transcription of this set of genes, RNA was isolated from E2A-deficient HPCs carrying the E47ER fusion protein cultured in the absence or presence of Notch ligand, DL1. mRNA derived from these populations was analyzed by real-time PCR for the expression of Hes1, Hes5, pTα, Notch1, Grg3, and Grg6 (Fig. 4 C). As expected Hes1, Hes5, Notch1, and Grg6 transcription was activated by both E47 and Notch-mediated signaling, although the activation of gene expression was not synergistic (Fig. 4 C). However, E47 and Notch-mediated signaling acted in synergy to induce the expression of pTα and CD7 (Fig. 4 C). These data indicate that E47 activity and Notch signaling in E2A-deficient HPCs act in parallel pathways to induce the expression of a common set of target genes.
Enforced expression of Notch ICN rescues developmental arrest in fetal-derived E2A-deficient thymocytes
The data described in the previous paragraph suggest that E47 activates the expression of several components of the Notch signaling pathway and acts in parallel with Notch signaling to induce a program of T lineage–specific gene expression. In vivo, thymocyte development in adult E2A-deficient mice is partially blocked at the DN1 cell stage (14). However, a substantial fraction of thymocyte has the ability to develop into the mature T cell lineages (14). In contrast to adult thymocyte maturation, E2A-deficient fetal thymocytes inefficiently develop beyond the DN3 cell stage (unpublished data).
To determine whether constitutive active Notch signaling has the ability to overcome the impairment of T cell development in E2A-deficient fetal progenitors, we used a retrovirus vectors expressing ICN in fetal thymocytes. Hematopoietic progenitors derived from either E2A+/+ or E2A−/− fetal livers were infected with the retrovirus and cocultured with the deoxyguanosine-treated fetal thymic lobes. After 17 d of culture, the cells were harvested and analyzed by flow cytometry. As expected, E2A+/+ progenitors infected with control (MigR1) virus differentiated to CD4+CD8+ DP thymocytes as well as CD3−NK1.1+ NK cells (Fig. 5). On the other hand, E2A−/− progenitors infected with MigR1 virus gave rise to NK cells but failed to differentiate into CD3+ and DP cells (Fig. 5). These data are consistent with the phenotype of fetal E2A-deficient thymocytes that are severely perturbed in their ability to undergo T-lineage developmental progression. In contrast, enforced expression of ICN in fetal liver progenitor cells restored the ability of E2A-deficient progenitors to develop into CD3+ and DP cells. Collectively, these results indicate that the overexpresion of ICN can restore the impairment of T cell development of E2A-deficient progenitors.
Notch signaling and E47 activity act together to promote T-lineage development at the expense of NK and myeloid cell maturation
The data shown in Fig. 4 indicate that Notch signaling and E47 have the ability to activate the expression of a common set of target genes. However, because the abundance of E47 expression from the retroviral construct is significantly higher than observed in wild-type thymocytes, the data do not provide unambiguous evidence that E47 and Notch signaling act in synergy to induce a T-lineage program of gene expression. To examine whether Notch signaling and E47 act in concert to promote thymocyte maturation, we cultured progenitors derived from E2A+/+, E2A+/−, and E2A−/− fetal liver in the presence of OP9-DL1 cells. Furthermore, to inhibit Notch-mediated signaling the cells were cultured in the presence of different concentrations of γ-secretase inhibitor IX.
E2A+/+ and E2A+/− progenitors efficiently developed into DN2 to DN3 stages in the absence of γ-secretase inhibitor (DMSO alone) after 7 d of culture (Fig. 5 A). In contrast, E2A−/− progenitors were severely impaired in their ability to differentiate into DN3 cells, raising the possibility that the ability of these cells to receive Notch signals is perturbed in the absence of E2A (Fig. 6 A). Consistent with previous observations, increasing concentrations of the γ-secretase inhibitor significantly perturbed thymocyte maturation, resulting at 10 μM concentration in a complete arrest at the DN1 stage (Fig. 6 A) (44). Interestingly, E2A+/− progenitors readily developed into the DN3 stage (54%) (Fig. 6 A). Similarly, a considerable proportion (23%) of E2A+/+ progenitors cultured in the presence of 3 μM γ-secretase inhibitor developed into the DN3 stage (Fig. 6 A). However, the majority of the E2A+/− cultured in the presence of 3 μM γ-secretase inhibitor was arrested at the DN1 stage and the fraction of DN3 cells (6.4%) was severely declined compared with wild-type thymocytes (23%) (Fig. 6 A). These results suggest that Notch signaling and E47 act in concert to promote developmental progression through the DN stage.
To further define the effect of Notch signaling and E47 activity in lymphoid development, we also examined the generation of NK cells. In the E2A+/+ cells, a small number of NK cells were found in the absence of the Notch inhibitor (Fig. 6 B). However, the proportion of NK cells was significantly increased when cultured in the presence of γ-secretase inhibitors as previously demonstrated (Fig. 6 B) (44). Interestingly, E2A+/− and E2A−/− progenitors gave rise to substantial proportions of NK cells even in the absence of Notch inhibitor (Fig. 6 B). Moreover, the proportion and the absolute number of the NK cells were synergistically increased in the presence of Notch inhibitor in E2A+/− and E2A−/− progenitors (Figs. 6, B and C). These results suggest that Notch signaling and E47 act in concert to initiate T-lineage specification and to restrain fetal hematopoietic progenitors to adopt a NK cell fate.
To examine the effect of myeloid lineage development by the combined absence of E2A and Notch signaling, we analyzed the generation of Mac1+ cells (Fig. 7). A substantial fraction of E2A-deficient fetal progenitors developed into myeloid cells (Fig. 7 A). Interestingly, E2A+/− fetal hematopoietic progenitor cells showed an increased ability to differentiate into myeloid cells (Fig. 7 A). Strikingly, culturing E2A+/− progenitors in the presence of γ-secretase inhibitor IX synergistically increased the number of Mac1+ cells (Fig. 7 B). We note that the number of Mac1+ cells was significantly higher in cells derived from fetal hematopoietic E2A+/− compared with E2A−/− progenitors, suggesting that the E2A proteins play an essential role in myeloid lineage commitment and/or progression (Fig. 7 B).
Collectively these data suggest, in conjunction with the data described in the previous section, that in fetal thymocyte development E47 acts upstream of an ensemble of genes involved in Notch signaling. In the absence of E47, fetal thymocytes do not have the ability to receive and/or integrate Notch-mediated signaling and fail to differentiate into T-lineage cells but allow the development of NK and myeloid cells. During subsequent stages of fetal thymocyte maturation, E47 acts in parallel with Notch-mediated signaling to promote T-lineage developmental progression.
Previous data have indicated that the initial stages of B- and T-lineage development require the activities of E proteins. These observations have raised the question as to how E proteins influence lineage specification in both B- and T-lineage cells. We propose that in the bone marrow, E proteins by themselves in the CLP allow the initiation of a B-lineage program of gene expression, whereas in the thymus they act in concert with Notch-mediated signaling to induce a T cell fate.
Consistent with a CLP deficiency in E2A-deficient mice, we propose that E2A is transcriptionally active in the CLP cell stage (Fig. 8 A) (45). Once E2A is activated it induces the expression of a subset of genes that are essential to promote B cell development. Among the critical target genes is IL7Rα. Specifically, we show that IL-7Rα gene transcription is modulated by E47 expression. We note, however, that IL-7Rα is expressed at substantial levels in E2A-deficient HPCs, consistent with its dependence on IL-7 for cell growth (unpublished data). Thus, other factors must contribute to the regulation of IL-7Rα expression as well. One such candidate is PU.1. PU.1 has been shown to regulate IL-7Rα gene expression (46). Because PU.1 is expressed at considerable levels in E2A-deficient HPCs, it is conceivable that E proteins and PU.1 act in concert to control the expression of IL-7Rα (Fig. 8 A). IL7Rα-mediated signaling, E2A and PU.1, in turn activates EBF transcription, which in turn activates Pax5 (Fig. 8 A) (47). Once activated EBF and Pax5 then act in concert to induce a B-lineage program of gene expression (Fig. 8 A) (48). Our data also show that Bcl11a transcript levels were modestly elevated by enforced E47 expression. How Bcl11a acts in relationship to EBF and Pax5 to promote B cell development is an interesting issue that needs to be addressed.
E47 also induces the expression of genes involved in the commitment of alternative cell fates, including pTα and Notch1. This brings about the question as to how the expression of these genes is antagonized in B-lineage cells. Pax5 has been shown to suppress the transcription of pTα and Notch1 (2, 49). Thus, Pax5 functions to both activate and maintain gene expression of a subset of B lineage–specific genes and, additionally, may function to repress transcription of non–B lineage–specific genes that we suggest are activated by at least in part by E2A. Collectively, we propose a genetic regulatory network in which E2A, Bcl11a, PU.1, EBF, and Pax5 act in linear and parallel pathways to initiate, repress, and maintain B lineage–specific gene transcription, promoting commitment of the CLP in the bone marrow environment to the B cell fate (Fig. 8 A).
The data demonstrate that E47 and the Notch signaling module interact at two distinct levels in fetal hematopoietic progenitors: (a) E47 acts to induce the expression of genes involved in Notch signaling, including Hes1, Hes5, Grg6, Notch3 and Notch1; (b) E47 acts in concert with Notch signaling to synergistically induce the expression of pTα and CD7 (Fig. 8 B). The induction of an ensemble of genes involved in Notch signaling by E47 is consistent with a block in thymocyte development before the onset of T-lineage commitment in E2A-deficient fetal thymocytes. We propose that this block is caused at least, in part, by an inability of these cells to receive Notch and/or integrate Notch signals. During later stages of fetal thymocyte development, E47 and Notch signaling act in a parallel pathway to promote T-lineage maturation. It will be important to determine whether similar mechanisms operate in adult thymocyte development.
Previous studies have suggested a direct link involving Notch and E2A. Notch signaling has been demonstrated to alter the transactivation activity of E47 (28, 34). Further studies have demonstrated that Notch-mediated signaling promotes the degradation of E47 upon MAP kinase–dependent phosphorylation (35, 36). Both studies suggest a linear pathway in which Notch-mediated signaling acts upstream of E-protein activity. However, we note that synergistic activation of pTα and CD7 expression by E47 and Notch signals is difficult to reconcile with a linear relationship. We note that binding sites for E47 and CSL have been identified in the regulatory elements of the pTα chain gene regulatory elements (50, 51). E47 has been demonstrated to bind directly to these enhancer elements using ChIP assays (19). How then can we reconcile a linear versus parallel relationship involving E47 and Notch-mediated signaling? Our data do not exclude the possibility that Notch signaling influences E2A activity in developing DN thymocytes. For example, it is conceivable that Notch signaling affects the transactivation activity of E47 while acting in parallel to modulate down-stream target gene expression. Likewise, it is plausible that Notch signaling in conjunction with MAP kinase signaling at the DN3 and DN4 cell stage acts to promote the degradation of E47, resulting in lower E47 proteins levels. We note that a recent report has documented that Notch signaling has the ability to overcome the block in β-selection in mice that carry mutations causing defects in pre-TCR signaling (52). Nevertheless, it will be important to assess the role of Notch signaling in modulating E2A-mediated transactivation and altered stability in the context of endogenous gene expression.
We show here that E2A proteins act to induce the expression of members of the Hes family, independent of Notch signaling. Several studies have provided evidence for a Notch-independent mechanism of Hes1 and Hes5 gene regulation. CSL-deficient mice express Hes1 and Hes5 levels in DN thymocytes that are similar to that of wild-type mice (53). Pax5-deficient pro–B cells also express substantial levels of Hes1 even in the absence of Notch signaling (43). Hes1 transcripts have been detected in fetal liver precursors, suggesting that Notch signaling was active prethymically (54). Because E47 is expressed at high levels in fetal liver progenitors, it is conceivable that Hes-1 expression in fetal liver progenitor cells is independent of Notch signaling. Thus, a role for Notch signaling in controlling Hes1 expression prethymically needs to be reevaluated. Similarly, Hes1 transcription in neuroepithelial cells is activated before the expression of Notch and Delta ligands, indicating a mechanism of regulation independent of Notch-mediate signaling (55). E47 is expressed in the neuroepithelium, and it is conceivable that also in the neuroepithelium Hes1 is regulated by E-protein activity (56).
The activation of Hes1 and Hes5 by the parallel activities of E47 and Notch signaling is intriguing because a similar regulatory network regulates Drosophila sensory organ development and notochord-specific gene expression in the ascidian Ciona intestinalis (38, 57). In flies, sensory organ development is mediated in part by Notch signaling and bHLH activity, which act in parallel to regulate the expression of the enhancer of split complex (Fig. 8 C) (38, 57). The bHLH activity in peripheral nervous system development is mediated by the proneural proteins, achaete and scute, that bind as heterodimers with daughterless, to sites present in an enhancer controlling the expression of the enhancer of split complex. We note that daughterless is closely related to the E2A proteins. Thus, the enhancer of split gene and its homologue in mammalian organisms, Hes1, are both regulated by the combined activities of Notch-mediated signaling and bHLH activity (57, 58). Notch signaling and HLH activity also regulate tissue-specific gene expression in the notochord-specific enhancer located in the Brachyuri gene of the ascidian Ciona intestinalis (57). It is plausible that an ancient basic mechanism underlying notochord-specific gene expression in ascidians and Drosophila sensory organ development has been conserved in mammalian organisms to promote T-lineage developmental progression.
Materials And Methods
RNA purification, microarray analysis, and real-time RT-PCR.
E2A-deficient HPCs were isolated from E2A-deficient bone marrow and cultured on S17 stromal cells in the presence of SCF, IL7, and Flt3L (R&D Systems), as described previously (37). E2A-deficient HPCs were transduced with E47ER retroviral vectors as described (37). Purification of hCD25+ cells was performed using MACS beads (Miltenyi Biotec). Total RNA was isolated using an RNeasy kit (Qiagen). Microarray analysis was performed in our core facility (BIOGEM). The Codelink microarrays were used in this analysis (BD Biosciences). For real-time PCR, cDNA synthesis was performed using Superscript III (Invitrogen) following the manufacturer's protocol. Real-time PCR was performed using SYBR Green Master Mix (Stratagene) and analyzed by Mx4000 Instrumentation (Stratagene). The reactions were performed in triplicate at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 55°C for 1 min and 72°C for 30 s. The primer sequences used are: Hes1 forward, TTGGCTGAAAGTTACTGTGG and rev ACTATTCCAGGACCAAGGAG; Hes5 forward TACCTGAAACACAGCAAAGC and reverse GCTGGAGTGGTAAGCAG; Notch1 forward ACAACAACGAGTGTGAGTCC and reverse ACACGTGGCTCCTGTATATG; Notch2 forward TGACTGTTCCCTCACTATGG and reverse CACGTCTTGCTATTCCTCTG; Notch3 forward AGATCAATGAGTGTGCATCC and reverse GCAGACTCCATGACTACAGG; NumbL forward TTCGAGATTGAACTGTAGCC and reverse AGTGAAATGGTTCCCTTAGC; Grg6 forward GATGACAACATTTGGACAGG and reverse TAAGATGGTGTTGTCCTTGG; Grg3 forward TGAGAAGAACCACCATGAAC and reverse TGAGAAGAACCACCATGAAC; p-Tα forward CTGCAACTGGGTCATGCTTC and reverse TCAGAGGGGTGGGTAAGATC; IL-7Rα forward TTACTTCAAAGGCTTCTGGAG and reverse CTGGCTTCAACGCCTTTCACCTCA; LAT forward AGAATGTGGATGCAGATGAG and reverse CGTAATCTTCACACGACTCC; ARP forward CGACCTGGAAGTCCAACTAC and reverse ATCTGCTGCATCTGCTTG.
OP9 cocultures and flow cytometric analysis.
OP9-GFP or OP9-DL1 stromal cells were cultured in minimum essential medium alpha (MEMα; Invitrogen), supplemented with 10% fetal bovine serum (Gemini), 100 U/ml penicillin, 100 μg/ml streptomycin, and l-glutamine (Invitrogen), and plated 1 or 2 d before use in a 24-well plate, as described (20). The differentiation medium consisted of MEMα supplemented with 10% FCS (Gemini), 50 μM 2-ME, 2 mM l-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 ng/ml IL-7, and 5 ng/ml Flt3L. Lineage marker (Lin)− progenitors isolated from E2A+/+, E2A+/−, and E2A−/− fetal liver by MACS purification were seeded in 1 ml of IL-7/Flt3L medium at 105 cells/well of a 24-well plate. Various concentration of γ-secretase inhibitor IX (Calbiochem) was added to the culture, as indicated (Fig. 6). At day 7 of culture, cells were harvested and analyzed by flow cytometry. FACS analyses were performed as described (37). All antibodies were purchased from BD Biosciences or eBioscience.
Retroviral transduction and fetal thymic organ culture.
Control MigR1 and ICN retroviral vectors were provided by M.J. Bevan (University of Washington, Seattle, WA). Retroviral transduction and fetal thymic organ culture were performed as described (6, 37). Lin− progenitors were isolated from E2A+/+ and E2A−/− fetal liver at day 14 of gestation and transduced with MigR1 and ICN retroviral vectors. After 24 h of transduction, 104 cells were cultured with a deoxyguanosine-treated fetal thymic lobe for 17 d. The culture medium was replaced by half every 5 d. The cells were harvested and analyzed for the expression of GFP, CD3, CD4, CD8, and NK1.1.
Luciferase reporter assay was performed as previously described (59). The 3′E-Hes1 region was amplified with the primers: forward CATGCCCCATTTCCAGGCAAG and reverse GAGCCAATTAAACAAAACAAA. E2A-deficient HPCs were used for the transfection with the various luciferase constructs.
Chromatin histone immunoprecipitation.
ChIP assays were performed essentially as described (59). 107-fixed cells from mice carrying the E2AGFP fusion protein as well as wild-type thymus were lysed in SDS lysis buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 1% SDS) on ice for 10 min. The lysates were sonicated and diluted with 9 parts of 50 mM Tris-HCl, pH 8.0, 167mM NaCl, 1.1% Triton X-100, and 0.11% sodium deoxycholate. 2 × 106 cells were used for each immunoprecipitation. Full-length A.V. polyclonal antibody (BD Biosciences) was used to label E2AGFP-DNA complexes. Immunocomplexes were bound to protein G-Sepharose beads and washed four times. The immunocomplexes were then eluted by incubating the beads at 65°C overnight in 10 mM Tris-HCl (pH 8.0), 5 mM EDTA, 300 mM NaCl, and 0.5% SDS. Eluted DNA was treated with RNase A and digested with proteinase K. DNA was purified using the PCR Purification kit (Qiagen). PCR was performed with the following primers: forward GGGTTGTCTCGGGTTTCAG and reverse CAGGGCTCGTGGTTTTCAT.
We thank Jim Posakony and members of the Murre laboratory for insightful discussions. We thank Juan Carlos Zuniga-Pflucker for OP9-GFP and OP9-DL1 cells and Yuan Zhuang for providing the E2AGFP knock-in mice. This work was supported by grants from the National Institutes of Health (to C. Muree).
The authors have no conflicting financial interests.
Abbreviations used: 4-OHT, 4-hydroxytamoxifen; ARP, acidic ribosomal protein; bHLH, basic HLH; ChIP, chromatin immunoprecipitation; CLP, common lymphoid progenitor cells; CHX, cycloheximide; DL1, delta-like 1; DN, double negative; DP, double positive; HLH, helix loop helix; HPC, hematopoietic progenitor cell; HSC, hematopoietic stem cell; MAP, mitogen-activated protein.

domain: biology


text:
Animal Planet Wild Animals With than gorgeous animal photos of lions giraffes ibexes flamingos hippopotamuses and many others Animal Planet Wild Animals is a fun habitat by habitat guide that provides kids in the fir
Wild Animals Animal Planet Watch video of wild animal footage on an encyclopedic level, from the plight of endangered species to the hope of adorable baby animals. Animal Planet Official Site Official Homepage for Animal Planet Watch Full Episodes FREE with your TV subscription Get Behind the Scenes with Your Favorite Shows Start Exploring Now Animal Planet Animal Planet is an American cable channel owned by Discovery, Inc First established on October , , the network is primarily devoted to series and documentaries about wild animals and Wild Planet Animal Scramble Toys Games Product description From Wild Planet Animal Scramble is a really fun game and we thank each customer for their honest feedback it was through your reviews that we learned how to improve the design of this toy. Category Animal Planet shows Pages in category Animal Planet shows The following pages are in this category, out of total This list may not reflect recent changes. Amazing Wild Animal Sanctuaries You Should Support Wild animals are often seen in a dangerous light, but they have souls too, many of which are in need of saving Sanctuaries are dedicated to wild animals for that sole purpose. How Life for a Circus Tiger Compares to the Life of a Wild Think tigers love life under the big top Well, when you compare that to the life they should be living in the wild, we re guessing their not to excited to be in the circus. Watch Animal Planet Presents Season Prime At the intersection of human and wild you ll find hugely entertaining stories with larger than life, but very real characters from the full range of the animal kingdom.
Title: Animal Planet Wild Animals
Author: Laaren Brown
With than 200 gorgeous animal photos of lions, giraffes, ibexes, flamingos, hippopotamuses, and many others, Animal Planet Wild Animals is a fun, habitat by habitat guide that provides kids in the first years of schooling with the perfect bite sized view of their favorite wild animals Arranged thematically with focus on animal behavior and family relationships, youngWith than 200 gorgeous animal photos of lions, giraffes, ibexes, flamingos, hippopotamuses, and many others, Animal Planet Wild Animals is a fun, habitat by habitat guide that provides kids in the first years of schooling with the perfect bite sized view of their favorite wild animals Arranged thematically with focus on animal behavior and family relationships, young readers will explore sections about animal bodies, baby animals, food, play time, conservation and Special book features designed for this age group include Quick Bites sidebars with cool animal facts, simple infographics, and illustrated maps of life in the African savannah and rain forest.For Animal Bites books, check out Animal Planet Farm Animals, Animal Planet Polar Animals, and Animal Planet Ocean Animals.A portion of the proceeds will benefit Animal Planet s R.O.A.R Reach Out Act Respond campaign that partners with leading animal organization to make the world a better place for domestic and wild animals.

domain: entertainment


text:
diabetes mainly characterized presence of high glucose levels chronic and persistent in the blood. By International Diabetes Federation (IDF), approx. 425 million people worldwideThis disease suffers from 20 to 79 years of age.
In the case of Peru, the National Institute for Statistics and Informatics (INEI), 3.3% of citizens older than 15 years were diagnosed with diabetes mellitus In 2017, 3 out of 100 people They suffer from this disease.
Diabetes heart attack, stroke, kidney failure, eye damage, slow recoverySome limbs form a high amputation risk.
When they encounter this scenario, Our eating habits are healthy. For example, from the National Fisheries Association blue fish consumptionThis is one of the foods that help to prevent the disease.
we share three tips to consider:
one. HEALTHY HEALTH CONSUMPTION. I mean dark meat fish, avocado, olives, extra virgin olive oil, coconuts, nuts, among others. These foods help reduce inflammation and keep in good condition our cardiovascular system This is important because people with diabetes are heart attack or stroke.
2nd. CONSUMER FIBER. The fiber helps regulate the absorption of glucose, thereby controlling blood levels. It must be included in the meal every day. Basically whole grains: seeds such as oats, brown rice, wheat, beans and even flax seeds, sesame and chia.
3. CHOOSE GOOD CARBONHYDRATES. Most of the time we are afraid to consume them because we believe that they will contribute significantly to glucose levels, but the fact is that as long as we prefer carbohydrates, we will consume it well. So, weekly menu Must include healthy and natural carbohydrates such as potatoes, sweet potatoes, brown rice, fresh fruitsamong others. Avoid refined carbohydrates free of fibers, such as white flour, processed biscuits, industrial cakes and refined sugar.

domain: health


Make easy monthly payments. Select Affirm at checkout.Learn more
Here's a beautiful and lightly played American Standard Strat, built in 2016 with a Bordeaux Metallic finish. This Strat is loaded with stock Custom Shop Fat '50s single-coil pickups, and has locking tuners installed. It's in overall excellent condition, and includes the Fender gigbag and tremolo arm.
Includes: Gigbag and Trem Arm
- Solidbody Electric Guitar
- Alder Body
- "C" Shape Maple Neck
- Rosewood Fingerboard
- 25.5" Scale Length
- 9.5" Fretboard Radius
- Custom Shop Fat '50s Single-Coil Strat Pickups
- 5-Position Pickup Switch
- Master Volume, Tone 1, and Tone 2
- 2-Point Tremolo
- Chrome Hardware
- Bordeaux Metallic Finish
- 7 lbs, 6 oz.
- .820" Neck Profile at 1st Fret
- .860" Neck Profile at 12th Fret
Since 1994 The Music Zoo has been a trusted source for musicians around the world. We've located guitars for Slash, sold guitars for Steve Miller, held performances by major artists like Steve Vai, built thousands of customer relationships, and helped countless enthusiasts find just the right instrument. Today we look forward to finding your next guitar!


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On October 6th, the WHS Golf team joined with other local area high school golf teams at the Great Hope Golf Course in Westover, MD to "Swing for a Cure."
Snow Hill High School, Snow Hill, MD $170
James M Bennett High School, Salisbury, MD $224
Pocomoke High School, Pocomoke MD $250
Washington High School, Princess Anne, MD
Stephen Decatur High School, Berlin, MD $775
For a grand total of $1840 which was donated to the National Cancer Research Center.
Way to represent JAG NATION Mr. Knopf and the WHS Golf Team!
This story can be found on Delmarvanow.com.

domain: news


text:
The delicate feeling of the pampered feminine look is given to you by this cute bracelet. It is a sublimation of beauty, and it is tied between your wrists, as the fickle leaf remains to impress with your attractive look.
- Gold plating.
- overlapping rings.
- For ladies and young women.
How gorgeous is this golden bangle when it glows on your wrist. It has an overlapping bead design with a cut-out leaf addition that continues to sparkle when you wear the bracelet.
Christmas gifts with your loved ones, And happy parties with this gift that women love, And girls alike. It is an ideal choice and matches with all sections of clothing.

domain: artistic


text:
San Francisco, California
London, United Kingdom
Hunter Barnes is a photographer who focuses his artistic gaze on the faces of groups of people who are consistently misrepresented in the modern American narrative. At MOBIA he will speak about his project and book, A Testimony of Serpent Handling, and share images and personal stories from his time spent in West Virginia. “I was invited by the families and congregation of my friend’s Serpent Handling churches in West Virginia to live and document their lives. The people in these photographs took me in as one of their own. I was shown a way of life sometimes unseen and able to share their story.” - Hunter Barnes
For more information about Hunter Barnes and his photograph, visit his website: http://www.hunterbarnes.com/
When & Where
Museum of Biblical Art
The Museum of Biblical Art celebrates and interprets art related to the Bible and its cultural legacy in Jewish and Christian traditions through exhibitions, education and scholarship.


Champions Oncology is a technology-enabled research organization that leverages an end-to-end range of research and development solutions to improve the productivity of oncology drug development. Using computational and experimental testing platforms, Champions Oncology improves decision making for effective therapeutic development. Our team is passionate and self-motivated working together to accelerate the challenges of oncology drug development and ultimately improve patients’ lives around the world.
Job location: Remote, US.
As a Bioinformatics Engineer working with Champions Oncology, you’ll have a unique opportunity to make a significant impact on the field. You’ll serve as an evangelist for our Lumin Bioinformatics SaaS platform, working with clients to help them leverage Lumin in their research. You’ll also work with our bioinformatics scientists to move workflows to production, be responsible for curating bioinformatics scripts and workflows, and work with our software engineering team to ensure the platform delights scientists worldwide. Duties and responsibilities include:
- Support Lumin clients with their use of Lumin Bioinformatics
- Provide subject matter expertise to the software development team
- Curate a growing library of scripts for common bioinformatics tasks and workflows
- Help move research tools to a production environment
- Document workflows and common tasks
- Help manage custom analysis projects for clients
Knowledge, Skills & Abilities
- MS or Ph.D. in Bioinformatics or related degree
- Significant experience with both R and Python
- Experience with NGS pipelines, particularly Nextflow
- 5+ years of professional bioinformatics experience
- Experience with Jira, AWS, Git, New Relic, Selenium desired
- Excellent communication skills
This description reflects management’s assignment of essential functions, it does not proscribe or restrict the tasks that may be assigned.
Champions Oncology is an Equal Opportunity/ Affirmative Action Employer. We celebrate diversity and are committed to creating an inclusive environment for all employees.
Read more here: Source link


text:
Choose your preferred language. We speak English (UK) and 41 other languages.
Most often used by people in the United Kingdom
Choose this apartmentSelf-catering with all the facilities you need
Via Croce Rossa 4, Chiaia, 80122 Naples, Italy – Show map
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591 properties in Naples
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189 verified reviews
The staff speak English
Residence Appartamenti Mergellina's staff speak: Italian, French, Spanish, English, German
Booking is safe
When you book with us your details are protected by a secure connection.
Canoeing, Hiking, Sauna, Beachfront, Bikes available (free), Library
Food & Drink
Bar, Breakfast in the room
Private parking is possible at a location nearby (reservation is not needed) and costs EUR 15 per day.
Shuttle service (surcharge), Airport shuttle (surcharge), Luggage storage, Concierge service, Babysitting/child services, Laundry
Safety deposit box, Non-smoking rooms, Family rooms, Lift, Heating, Air conditioning
Italian, French, Spanish, English, German
09:30 - 21:00 hours
Until 11:00 hours
Cancellation and prepayment policies vary according to apartment type. Please enter the dates of your stay and check the conditions of your required room.
Children and extra beds
All children are welcome.
One child under 5 years stays free of charge when using existing beds.
One older child or adult is charged EUR 10 per person per night in an extra bed.
The maximum number of extra beds in a room is 1.
There is no capacity for cots in the room.
Supplements are not calculated automatically in the total costs and will have to be paid for separately during your stay.
Pets are not allowed.
Cards accepted at this property
Hover over the cards for more info.
Based on 189 reviews
Value for money
Show reviews from:
11 April 2015
The bed was a bit hard and the stairs in the loft was a bit akward if you have a husband who is 6 foot tall and a child. But other than that it was great.
The apartment was 10 minutes walk from Piazza Amadeo metro, and 15 minutes walk from Mergellina metro station. So you have two choices. The staff were friendly and helpful all of them. They make sure that your stay is great. The apartment has a kitchen and good wifi. Its clean as they clean it everyday. We will definiteley stay back incase we go to naples. And by the way go to Chiaia its near the apartment with great food and drinks. Very affordable 2 euros for a beer in the kebab place on the corner of Louis Vuitton.
25 October 2014
1. The apartment was dirty and shabby (unlike the photos they provide), especially the bathroom 2. The windows look onto the busiest street, even at night (they don’t say anything about this on their page) - virtually impossible to sleep because of noise 3. In a centimeter-high layer of dirt under the bed there was a USED CONDOM WRAPPING!! What an outrage!
23 October 2014
The actual city location of the apartment wasn't made clear when booking, as it's not in Mergellina. The apartment is facing onto a very busy road and therefore there is loud traffic noise. The upstairs sleeping/bed area is very cramped.
Friendly and helpful staff. Fairly Central location. Near to public transport. Local shops good for cheap food. Fair price for Italy.
21 October 2014
The people next door were very loud so I could not sleep during the night. I could hear neighbours talking and walking clearly.
Hotel owners are friendly and nice
14 October 2014
Very nice personal.
8 October 2014
The apartment was very nice and with a lot of space in one of nicest areas of Naples, called Chiaia. It was clean and for us with two children perfect. The staff is absolutely fabulous and very welcoming. It's close to the Metro and close to the nice promenade on the seaside. Everything was great! We will come again!
25 September 2014
Pillows/mattress - awful, almost useless with only option to use dirty spare pillows without pillowcases that were in the cupboard. Bad nights' sleep on very hard and lumpy mattress. No optional throw on bed, just a sheet. Dirty Floor - the floor was filthy, meaning that walking around barefoot after a shower made feet black again within minutes! Towels - only 1 bath towel and 1 hand towel left per person, for 6 nights. Noise - noise was expected with the busy roads, and the apartment was on a very busy main road. However, the double doors did little to shut out the noise. Shower - only gave hot water for a matter of minutes meaning that shower time had to be done as quick as possible before it went cold.
It's a lovely little apartment. Ciro was very welcoming and eager to help although we weren't able to get a response by phone with one of our requests so therefore didn't contact any further regarding other issues (bad pillows) Very central location not far from the Archaeological Museum. Nice bathrooms. Wi-fi. Easily arranged personal airport transfers.
25 September 2014
Bad smelling toilet room, dirty bed.
8 September 2014
Area was rough and no parking as stated. We had to pay at local parking garage 15 euros per day. Didn't feel safe in area. Very busy and run down area. Lots of rubbish out front of building that the B and B is in which is like flats or private apartments.
Clean and modern, the staff were really friendly and helpful.
4 September 2014
The appartment manager, Ciro was excellent! Helpful and welcoming. Always there to answer any questions and offer information on Naples. The appartment was clean and of a good size. The area was fine and just a few minutes from the sea front.We loved our time in Naples and at the appartment! Thank you
29 August 2014
We had to ask for fresh towels some times. I'm not sure if they normally do it automatically or if there's a specific way of indicating you like fresh towels! We got them by asking in the end so it wasn't anything major! Definitely didn't put us off coming back!!
Firstly, the staff were incredibly helpful. The manager Gaia gave her number to us so we could get in contact at any time, which was very helpful in recommending places to see and eat. Ciro manned the office most days and again was incredibly helpful on our trip (his English is better than he gives himself credit for!). The apartment was great for the price, petite but good for 2 people. A great advantage having a kitchenette if you fancy saving a little money some nights! Although, the price was so good, you probably won't have to! The location was great! Far enough from the main Neapolitan hustle and bustle so you can get some rest but also very close to everything you want. It was a few minutes walk from the local shops and restaurants, a few minutes from the seafront and a short walk to the nearest Metro station to get to other parts of the city. All in all, a great place to stay at if you want to explore Naples!!
26 August 2014
We really enjoyed our stay at this apartment! Everything was as described on the site. The staff was very helpful and attentive to our needs. Lots of thanks to Ciro for his assistance and amazing hospitality!
22 August 2014
Very basic (but clean) Needs modernising and redecorating Had to pay cash, as credit card machine not working Walking up 4 flights of stairs when we had no 10 cent coins for the lift!
Staff all very helpful and tried very hard to make our stay a good one Not far from Metro station (10 mins) Close to sea/beach (5 mins) Not far from amenities and restaurants Felt 'secure' at night as main outer courtyard doors locked
11 August 2014
Could've had a few more basic facilities in the apartment like a full length mirror, tea/coffee, a kettle.
Layout location safety staff light and airy air conditioning
15 July 2014
No proper directions to get there... Its in back street. Staff knew nothing, maybe because it was Sunday so proper staff was not there.
Location, cleanliness, space.. Very close to US embassy and mergellina. Good eating places are nearby.
7 July 2014
- never met the owners - had to wait about 30 mins to check in (no answer at the door when we rang the bell)
We got to Naples and found the place fairly quickly (google maps helps). The rooms were very clean and very comfortable. TV and aircon worked fine. Oddly enough we never met the owners, just the manager once who wa lovely. It's a very close walk to the port and restaurants. Loved the place
1 July 2014
10cent for the lift up and down was a bit of a piss take, otherwise everything was good
I liked everything about the accomondation. ciro the manager is such a wonderful human being. he made sure we were comfy as soon as we arrived and for each day we stayed, it became more like home. ciro is one of the best people you could ever wish to meet. He made sure we had everything and I wouldn't hesitate to go back there when we visit naples next. I strongly recommend this apartment.tell ciro Ed said he is a rock star
29 June 2014
Be careful which apartment you get as ours was quite far from all things central, a good half an hour walk if not more and all up hill on the way back. Our apartment could do with a bit of an upgrade, it's obviously one of the older ones.
Ciro couldn't have been more accommodating and the apartment was fine for the 2 nights we stayed for. Clean and comfortable.
13 June 2014
people working in the hotel were very welcoming, attentive and ready to provide their assistance.
10 June 2014
No air conditioning or reasonable ventilation made the room very warm.
The person we had the most contact with was very helpful.
10 June 2014
The very pleasant apartment, located just outside the historic centre of Naples in the quiet and safe street. The apartment offers you comfortable conditions and friendly atmosphere. You will spend inspiring time there.
1 June 2014
We couldn't figure out how to work the radiator in the bathroom, and we like a warm towel when we get out of the shower (doesn't everyone?!). Clearly, i have survived that ordeal ;)
The apartments have clearly just been renovated to an extremely high standard, with a new kitchen and bathroom. The apartments are so comfortable and really are a home from home. The staff are wonderful and very welcoming, especially Ciro who is so friendly, but only speaks a little English :) The apartment is in a very nice neighbourhood with some gorgeous architecture and is ideally located; only a 4 minute walk to the nearest metro stop. A 4 minute walk in the opposite direction takes you down to the sea to some fantastic views and the promenade. A 30 minute walk along the promenade takes you to Castle st. Angelo, a castle out in the sea, and another 5 minutes from there lands you in piazza del plebiscito at the bottom of the main shopping street, via Toledo. A 10 minute walk towards Chiaia from the apartment lands you in the mid of a huge array of restaurants and there are two good sized supermarkets close to the apartment. I can't quite believe how good the price is considering how amazing this place is! I wouldn't be surprised if they increased the price TBH!
27 May 2014
The flat is located just a few steps from the waterfront gardens and the seaside promenade, nearby many lively bars, restaurants and other lively spots, nevertheless the building is very quiet and peaceful. The apartment is tidy, airy, properly-lighted and spacious - for a studio; plus it is very well furnished and fully equipped.
13 May 2014
The apartment is hidden behind the maze of confusing streets and you cant reach there without using online maps and superb driving skills. There was no one at the reception, you have to pay on your own for parking. In my 2 days of stay there was no breakfast, no drinking water. I would advise people to avoid this place, you might get something more reliable at similar prices.
10 April 2014
decor, noise, freezing cold.....
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domain: travel


U.S. Rep. Jackie Walorski (R-Ind.), a pro-life leader in Congress, today voted in opposition to the so-called Women’s Health Protection Act and the Ensuring Access to Abortion Act.
“At Speaker Pelosi’s urging, House Democrats once again voted to greenlight abortion at any time in pregnancy, anywhere in the country, and bankrolled by American taxpayers. Abortion on demand is a direct affront to pro-life values and Americans’ conscience rights,” said Congresswoman Walorski. “This radical anti-life agenda goes beyond Roe v. Wade to not only encourage – but also celebrate – abortion and the destruction of life. Democrats’ extreme abortion priorities are wildly out-of-touch with Americans’ values. As pro-abortion activists push dangerous abortion schemes – including unfettered access to abortion by mail – we must double down on our mission to protect women and children’s lives.”
Earlier this week, Congresswoman Walorski joined Family Research Council’s Washington Watch with Tony Perkins to preview these votes. Watch more here.
Walorski represents the 2nd Congressional District of Indiana, serving as a member of the House Ways and Means Committee and the Ranking Member of the House Ethics Committee.
Article provided by Rep. Walorski’s office


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Celebrating Adorably Cute Baseball Players: Where Skill Meets Charm
In the world of sports, athleticism and charm often go hand in hand. Baseball, a sport steeped in tradition and passion, is no exception. Beyond their impressive skills on the field, cute baseball players capture the hearts of fans with their endearing smiles, charisma, and genuine love for the game. In this article, we explore the appeal of these players who bring a delightful dose of cuteness to America’s pastime.
The Allure of Charm and Talent
Cute baseball players possess a unique blend of athleticism and undeniable charm. Their infectious smiles and friendly demeanor add a layer of relatability that resonates with fans of all ages. These players prove that being cute doesn’t overshadow their skill; rather, it enhances their appeal both on and off the diamond.
Icons of Approachability
Cute baseball players create an approachable atmosphere that bridges the gap between the fans in the stands and the players on the field. Their interactions with supporters, from signing autographs to taking selfies, create unforgettable memories for fans who feel a personal connection with their favorite players.
A Heartwarming Fan Base
Cute baseball players attract a dedicated fan base that’s as passionate about the players’ personalities as they are about their athletic prowess. Social media platforms light up with hashtags dedicated to these players, and fan clubs spring up to celebrate every home run, amazing catch, and charming post-game interview.
Balancing Athletics and Charisma
While their cuteness may garner attention, cute baseball players are serious athletes who dedicate themselves to honing their skills and contributing to their team’s success. Their on-field achievements are equally impressive, demonstrating their commitment to the sport they love.
Fans cherish the memorable moments created by cute baseball players. Whether it’s a player sharing a smile with a young fan, participating in community events, or celebrating a victory with exuberance, these moments stay etched in the hearts of supporters, enhancing their bond with both the player and the game.
Breaking Stereotypes and Inspiring
Cute baseball players challenge stereotypes by proving that strength, determination, and charm can coexist. Young athletes aspiring to reach the big leagues find inspiration in these players, who demonstrate that passion, hard work, and a positive attitude are the keys to success.
Spreading Joy Beyond the Diamond
Cute baseball players don’t just spread joy on the field; their positivity and warmth extend to their interactions off the diamond as well. They visit hospitals, engage in charitable initiatives, and use their platform to make a positive impact on their communities, leaving an indelible mark beyond the confines of the ballpark.
The Power of Role Models
Cute baseball players are more than just role models for their skills; they’re role models for being genuine, kind, and relatable individuals. Their authenticity resonates with fans, who are drawn to their personalities just as much as their performances.
A Love for the Game and Life
At the core of their appeal, cute baseball players exude a love for both the game and life itself. Their joy and enthusiasm are infectious, reminding us all to embrace the simple pleasures and celebrate every moment with a smile.
Cute baseball players bring an irresistible blend of skill, charm, and authenticity to the world of sports. Their ability to captivate fans through both their athletic feats and their endearing personalities makes them a beloved presence on and off the field. These players embody the heart and soul of baseball, reminding us that while the sport may be about competition, it’s also about forming connections, making memories, and celebrating the love of the game in the most adorable way possible.

domain: game


text: – In this study, Zinc Telluride (ZnTe)-based solar cells, which are metallic dichalcogenide materials, are used as a solar cell absorbent with the formation appropriate for solar cell use. The data has been analyzed by SCAPS-1D structures software. The replacement of Cadmium Sulfide CdS (buffer) layer by other green and save suitable materials has been investigated. The substituted buffer layers have been ZnSe, ZnS, CdSe, and In2S3. The higher device performance efficiency parameters have been found out when using CdS and ZnSe as buffer layers. SCAPS-1D shows that the optimal p-n junction device eff]iciency parameters have been achieved when the ZnTe (absorber) layer thickness is between 1200-1500 nm, while the ZnSe (buffer) layer thickness is between 20-60 nm, and the thickness of ZnO:Al (window) layer is 25 nm. The results of the simulation provide important hints that may enhance the performance of the cell with empirical studies useful in practical implementation. © 2021 The Authors. Published by Praise Worthy Prize S.r.l.

domain: design


“Never can say goodbye. No no no no, I Never can say goodbye.”
Gordon, who has appeared in ‘Spider-Man 3,’ ‘Serendipity’ and ‘Frost,’ was found dead on Wednesday. She hung herself following a friend’s recent suicide.
“I’d like to offer an apology to remarks I made recently…. most people understood that this was a joke…. However, I do apologize to anyone who took offense.”
GABRIELA-USA is calling for a public apology from Alec Baldwin who joked on The Late Show about ordering himself a mail-order bride from the Philippines.


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Reinvigorate your walls with the Light-Blue Metal Wall Planter by Stratton Home Decor. This contemporary hexagon shape planter is hand-painted in a modern hue that complements any decor. Toss a bunch of colorful faux flowers or an artificial ivy plant into this sleek planter that measures 9.00" W x 4.00" D x 10.25" H and create an eye-catching garden on any interior wall.


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What is the deductible on your car insurance?
Your car insurance deductible is the amount of money you agree to pay out of your pocket before the insurance company will pay towards a claim. For example, let's say you have a claim and it will cost $3,500 to fix your car. Your will pay the body shop $500 and the insurance company will pay the body shop $3,000. The higher your deductible, the less expensive your insurance costs. This is because you will be paying more out of pocket when you have a claim. There are two types of deductible on your auto insurance, a comprehensive deductible and a collision deductible.
What is a typical auto insurance deductible?
The most common deductible we see insureds carry is a $500 comprehensive and $500 collision deductible. Since comprehensive claims cover accidents that typically aren't your fault, like hail, theft, or vandalism, some people like to carry lower comprehensive deductibles like $100 or $250 while keeping their collision deductibles higher.
Still have questions? Give the expert agents at Pearson & Company Insurance a call at 601-482-6699. Want to find out how changing your deductible impacts your rates? We would be glad to quote you with different options- fill out this quote sheet and we will be in touch.

domain: finance


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Haifa Abdullah Alharbi, a Saudi businesswoman in Abu Dhabi, said the country is on the right path to modernisation.
Saudis in the UAE have praised their country’s modernisation drive, including boosting gender balance, as it celebrates its 90th National Day today.
Haifa Abdullah Alharbi, a Saudi businesswoman in Abu Dhabi, said the country is on the right path to modernisation. “The Saudi leaders have issued laws that empowered women and bridged the gender gap. Women are now feeling strong and empowered because they have equal opportunities with men,” she said.
Women now have access to good education and can obtain all government documents they want without the consent of a guardian unlike before, she added.
“Women have been allowed to drive cars, something that has eased family life. Also, Saudi Arabian women are now in various jobs unlike before. Women have been allowed to practise the legal profession and are now lawyers and advocates,” said Alharbi.
In 2017, the Custodian of the Two Holy Mosques, King Salman bin Abdulaziz of Saudi Arabia, ordered that women be allowed access to government services such as education and healthcare without the need for consent from a guardian. He also issued a decree allowing women to drive, lifting the world’s only ban on female drivers.
Moutaz Ozjan, 24, a media student at the University of Sharjah, said a lot of positive changes have been witnessed in Saudi Arabia over the recent years. “Many jobs have been created for the youths. Quality education and healthcare has been extended to all Saudi nationals, thanks to the relentless efforts by Saudi Crown Prince Mohammed bin Salman bin Abdul Aziz, Deputy Prime Minister and Minister of Defence.
“We are witnessing tremendous growth in tourism as the country has opened up and many changes have been made to the entertainment sector. New cinemas are opening up in Riyadh. Things are just getting better day by day.”
Effa Al Dabbagh, a designer, said she was “especially excited” about the support and opportunities created for the youth, and in particular, the creative industries.
“The leaders have created job opportunities for the youth – a clear signal that the country is on the path to modernisation,” she said.
‘We feel at home here in the UAE’
Moutaz Ozjan, a student, said the brotherly ties between the UAE and Saudi Arabia have enabled citizens of both nations feel at home in either country.
“When I am here, I feel at home. I don’t miss my home country because I am treated like a son here. All Emiratis I meet treat me like a brother,” he said.
For Effa Al Dabbagh, a designer, the UAE has been a home away from home. “Thirteen years ago, I started my Effa brand of Abayas here, and have always felt supported and honoured by my Emirati brothers and sisters. This is just one example of the brotherly ties between the UAE and Saudi Arabia.”
Click here to read more news from @khaleejtimes

domain: sociology


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FG has resolved to ensure rigorous and strict enforcement of certain interventions following recent surge in COVID-19 cases.
Eswatini Prime Minister, Ambrose Mandvulo Dlamini has died after contracting COVID-19 four weeks ago.
The Governor of Lagos State, Babajide Sanwo-Olu has tested positive for Coronavirus.
The Minister said that the ministry plans to increase its capacity to battle the disease through personnel supervision.
The ECOWAS COVID-19 daily update shows Nigeria placed on the 6th position as regards the recovery rate.
A new study suggests that the Coronavirus may have spread from China to other parts of the world earlier than previously thought.
The lowering of guard and change in weather conditions have been suggested to have contributed to the spike in COVID-19 cases.
Donald Trump's doctor, Sean Cornley has admitted that the President was given supplementary oxygen.
Twitter has warned users that tweets wishing Trump dead will be removed from its platform.
Madrid has headed back into lockdown after rising caseloads of COVID-19 caseloads.


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I’ve not been spending much time making art these days. I miss it, but I’m no good at multi-tasking so I accept my limitations and instead I’ve thrown myself into a period of deep work, creating The Thing.
The Concertina Retreat from Lockdown 1 was hastily thrown together. It was the best it could be given the short space of time and the limited resources, both in terms of making do with what I had available during confinement and my limited techie skills. But hey, it was free so it was good enough.
After then, I percolated an idea of how to deepen and improve the basic retreat into something expansive, nurturing and genuinely helpful. And here we now find ourselves in France’s Lockdown 2, and I feel pretty sad for many reasons. One is that my Big Dream of having retreats here in the crooked house, lots of books, vegan cakes and a squashy green sofa seems to slip further and further away from me …
… So I’m just going to have to bring the books, cakes and squashy sofa into the virtual realm.
Thus The Thing I’ve been working on is almost ready to fly off into the world. For ages I’ve been referring to it as the accordion retreat but I always knew that was a working title. Eventually, if I was patient, something would pass by my way and I would be ready to catch hold of it.
I love Rebecca Solnit and her book The Faraway Nearby passed by my way and I caught it. It seems to capture perfectly the spirit of this new retreat-cum-workshop.
It feels like I’ve been working on this for ever, it’s deep work but I love doing it – drawing on conversations and questions I’ve had, valuable feedback from the Concertina Retreat and my own personal, nurturing process.
This is so exciting and inspiring, while also calm and thoughtful. Going inward, outward, up and beneath; uncovering all the hidden bits. You will discover that you can actually feel ok with imperfection and if you can draw ‘properly’. Instead, we use observation as a doorway into the realms of what is possible, by providing the tools you need to cultivate your imagination, intuition and playfulness…
UPDATE: FARAWAY NEARBY IS NOW AVAILABLE. DETAILS ARE HERE


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Sneak Peek: Lisa Vanderpump Doesn’t Invite Adrienne Maloof To Her Party; Adrienne Says Lisa Is Being A ‘Mean Girl’
In this sneak peek footage from the all new season of The Real Housewives of Beverly Hills, Adrienne Maloof is not invited to a party by Lisa Vanderpump and feels really hurt! Check out the sneak peek below
You can’t help but feel bad for Adrienne. Do you think it was wrong of Lisa not to invite Adrienne?


OKLAHOMA CITY (KFOR) – Charges have been filed against a man accused of causing a lockdown at Mercy Hospital earlier this month.
Officers were called to Mercy Hospital after police say 22-year-old Cristo Martinez was trying to get into the labor and delivery wing.
“A woman was up there. The two had been in a relationship. He was wanting to be in the room. She did not want him in there, made it very clear she didn’t want him in there. He refused to leave the premise,” MSgt. Gary Knight said at the time of the incident.
Court documents say Martinez “became enraged and broke through the heavy security door, damaging the door and lock assembly.”
The amount of damage was about $3,300.
“He didn’t shatter the glass, but he basically broke the way the door functions,” Knight said.
Records say he went to the family’s room and was “yelling and being belligerent.”
Security guards de-escalated him, and he left.
After leaving, he “posted a photo to his Snapchat with him holding a gold handgun in his lap with the text ‘let’s see if I can go in this time.'”
He was arrested after officers went to his home. He is now facing three counts: destruction of property, threatening to perform an act of violence, and breaking and entering.
“It was very frightening for some of the people in there, but in the end, everyone ended up okay and he ended up in jail,” Knight said.


1 of 1 people found this review helpful
By David on
Vehicle2006 Lexus GS 300 4dr Sedan (3.0L 6cyl 6A)
ReviewI used to drive a lot for business. Problems started when the warranty expired. It was like clock work! First the cruise control went dead. Took it to the neighborhood Lexus dealer. They kept my car for a 4 hours and could not find the problem. They were going to charge me $400.00 ($100 per hour) for diagnostics. Naturally, I didn't. Then the speedometer went crazy, going back and forth to not working at all, the car wouldn't shut off. Then nothing, everything would work fine the next day. The dealer told me it's a sensor problem and would cost $ 2000.00 + to repair!
Best FeaturesBody design, smooth ride, and handling. It's a great car minus the electronic issues!
Worst FeaturesI feel the electrical components in this model, being that it is the 1st year of the new body style and most likely newly designed parts for this model, there was a lack of testing on the part of the car maker.
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Any kind of kidney disease can be painful, cause serious complications, and require great deals of money to treat. This can make many patients unable to work and earn the income that they are used to. Because of this, the Social Security Administration (SSA) offers disability benefits for Kidney Failure that can be used to deliver needed financial resources.
Evaluation for Social Security Disability
The Social Securrity Administration (SSA) BlueBook lists conditions that may make someone eligible for disability benefits. It also goes into the symptoms and test results required for the condition to be a disability. Meeting or exceeding at least one criteria is typically needed to qualify for benefits. The Blue Book puts kidney failure under Genitourinary Disorders in Section 6.
Social Security Disability Insurance and Chronic Kidney Disease
For your kidney disease to be considered a disability, you must qualify for one of the below:
- Your kidney disease is chronic and requires the use of dialysis
- You have chronic kidney disease and have had a kidney transplant in the last year
- Your chronic kidney disease has led to at least three hospital visits in the last year
- Your kidney disease results in reduced kidney function that causes any of the things below
- Renal osteodystrophy
- Peripheral neuropathy
- Fluid overload syndrome
- Anorexia with weight loss by a BMI of 18 or less
Kidney Disease or Failure and Nephrotic Syndrome
Another situation that might make you capable of getting disability benefits for kidney failure is known as nephrotic syndrome. Essentially, this means that protein is being lost during urination. There are specific requirements for this syndrome to be approved for benefits. The syndrome needs to have been seen twice in tests in the same year but at least three months apart. It also must have resulted in skin swelling for 90 days or longer. To ensure you meet all requirments it is adviseable that you contact an attorney for disabled such as the Disability Help Group Arizona.
Kidney Failure or Disease Complications
Several different complications can occur from kidney disease. Those who have not had a kidney transplant and are not undergoing dialysis need to prove a few other things. Either you must have low serum creatinine levels over 90 days, a clearance level of 20ml or lower per minute, or one of the following complications:
- Chronic fluid overload syndrome accompanied by anorexia, diastolic hypertension, or vascular congestion
- Sensory or motor nephropathy
- Renal osteodystrophy
Medical Evidence for Social Security Disability (SSDI) and Kidney Failure
The SSA uses a particular set of medical guidelines, called the Blue Book, to determine whether a person‘s case qualified them for disability benefits. In some cases, even though you have kidney disease, the SSA will decide you can still work and should not receive benefits.
However, if the condition does make it impossible to work, the SSA will ask for a residual functional capacity form. A doctor fills out and submits the form to ensure the information you filled out in your application is accurate. As the doctor knows about your condition and takes care of the treatments, they also understand what you are capable of and what you aren‘t.
Kidney Failure or Disease Transplants
An individual who has had a kidney transplant will automatically get 12 months of disability. However, once that time is up, Social Security will decide whether the person continues to be eligible based on residual impairments. When it comes to a kidney transplant, the following items are considered:
- Systemic infections
- Kidney rejection situations
- Side effects of corticosteroid treatment
- Renal infection treatment
The SSA will also consider whether a person no longer has signs, symptoms, and lab findings showing kidney failure when deciding whether to approve or reject an application for benefits.
Kidney Failure or Disease Functional Limitations
Even when kidney disease doesn‘t meet all the listing requirements, functional limitations play a part in deciding on benefits. This involves looking at symptoms and whether they preclude someone from working.
If you have kidney disease or kidney failure, Disability Help Group Arizona offers a free disability evaluation. Our team can also help you work towards a claim to get the benefits that you deserve.
Disability Help Group Arizona
18444 N 25th Ave, Suite 420
Phoenix, AZ 85023

domain: medical


A central Alberta animal rescue organization is hosting an online telethon to support the wellbeing of its animals.
Saving Grace Animal Society is hosting its second annual telethon March 26 to raise money so the society can proceed with plans to construct a new veterinarian clinic this year at its facility in Alix.
“We have been very fortunate with our clinic fundraising efforts so far, and we are so close to hitting our goal that it’s getting very hard to contain our excitement,” said Saving Grace co-executive director McClughan, who will co-host the Facebook Live broadcast of this year’s telethon from Blindman Brewing in Lacombe.
“The Betty White Challenge earlier this year in memory of prolific animal lover and all around celebrity sweetheart Betty White provided an unexpected but very welcome boost to our fundraising goals.”
More than $100,000 was raised from that campaign, helping to push the society’s fundraising total even closer to their $500,000 goal.
“Although the finish line is in sight, we still have a way to go,” she said, adding this year’s telethon fundraising goal is $75,000.
A new vet clinic would allow the animal rescue and rehabilitation team to save time and resources to better serve the animals in their care.
“Last year our medical bills averaged $65,000 per month and we have projected savings of 50-60 per cent by having our own veterinary clinic on site and ready to serve the animals we take in,” said McClughan.
The organization has experienced tremendous strain in keeping up with rescues, animal overflow and higher-than-normal medical trauma cases in recent months, McClughan added.
“A vet clinic is the next step in improving our ability to follow our vision: to rescue, rehabilitate and re-home any animal in need. Rescue them all, big or small.”
Saving Grace is putting out a call for families, individuals and businesses to register as “Dream Team,” with up to five members.
“Leading up to the telethon, our dream teams will be encouraged to reach out to their friends, neighbours and networks to help raise funds for Saving Grace and have an opportunity at prizes too,” said McClughan.
“Teams can raise the fund however they wish to and the top teams will be awarded prizes by the society including branded swag and private time with the animals.”
For more information, visit www.savinggracecanada.com/store/dreamteam.


Quantum Discord of Certain Two-Qubit States
Quantum discord is an effective measure of quantum correlation introduced by Olliver and Zurek. We evaluate analytically the quantum discord for a large family of non-X-states. Exact solutions of the quantum discord are obtained for four parametric spaces of non-X-states. Dynamic behavior of the quantum discord is also explored under the action of the Kraus operator.
KeywordsQuantum discord Quantum correlations Bipartite quantum states Optimization on manifolds
The corresponding author gratefully acknowledges the partial support of NSFC grants 11426116, 11501251 and 11871325 during this work. The third author is supported by NSFC grant 11531004 and Simons Foundation grant 523868.


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Cannabis advocates around Minnesota recently celebrated a small victory in their fight for broader access to commercial marijuana across the state. On Thursday, June 2nd, Gov. Tim Walz (D) signed a bill that allows hemp businesses to legally cultivate and commercialize marijuana products.
In an April 2022 tweet, Walz said, “It’s time to legalize adult-use cannabis and expunge cannabis convictions in Minnesota.” This legislation seeks to create provisions for CBD-infused foods and beverages, as Walz mentions in his tweet.
Despite his recent comments on the legalization and decriminalization of marijuana in the state, Walz chose not to comment on the cannabis provisions specifically in a recent press conference to address the bill. The governor claimed that the legislation targeted issues brought on by the current opioid crisis.
Hemp, which was legalized in a 2018 Minnesota Farm Bill, is now approved for commercialization in the state, according to the recent bill. This legislation accounts for an issue with the 2018 bill, which made little to no provisions for the marketing of hemp.
The new legislation prohibits the sale of cannabinoids to anyone younger than 21. Additionally, the bill introduces new requirements for how hemp products are tested, packaged, and labeled.
In terms of CBD provisions, the bill allows all hemp-derived cannabinoids to be sold commercially if they are below the federal limit of 0.3 percent THC. This includes all foods, topical treatments, beverages, and other CBD products. The legal limit for food and beverages infused with CBD is 50 mg THC per package and 5 mg per serving.
This will remain the federal limit for all THC compounds, which have, until now, been given little legislative provision. With this bill’s signing and the 2018 Farm Bill, federal allowances now extend to psychoactive substances such as delta-8 and delta-9 THC.
Although broader marijuana policy reform has not been achieved in the Senate, this bill and others like it represent a genuine step in the journey towards marijuana legalization. Democratic leaders around the country continue to work towards a vote on more widespread marijuana legalization.
Similar measures have been introduced to expand Minnesota’s approach to marijuana decriminalization. Advocates have attempted to include non-flower forms of cannabis in recent legislation, meeting difficulties and opposition along the way. The passing of the recent bill indicates a minor success as cannabis advocates look toward more significant reform in the 2023 session.
Minnesota Democrats have faced many challenges in their efforts toward legalization, including an attempt to highlight opponents to Democratic support on the ballot by changing the name of the third party to one that would appeal to the conservatives who would seek to withhold their votes for pro-marijuana legislation in upcoming elections.
In years past, Gov. Walz has been less aggressive in his budget request for marijuana policy reform, with more of his efforts taking shape in the form of expanded access to cannabis products.
In 2019, the governor signed a bill that worked to expand Minnesota’s approach to medical marijuana policy by giving patients access to medically-approved smokable hemp products. This was one of several of Walz’s efforts to implement reform ahead of legalization in the state.
In this session, however, the governor has worked to introduce a market for adult-use marijuana that aligns with the bill passed in the House earlier this year. In his January 2022 budget request, the governor included specific funding to make legislation like this bill a reality. Walz’s recent legislation is the product of a more protracted fight for broader marijuana reform in Minnesota.
The state has historically been more progressive in decriminalization and legalization; marijuana was decriminalized in 1976, and early allowances were made in 2014 for the use of medical marijuana, with expanded access to medicinal hemp products introduced in 2019.
A 2019 survey conducted by the Minnesota congressional chamber indicated that 56 percent of the state’s residents favor legalization. A more recent survey earlier this year suggested that this number has risen to 58 percent of Minnesota residents in favor of legalization.
In May of 2022, Minnesota Senate Minority Leader Melisa López Franzen (D) proposed a procedural mechanism that would create space for broader decriminalization and legalization. A similar companion to the mechanism passed in the House chamber last year. The proposal would have required a supermajority of 41 votes to pass, and despite López Franzen’s best efforts, it failed.
Minnesota House of Representatives Majority Leader Ryan Winkler has proposed similar legislation, most notably in the form of HF600, Minnesota’s cannabis legalization bill, which seeks to legalize and regulate cannabis products while simultaneously expunging most cannabis convictions.
Winkler has sought to expand commercial access to cannabis products in many additional ways, claiming that each bill lawmakers propose is the “product of hundreds of hours of work involving thousands of people’s input, countless hearings, and public listening sessions.”
In an interview with the RAND Drug Policy Research Center, Winkler addressed the challenges associated with legalizing marijuana products and the disparities in equitable access across the board.
“The criminal justice system approach to dealing with cannabis creates more harm than cannabis does itself,” Winkler said. “We can do all of the good to address the harms of cannabis without incarcerating people, without using the criminal justice system to lock people up, or provide collateral consequences that can last a lifetime for a product that most people think should be legal.”
In 2020, Winkler commented on a vote to increase access to marijuana products, noting that if Minnesota Senate Republicans do not pass legislation, he hopes the issue will be in the hands of the voters on the 2022 ballot.
Whether such a vote will be on the ballot is unclear. Still, legislation like last week’s landmark bill is key to expanding commercial access to marijuana in Minnesota.

domain: law


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A south-west business owner says he has been left with no choice but to walk away from the caravan park he invested everything in.
Lloyd Phelan will close Port Fairy's Catalina Caravan Park and Take Away for good at the end of August.
Mr Phelan and his wife Janet, who took over the caravan park 12 years ago, are leaving Port Fairy and hope to secure jobs.
"The business - since COVID - it hasn't come back," Mr Phelan said.
"You can't keep going when you're not making any money."
Mr Phelan said he was bitterly disappointed he had to walk away from the business.
"We're just walking away," he said.
"We're not even trying to sell it."
Mr Phelan said it had also become increasingly difficult to find staff because of a lack of affordable housing options in Port Fairy.
"We can't get staff," he said.
"We used to have people coming in all the time asking for a job but now there's nobody."
Mr Phelan said the COVID-19 pandemic had created a "perfect storm".
"We got some money from the government but it stopped and people haven't come back," he said.
"We opened (the take away shop) on Thursday and we took $42 for the day.
"It was a really good business before COVID but not anymore.
"The fuel prices are through the roof, so that doesn't help.
"Who is going to travel when the prices are so high?"
Mr Phelan said he wouldn't sell the business, even if he had an offer.
"I wouldn't wish it even on my worst enemy," he said.
"You just can't make money over a 12 month period - you can make money for three months but that's not enough to carry you for the whole year."
Mr Phelan said there were 14 tenants who lived permanently at the caravan park.
"We've given them the proper notice but I feel for them because there is nothing to rent in Port Fairy."
An online search for rentals in the town resulted in six listings, with a three-bedroom house the cheapest at $490 a week.
In recent months, south-west organisations say they have been flooded with requests for help from people seeking affordable housing.
Brophy Family and Youth Services housing support and linkages programs team leader Leah McDonald said it was an issue across the region.
"We have seen a large increase of people in the community who wouldn't normally be accessing homelessness services, contact us for support to obtain private rentals," she said.
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domain: economics