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IκB-ζ [inhibitor of NF-κB (nuclear factor κB) ζ] is a nuclear protein that is induced upon stimulation of TLRs (Toll-like receptors) and IL (interleukin)-1 receptor. IκB-ζ harbours C-terminal ankyrin repeats that interact with NF-κB. Our recent studies have shown that, upon stimulation, IκB-ζ is essential for the induction of a subset of inflammatory genes, represented by IL-6, whereas it inhibits the expression of TNF (tumour necrosis factor)-α. In the present study, we investigated mechanisms that determine the different functions of IκB-ζ. We found that co-expression of IκB-ζ and the NF-κB subunits synergistically activates transcription of the hBD-2 (human β-defensin 2) and NGAL (neutrophil gelatinase-associated lipocalin) genes, whereas it inhibits transcription of E-selectin. Reporter analyses indicated that, in addition to an NF-κB-binding site, a flanking C/EBP (CCAAT/enhancer-binding protein)-binding site in the promoters is essential for the IκB-ζ-mediated transcriptional activation. Using an artificial promoter consisting of the NF-κB- and C/EBP-binding sites, transcriptional activation was observed upon co-transfection with IκB-ζ and NF-κB, indicating that these sequences are minimal elements that confer the IκB-ζ-mediated transcriptional activation. Chromatin immunoprecipitation assays and knockdown experiments showed that both IκB-ζ and the NF-κB subunits were recruited to the NGAL promoter and were essential for the transcriptional activation of the hBD-2 and NGAL promoters on stimulation with IL-1β. The activation of the NGAL promoter by transfection of IκB-ζ and NF-κB was suppressed in C/EBPβ-depleted cells. Thus IκB-ζ acts as an essential transcriptional activator by forming a complex with NF-κB on promoters harbouring the NF-κB- and C/EBP-binding sites, upon stimulation of TLRs or IL-1 receptor.
- CCAAT/enhancer-binding protein (C/EBP)
- inflammation
- inhibitor of nuclear factor κB ζ (IκB-ζ)
- innate immunity
- nuclear factor κB (NF-κB)
- transcription
Cells alter gene expression profiles in response to environmental changes. The expression of genes is regulated not only by initiation of transcription, but also by processing of transcripts, transport to the cytoplasm and translation of mRNA. Nevertheless, the initiation of transcription is the major step controlling the expression levels for most genes, and precise regulation of the activities of transcription factors is vital to accomplish specific tasks in cells that are essential to maintain life.
The transcription factor NF-κB (nuclear factor-κB) plays key roles in the innate and adaptive immune systems as well as in apoptosis, cell proliferation and differentiation [1–3]. It consists of homo- and hetero-dimers of five subunits, p65/RelA, RelB, c-Rel, p50/NF-κB1 and p52/NF-κB2. All subunits bind to DNA through the conserved Rel homology domain. The p65, RelB and c-Rel subunits contain a transcriptional activation domain at their C-terminal regions, which is lacking in p50 and p52. In resting cells, NF-κB localizes in the cytoplasm via association with cytosolic IκB (inhibitor of NF-κB) proteins such as IκB-α, -β and -ϵ. Upon stimulation, the cytosolic IκB proteins are phosphorylated and degraded by the ubiquitin–proteasome system. The liberated NF-κB dimer translocates into the nucleus, where it binds to its cognate κB enhancer elements and interacts with the basal transcription machinery or transcriptional coactivators to stimulate gene expression.
The activity of NF-κB is also modulated in the nucleus [1,2,4]. NF-κB activation, in turn, leads to transcription of IκB-α, and re-synthesized IκB-α binds to the nuclear NF-κB to export it to the cytoplasm, thereby attenuating its activity. Reversible acetylation of NF-κB regulates the duration of its action in the nucleus via modulation of its interaction with IκB-α [5]. In addition, phosphorylation of p65 allows its association with coactivators such as CBP [CREB (cAMP-response-element-binding protein)-binding protein]/p300, while unphosphorylated nuclear p65 associates with a histone deacetylase in the nucleus and is unable to bind to DNA [6]. As well as the coactivators or histone deacetylase, other nuclear proteins can interact with NF-κB and modulate its activity positively or negatively. The transcription factor Twist [7] and glucocorticoid receptor [8] interact directly with p65 and repress p65-mediated transcriptional activation. Reportedly, c-myc [9], p202a [10] and prostaglandins [11] also negatively regulate NF-κB in the nucleus. On the other hand, FBI-1 (factor that binds to inducer of short transcripts 1) [12] or TLS (translocated in liposarcoma) [13] have been reported to augment the transcriptional activity of nuclear NF-κB. Bcl-3 also binds to NF-κB in the nucleus, but its effects on the activity of NF-κB remain elusive and appear to differ for different target genes [14–16].
During screening for genes that are induced by pro-inflammatory stimuli, we identified a nuclear protein with ankyrin repeats that is strongly induced in response to bacterial LPS (lipopolysaccharide), which stimulates TLR (Toll-like receptor) 4 [17]. This protein, designated IκB-ζ, is barely detectable in resting cells, but is induced not only by LPS, but also by other microbial components that stimulate TLRs such as peptidoglycan, bacterial lipopeptide and CpG DNA [17–19]. The induction is also observed upon stimulation with IL (interleukin)-1β, but not by another pro-inflammatory cytokine, TNF (tumour necrosis factor)-α [17,20]. The protein IκB-ζ was also identified by other groups as MAIL (molecule possessing ankyrin repeats induced by LPS) [21] or INAP (IL-1-inducible nuclear ankyrin repeat protein) [22]. In a recent study, we showed that the stimulus specificity of IκB-ζ induction is determined at the post-transcriptional level, but not at the transcriptional level, on the basis of the finding that the stability of IκB-ζ mRNA is specifically up-regulated by stimulation with LPS or IL-1β, but not with TNF-α [20].
At least two major IκB-ζ variants are generated by alternative splicing [17,21]. We designated the longer form IκB-ζ(L) and the shorter form IκB-ζ(S), which lacks the N-terminal 99 amino acids of IκB-ζ(L). Both forms of IκB-ζ are induced upon stimulation, and IκB-ζ(L) is the predominant form in macrophages stimulated by LPS [20]. In contrast with the typical cytosolic and constitutively expressed IκB proteins, IκB-ζ is localized in the nucleus, where it preferentially binds, via the C-terminal ankyrin repeats, to the NF-κB p50 subunit rather than to the p65 subunit [17].
Although IκB-ζ was initially characterized as a negative regulator of NF-κB by reporter analyses [17], subsequent studies demonstrated that IκB-ζ also acts as a positive regulator of NF-κB [23]. Analyses using GAL4-fusion proteins of IκB-ζ revealed that its N-terminal region exhibits transcriptional activation activity, after association with the NF-κB p50 subunit. Overexpression of IκB-ζ augmented IL-6 production in response to LPS, but inhibited TNF-α production, indicating specific target gene activity of IκB-ζ. IκB-ζ-deficient mice exhibited chronic inflammation in the skin and on ocular surfaces [19,24], suggesting anti-inflammatory roles for IκB-ζ. However, prominent phenotypes of IκB-ζ-deficient cells were observed when the responses of macrophages following stimulation with LPS were analysed [19]. Peritoneal macrophages from IκB-ζ-deficient mice exhibited severe impairment in production of a subset of inflammatory genes, represented by IL-6, in response to LPS. The impaired induction of the genes was also observed upon stimulation with other microbial ligands for TLRs as well as IL-1β, which elicit the induction of IκB-ζ. This indicated that IκB-ζ is indispensable for the induction of the subset of inflammatory genes activated through TLR/IL-1 receptor signalling pathways. As expected from the preferential binding of IκB-ζ to the p50 subunit [17] and the role of p50 in mediating transcriptional activation via IκB-ζ [23], the genes whose induction was dependent on IκB-ζ were not induced in the p50 subunit (NF-κB1)-deficient cells [19]. Interestingly, IL-6 production in the IκB-ζ-deficient cells was not affected on stimulation with TNF-α, which does not induce IκB-ζ [19].
Thus IκB-ζ exhibits dual roles in NF-κB-mediated transcription, but the mechanism that determines the opposite actions of IκB-ζ on different genes remains to be clarified. In the present study, we analysed the molecular mechanisms of the IκB-ζmediated transcriptional activation. We focused on hBD-2 (human β-defensin 2) and NGAL (neutrophil gelatinase-associated lipocalin), both of which are preferentially induced by stimulators of TLR/IL-1 receptor signalling pathways, rather than by TNF-α [25–30]. We found that the promoters of both genes are synergistically activated by co-transfection of IκB-ζ and NF-κB. Promoter analyses of these genes identified a cis element that is critical for the observed induction. ChIP (chromatin immunoprecipitation) assays and RNAi (RNA interference) experiments indicated that IκB-ζ acts as an essential transcriptional activator by forming a complex with NF-κB on the promoters with an NF-κB-binding site and the cis element, upon stimulation of TLRs or IL-1 receptor.
Cell culture, reagents and antibodies
The human embryonic kidney cell line HEK-293 and the human lung adenocarcinoma cell line A549 were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% heat-inactivated fetal calf serum, 100 units/ml penicillin and 100 μg/ml streptomycin at 37 °C under 5% CO2. Mouse bone-marrow-derived macrophages were obtained by culturing femoral and tibial bone marrow from sex- and age-matched wild-type (C57BL/6) and IκB-ζ-deficient mice [19] in the same medium supplemented with L929 cell-conditioned medium as a source of macrophage CSF (colony-stimulating factor). LPS from Escherichia coli 0111:B4 was purchased from List Biological Laboratories. IL-1β and PMA were purchased from Genzyme Techne and Sigma–Aldrich respectively. Anti-(human IκB-ζ) polyclonal antibody was raised as follows. A cDNA fragment for amino acids 189–718 of human IκB-ζ was subcloned into pGEX-2T (GE Healthcare) or pMALg [17], and the recombinant protein was expressed in bacteria as a GST (glutathione S-transferase)- or a MBP (maltose-binding protein)-fusion protein. Antiserum was raised following immunization of a rabbit with the MBP-fusion protein and was subjected to affinity purification using Affi-Gel-10 resin (Bio-Rad Laboratories) coupled with the GST-fusion protein. Antibodies against the NF-κB p65 subunit (C-20) and the p50 subunit (NLS) were purchased from Santa Cruz Biotechnology.
Expression plasmids were constructed by subcloning a DNA fragment obtained by PCR into pcDNA3 (Invitrogen) with or without an N-terminal FLAG tag. Reporter plasmids for E-selectin, hBD-2 or NGAL were created by subcloning a human genomic fragment amplified by PCR into pGL3-basic vector (Promega). The mutations introduced into the hBD-2 promoter were as follows: TGGGGTTTCC (NF-κB-binding site 1) to TCGGGTTTCC; GGCATTTTCT (NF-κB-binding site 2) to GCCATTTTCTT; TTTGCATAAG [C/EBP (CCAAT/enhancer-binding protein)-binding site 1] to TAACCATAAG. The mutations in the NGAL promoter were as follows: GGGAATGTCC (NF-κB-binding site) to AATAATGTCC; ATTGCCTCAC (C/EBP-binding site 1) to AAACCCTCAC; GTGCAGAAAT (C/EBP-binding site 2) to GTGCAGGTTT; CTTGCCCAAT (C/EBP-binding site 3) to CAACCCCAAT (mutated nucleotides are underlined in all sequences). Plasmid pNF-κB-Luc harbouring four tandem NF-κB-binding sequences (GGGAATTTCC×4) was purchased from Clontech, and the phRL-TK internal control reporter plasmid expressing Renilla luciferase was from Promega. Plasmid phBD-2-κB-Luc was generated by insertion of a double-stranded oligonucleotide containing two copies of the sequence spanning from −206 to −187 bp of the hBD-2 promoter (5′-AGGGATTTTCTGGGGTTTCC-3′) into the NheI and BglII sites of pNF-κB-Luc, resulting in replacement of the tandem NF-κB-binding sequences. All mutated plasmids were sequenced to confirm correct sequences and orientation.
Quantification of mRNA by real-time RT (reverse transcription)–PCR
HEK-293 cells (5.0×105 cells) were transfected with the indicated expression plasmids by the calcium phosphate method [31]. At 24 h after transfection, total RNA was extracted using TRIzol® (Invitrogen) according to the manufacturer's instructions. Bone-marrow-derived macrophages were stimulated with 20 ng/ml LPS for the indicated periods, and total RNA was extracted. A 5 μg sample of the total RNA was reverse-transcribed using an oligo(dT) primer and the ReverTra AceR reverse transcriptase (Toyobo). Real-time RT–PCR was carried out with the reverse-transcribed cDNAs using SYBR Premix Ex Taq (Takara Bio) by a Light Cycler (Roche Diagnostics). The primers used were 5′-GCCATGAGGGTCTTGT-3′ and 5′-AGCCCTTTCTGAATCCG-3′ for hBD-2, 5′-CAGTTCCGGGAAAGATCA-3′ and 5′-ATGGTGCTAATGTCAGG-3′ for human E-selectin, 5′-AGCGACGAGTACAAGATCCG-3′ and 5′-AGCTGCTCCACCTTCTTCTG-3′ for human C/EBPβ, 5′-GCGAGCGCAACAACATC-3′ and 5′-CGACAGCTCCACCAACTTCT-3′ for human C/EBPδ, 5′-TCGGAGTCAACGGATTT-3′ and 5′-CCACGACGTACTCAGC-3′ for human GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 5′-CGTTTCACCCGCTTTG-3′ and 5′-AATAAGAAGAGGCTCCAG-3′ for mouse NGAL, and 5′-GATGACCCAGATCATGTTTGA-3′ and 5′-GGAGAGCATAGCCCTCGTAG-3′ for mouse β-actin.
Luciferase reporter assay
HEK-293 cells (1.25×105 cells) were transfected with 100 ng of the indicated reporter plasmid together with 5 ng of the expression plasmid for the NF-κB subunit p65 or p50, 100 ng of the IκB-ζ(L) expression plasmid and 2.5 ng of the internal control plasmid phRL-TK using Lipofectamine™ 2000 (Invitrogen) according to the manufacturer's protocol. A549 cells (1.25×105 cells) were transfected with 100 ng of the indicated reporter plasmid together with 10 ng of the expression plasmid for the NF-κB subunit p65 or p50, 10 ng of the IκB-ζ expression plasmid and 2.5 ng of the internal control plasmid phRL-TK using Lipofectamine™ 2000. Cells were lysed 24 h after transfection. To analyse the responses to IL-1β stimulation, A549 cells (1.25×105 cells) were transfected with 200 ng of the hBD-2 reporter plasmids or 50 ng of the NGAL reporter plasmids, together with 2.5 ng of the internal control plasmid phRL-TK. At 24 h after transfection, cells were stimulated with 1 ng/ml IL-1β for 5 h, and the cells were then lysed. The luciferase activity was measured using the dualluciferase reporter system (Promega) according to the manufacturer's instructions. The transfection efficiency was normalized by the Renilla luciferase activity derived from phRL-TK. The data shown are the means±S.E.M. for duplicate samples, representative of at least two independent experiments.
ChIP assay
The ChIP assay was performed according to a described protocol (Upstate Cell Signaling Solutions) with some modifications. A549 cells (5×106 cells) were stimulated with 1 ng/ml IL-1β for 4 h, and fixed with 1% formaldehyde for 10 min at room temperature (25 °C). Cells were washed twice with ice-cold PBS, collected by centrifugation at 500 g for 5 min, and resuspended in the lysis buffer containing 50 mM Tris/HCl (pH 8.1), 1% SDS and 10 mM EDTA supplemented with 1× Complete™ protease inhibitor cocktail (Roche Applied Science) on ice for 20 min. The cell lysate was sonicated 10 times for 30 s with 1 min intervals using a Bioruptor (Cosmo Bio). Debris was removed by centrifugation at 15000 g for 10 min, and the supernatant was diluted 10 times with the buffer containing 16.7 mM Tris/HCl (pH 8.1), 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 167 mM NaCl, supplemented with 1× Complete™. Extracts were pre-cleared for 1 h with 80 μl of Protein A–Sepharose 4 Fast Flow (GE Healthcare) 50% slurry containing 0.2 mg/ml yeast tRNA (Ambion). Immunoprecipitation was performed at 4 °C overnight with 2 μg each of specific antibodies. Immune complexes were collected with 50 μl of Protein A–Sepharose 4 Fast Flow 50% slurry with yeast tRNA. Sepharose beads were washed sequentially in the following buffers: low-salt wash buffer (20 mM Tris/HCl, pH 8.1, containing 150 mM NaCl, 0.1% SDS, 1% Triton X-100 and 2 mM EDTA), high-salt wash buffer (the same buffer, but with 500 mM NaCl), LiCl wash buffer (10 mM Tris/HCl, pH 8.1, 0.25 M LiCl, 1% sodium deoxycholate, 1% Triton X-100 and 1 mM EDTA), and twice with Tris/EDTA buffer (10 mM Tris/HCl, pH 8.0, and 1 mM EDTA). Immune complexes were extracted from the beads with 1% SDS and 0.1 M NaHCO3, and cross-linking was reversed by heating at 65 °C overnight. Proteins were digested with proteinase K at 50 °C for 1 h, and DNA was purified by phenol/chloroform extraction and precipitated with ethanol. The immunoprecipitated DNA thus obtained was used for quantitative real-time PCR. The primers used were 5′-TTCTTTCCTTTCTGTGGGTGTG-3′ and 5′-CAACTCCTGCGGAAACACTT-3′ for the κB site in the NGAL promoter, 5′-CCATCACCCTCATATCCACC-3′ and 5′- TGTGTCTTCACTCTGCAGCC-3′ for a 3′-gene segment in the NGAL gene and 5′-GTTGTAGTATGCCCCCTAAGAG-3′ and 5′-CTCAGGGCAAACCTGAGTCATC-3′ for the κB site in the IL-8 promoter.
siRNA (small interfering RNA) transfection experiments
Duplexed modified RNA oligonucleotides (Stealth RNAi) were synthesized by Invitrogen. The sequences of the sense strands of the siRNAs were as follows: 5′-UCACACAGUAGGAAGAUCUCAUCCC-3′ for the NF-κB p65 subunit, 5′-GCACGAAUGACAGAGGCGUGUAUAA-3′ for the NF-κB p50 subunit, 5′-UAUGAAGGAACGUGUCACCAUCUGC-3′ for IκB-ζ, 5′-AGCUCCAGGACCUUGUGCUGCGUCU-3′ for C/EBPβ, and 5′-UUUAGUGGUGGUAAGUCCAGGCUGU-3′ for C/EBPδ. Stealth RNAi negative control duplexes (Invitrogen) were used as controls. A549 cells (3.0×105 cells) were transfected with 40 pmol of siRNA using Lipofectamine™ 2000. At 24 h after transfection, the cells were stimulated with 1 ng/ml IL-1β for 4 h. Cell lysate was prepared and subjected to Western blotting analysis with specific antibody. Reacting proteins were visualized by chemiluminescence using Lumi-Lightplus Western blotting substrate (Roche Diagnostics). In reporter analyses, A549 cells (7.5×104 cells) were transfected with 10 pmol of siRNA, together with 200 ng of a reporter plasmid using Lipofectamine™ 2000. At 24–36 h after transfection, the cells were stimulated with 1 ng/ml IL-1β for 5 h or with 10 ng/ml PMA for 18 h. HEK-293 cells (5.0×105 cells) were transfected with 40 pmol of siRNA. At 24 h after transfection, total RNA was extracted, and mRNA expression levels were quantified by real-time RT–PCR. In reporter analyses, HEK-293 cells (1.25×105 cells) were transfected with 10 pmol of siRNA, together with 100 ng of indicated reporter plasmid and 2.5 ng of the internal control plasmid phRL-TK using Lipofectamine™ 2000. At 24 h after transfection, the cells were transfected with 5 ng of the expression plasmid for the NF-κB subunit p65 or p50 and 100 ng of the IκB-ζ(L) expression plasmid. Following further incubation for 24 h, the cells were then lysed, and the luciferase activities were measured. The amount of transfected DNA was kept constant by using an empty vector.
Co-expression of IκB-ζ and the NF-κB subunit increases expression of hBD-2
Our previous studies have shown that IκB-ζ has dual opposite functions on expression of different inflammatory genes activated by NF-κB [17,19,23]. To identify genes whose expression is regulated by IκB-ζ, we examined the induction levels of several NF-κB target genes in HEK-293 cells transfected with the NF-κB p65 or p50 subunit and IκB-ζ. Because IκB-ζ(L) is the predominant form in LPS-stimulated cells [20], this form was used in the experiment. Quantitative real-time RT–PCR indicated that expression of E-selectin was strongly induced by transfection of the NF-κB p65 subunit as expected, whereas induction of hBD-2 was modest (Figure 1A). Transfection of the NF-κB p50 subunit or IκB-ζ alone did not elicit significant induction of either genes. Co-transfection of IκB-ζ dose-dependently inhibited the p65 subunit-induced expression of E-selectin, confirming our previous report that IκB-ζ is a negative regulator of the NF-κB-mediated transcription of this gene [17]. In contrast, co-transfection of IκB-ζ augmented expression of hBD-2 induced by the p65 subunit. Interestingly, co-transfection of IκB-ζ and the p50 subunit, either of which alone did not affect the expression, resulted in robust induction of hBD-2. The different actions of IκB-ζ on the two genes are attributable to the different promoter structures, since similar effects of IκB-ζ were observed in the reporter analyses using the E-selectin and hBD-2 promoters (Figure 1B). These results indicate that the actions of IκB-ζ on NF-κB-mediated transcription differ and could exhibit opposite effects against different genes.
NF-κB-binding sites in the hBD-2 promoter are essential, but not sufficient, for the IκB-ζ-mediated transcriptional activation
Luciferase reporter analyses indicate that co-transfection of IκB-ζ with the NF-κB subunits activates transcription of the promoter of the hBD-2 gene spanning from −1050 to +50 bp (Figures 1B and 2B; the nucleotide numbering refers to the transcription initiation sites of genes as the base +1). This region of the hBD-2 promoter contains four NF-κB-binding sites, which consists of two tandem consecutive binding sites (Figure 2A).
To identify cis-acting element(s) in the hBD-2 promoter responsible for the IκB-ζ-mediated transcriptional activation, we generated a series of 5′-deletion mutant constructs. The transcriptional activation by IκB-ζ and either the NF-κB p65 or p50 subunit was observed with a promoter fragment beginning at −577 bp, indicating that the distal NF-κB-binding sites (NF-κB-3 and -4 in Figure 2A) are dispensable (Figure 2B). Even a smaller fragment of the promoter, from −205 bp, responded to the co-transfection of IκB-ζ, although the induction levels were lower (Figure 2C). However, further deletion of the proximal NF-κBbinding sites (−185 to +50) severely impaired the IκB-ζ-mediated transcriptional activation, suggesting the importance of the NF-κB-binding sites in this region (NF-κB-1 and -2). To elucidate further the role of the NF-κB-binding sites, we introduced point mutations into the tandem NF-κB-binding sequences. Consistent with the results of the deletion constructs, introduction of mutations at NF-κB-binding sites 1 and 2 in the promoter from −577 to +50 bp significantly damaged not only the induction by p65, but also the augmentation by co-expression of IκB-ζ (Figure 2B, bottom panel). Thus the proximal NF-κB-binding sites 1 and 2 from −205 to −186 bp are essential for the IκB-ζ-mediated transcriptional activation.
To examine whether the sequence of the NF-κB-binding site determines the specific action of IκB-ζ in the observed transcriptional regulation, we constructed a reporter plasmid in which the duplicated sequences of the proximal NF-κB-binding sites 1 and 2 of the hBD-2 promoter were used to replace the tandem canonical NF-κB-binding sequences in the NF-κB reporter plasmid pNF-κB-Luc (phBD-2-κB-Luc). As observed with the E-selectin promoter reporter (Figure 1B), co-transfection of IκB-ζ resulted in the inhibition of p65-mediated activation of the pNF-κB-Luc. Similarly, phBD-2-κB-Luc was activated by p65, but the activity was suppressed by co-transfection of IκB-ζ (Figure 2D). Synergistic transcriptional activation by co-transfection of IκB-ζ, and the p50 subunit was minimal with pNF-κB-Luc or phBD-2-κB-Luc as with the E-selectin promoter reporter. Therefore the NF-κB-binding sites are essential, but the sequences of the sites themselves do not confer the IκB-ζ-mediated transcriptional activation to the promoter.
Critical roles for the C/EBP-binding site in the hBD-2 promoter in the IκB-ζ-mediated transcriptional activation
To identify another critical cis element(s) in the hBD-2 promoter for the IκB-ζ-mediated transcriptional activation, we created several internal deletion mutants (Figure 3A). The hBD-2 promoter of −205 to +50 bp responded to the co-expression of IκB-ζ to exhibit the augmented transcription as mentioned above. The response to IκB-ζ was markedly reduced by deleting a region of −185 to −75 bp (Δ−185/−75), which harbours a C/EBP-binding site (C/EBP1 in Figure 2A) and an AP-1 (activator protein 1)-binding site. A mutant with a shorter deletion from −185 to −164 bp (Δ−185/−164), a region containing the C/EBP-binding site, also minimally responded to the co-transfection of IκB-ζ. However, transcription of another deletion mutant lacking −163 to −106 bp (Δ−163/−106), and therefore without the AP-1-binding site, was stimulated upon the co-transfection of IκB-ζ. These results indicate that the region from −185 to −164 bp, which contains a potential C/EBP-binding sequence, GATTTGCATAAGAT, is important for the function of IκB-ζ. To evaluate the role(s) of this sequence, we introduced mutations at conserved nucleotide residues of the C/EBP-binding site. The hBD-2 promoter of −577 to +50 bp harbouring the mutated C/EBP-binding site with a sequence of GATAACCATAAGAT (mutated nucleotides are underlined) did not respond to the co-transfection of IκB-ζ (Figure 3B). Thus the C/EBP-binding site-1 in the hBD-2 promoter is essential for the effects of IκB-ζ.
Furthermore, we found that an internal deletion mutant (Δ−163/−33), consisting of only the TATA box and the region of −205 to −163 bp of the hBD-2 promoter, which harbours NF-κB-binding sites 1 and 2 followed by C/EBP-binding site 1, showed the maximum degree of transcriptional activation upon the co-transfection of the NF-κB subunit p65 or p50 and IκB-ζ (Figure 3C). Therefore the NF-κB- and C/EBP-binding sites are core elements for the IκB-ζ-mediated transcriptional activation of the hBD-2 promoter.
NF-κB- and C/EBP-binding sites are essential for the IκB-ζ-mediated transcriptional activation of the NGAL promoter
Our previous study using microarray analyses showed that a subset of LPS-inducible genes was not induced in IκB-ζ-deficient macrophages [19]. Quantitative RT–PCR showed that NGAL mRNA was induced in LPS-stimulated bone-marrow-derived macrophages of wild-type, but not of IκB-ζ-deficient mice (Figure 4A). Thus NGAL is one of the genes that require IκB-ζ for its induction, and we therefore analysed the promoter of this gene. The NGAL promoter contains an NF-κB-binding site and four C/EBP-binding sites (C/EBP1–4) in addition to AP-1-binding sites (Figure 4B). A reporter plasmid containing the NGAL promoter from −900 to +51 bp [pNGAL(−900)-Luc] was stimulated by co-expression of IκB-ζ and the NF-κB p50 subunit (Figure 4C). We focused on the roles for the NF-κB-binding site and the proximal C/EBP-binding sites (C/EBP1–3), and introduced point mutations into these sites. We found that the IκB-ζ-mediated transcriptional activation was abolished by mutations of the NF-κB-binding site and was substantially decreased by mutations of one of the C/EBP-binding sites (C/EBP1) (Figure 4C, left-hand panels). Introduction of mutations at all the three C/EBP-binding sites (C/EBP1–3) resulted in the minimal induction by IκB-ζ, as the mutation of C/EBP-binding site 1. We carried out similar experiments by using the human lung adenocarcinoma cell line A549, which is very sensitive to IL-1β to elicit induction of NGAL [30] (Figure 4C, right-hand panels). In the cells, transfection of IκB-ζ alone induced the activation of the NGAL promoter, which was abolished by the mutation of the NF-κB-binding site. The induction was severely impaired by the mutation of the C/EBP-binding site 3 and was suppressed further by the mutations of all three C/EBP-binding sites. These results indicate that the NF-κB- and C/EBP-binding sites are also essential for the IκB-ζ-mediated transcriptional activation of the NGAL gene, supporting further the results obtained with the hBD-2 promoter.
NF-κB- and C/EBP-binding sites are essential for IL-1β-induced transcriptional activation of the hBD-2 and NGAL promoters
Next, we examined whether the NF-κB- and C/EBP-binding sites are also required for induction of the hBD-2 and NGAL genes upon stimulation by IL-1β. Reporter analyses clearly indicated that both NF-κB- and C/EBP-binding sites in the hBD-2 promoter are required for efficient transcriptional activation following stimulation of A549 cells with IL-1β, which elicits induction of IκB-ζ (Figure 5A). As in the hBD-2 promoter, the NF-κB-binding site in the NGAL promoter was also essential for the IL-1β-induced transcription. We found that mutations at C/EBP-binding site 3 substantially reduced the transcriptional induction via IL-1β, and the mutations of all the C/EBP-binding sites suppressed further the induction, as in the IκB-ζ-mediated induction shown in Figure 4C (right-hand panels). Thus the NF-κB and C/EBP-binding sites in the hBD-2 and NGAL promoters are also crucial for activation of these promoters following physiological stimulation (via IL-1β) that leads to induction of IκB-ζ.
NF-κB and IκB-ζ are recruited to the NGAL promoter upon stimulation with IL-1β
To investigate recruitment of IκB-ζ and/or the NF-κB subunits to the promoters of the genes, we performed ChIP assays using specific antibodies against IκB-ζ or the NF-κB subunits. In unstimulated A549 cells, neither IκB-ζ nor the NF-κB subunits were recruited to the NGAL or IL-8 promoters. However, on stimulation of the cells with IL-1β, the NF-κB p65 subunit was found to be recruited to the NF-κB-binding sites of the NGAL and IL-8 promoters (Figure 6). In contrast, IκB-ζ and the NF-κB p50 subunit were specifically recruited to the NGAL promoter, but not to the IL-8 promoter, in the stimulated cells. None of the three proteins was recruited to the 3′-flanking region of the NGAL gene, thus showing the specificity of the assay. These results demonstrate that upon stimulation with IL-1β, IκB-ζ and both the NF-κB p65 and p50 subunits are recruited to and remain bound at the NF-κB-binding site of the NGAL promoter.
NF-κB and IκB-ζ are essential for transcriptional activation of the NGAL and hBD-2 promoters upon stimulation with IL-1β
We next examined whether NF-κB and/or IκB-ζ are required for the induction of the genes upon stimulation. Transfection of siRNAs for p65, p50 or IκB-ζ specifically knocked down the expression of corresponding endogenous protein, verifying the specificity (Figure 7A). We knocked down the expression levels of the endogenous NF-κB or IκB-ζ in A549 cells by introducing the siRNA, and examined the activities of the NGAL or the hBD-2 promoter reporter on stimulation with IL-1β. The siRNA for the p65 subunit or IκB-ζ strongly repressed induction of both NGAL and hBD-2 promoters in response to IL-1β (Figures 7B and 7C). The siRNA for the p50 subunit of NF-κB also substantially inhibited the activation of both genes. In contrast, none of the siRNAs for the p65 or p50 subunits or IκB-ζ affected PMA-mediated activation of an AP-1 reporter plasmid, pAP-1-Luc, in the same cells (Figure 7D). Thus it was strongly suggested that the NF-κB p65 and p50 subunits and IκB-ζ are essential for the transcriptional activation of the NGAL and the hBD-2 promoters upon stimulation with IL-1β.
Activation of the NGAL promoter by transfection of the NF-κB p50 subunit and IκB-ζ was suppressed in C/EBPβ-depleted HEK-293 cells
In order to examine whether C/EBPs are required for the IκB-ζ-mediated transcription, we attempted to knock down C/EBPβ and C/EBPδ. Introduction of siRNAs for C/EBPβ and C/EBPδ into HEK-293 cells successfully suppressed their respective mRNAs (Figure 8A). HEK-293 cells treated with siRNA for C/EBPβ or C/EBPδ were transfected with NF-κB and/or IκB-ζ, and we examined their effects on the NGAL promoter. As shown in Figure 8(B), the IκB-ζ-mediated activation of the NGAL promoter was specifically inhibited in the C/EBPβ-depleted cells, whereas the activation was not affected in the C/EBPδ-depleted cells. Another siRNA for C/EBPβ with a different sequence exhibited similar effects (results not shown). Both siRNAs for C/EBPβ and C/EBPδ did not affect the activity on the NF-κB reporter. Thus C/EBPβ is required for the IκB-ζ-mediated transcriptional activation of the NGAL promoter in HEK-293 cells.
Our previous studies have shown that IκB-ζ exhibits dual opposite activities on the expression of different subsets of inflammatory genes [17,19,23]. Consistent with these results, the present study also showed that exogenous expression of IκB-ζ augmented the NF-κB-mediated expression of hBD-2, whereas it inhibited that of E-selectin (Figure 1). In addition to IκB-ζ(L) used in this study, another isoform, IκB-ζ(S), exhibited qualitatively similar effects (results not shown). IκB-ζ alone promoted marginal levels of the transcription of the hBD-2 and NGAL genes, and it required NF-κB for robust transcriptional activation in HEK-293 cells. As expected from this requirement for NF-κB, reporter analyses indicated that NF-κB-binding sites in both promoters are essential for the IκB-ζ-mediated transcriptional activation (Figures 2 and 4C, left-hand panels). The sequence of the NF-κB-binding sites, however, was not sufficient for the activation, and a C/EBP-binding site was also essential (Figures 2, 3 and 4C, left-hand panels). Although NGAL transcription was induced by transfection of IκB-ζ alone in A549 cells, NF-κB is probably involved in the induction since the NF-κB-binding site, as well as a C/EBP-binding site, is also essential for the induction as in the hBD-2 promoter (Figure 4C, right-hand panels). Furthermore, in addition to IκB-ζ, NF-κB is essential for the induction of NGAL transcription in A549 cells (Figure 7). Endogenous nuclear NF-κB (possibly the p50 subunit) levels might be higher in this adenocarcinoma cell line, which is highly sensitive to IL-1β.
Since robust transcriptional activation by IκB-ζ was observed on an artificial small promoter consisting of the NF-κB- and C/EBP-binding sites and TATA box (Figure 3C), these two sites appear to constitute the minimal element that allows the IκB-ζ-mediated transcriptional activation. Since IκB-ζ acted as a negative regulator of NF-κB-mediated activation on the promoters constituted only with the NF-κB-binding sites of the hBD-2 promoter (Figure 2D), the presence of the C/EBP-binding site could convert the action of IκB-ζ. It should be noted that promoters of genes that exhibit IκB-ζ-dependent induction harbour NF-κB- and C/EBP-binding sites, including IL-6, the IL-12 p40 subunit, granulocyte/macrophage CSF and granulocyte CSF.
Among the three proximal C/EBP-binding sites in the NGAL promoter, different sites seemed to be used in HEK-293 cells and A549 cells for the IκB-ζ-mediated inductions (Figure 4). This may reflect that, in the two cell lines, IκB-ζ and NF-κB could form stoichiometrically different transcription complexes with different sizes, which would utilize C/EBP-binding sites located at different distances from the NF-κB-binding site. Multiple C/EBP-binding sites in the NGAL promoter might guarantee the maximum induction in both cell types.
Crucial roles for both NF-κB- and C/EBP-binding sites were also demonstrated in the induction of the hBD-2 and NGAL genes in response to IL-1β (Figure 5). IκB-ζ expression, which is elicited by IL-1β, was essential for the IL-1β-dependent induction of the two genes (Figure 7). In addition to the NF-κB p50 subunit, the p65 subunit was also recruited to the NGAL promoter upon IL-1β stimulation, and was required for the expression of the NGAL gene (Figures 6 and 7). Although preferential binding of IκB-ζ to the p50 subunit has been shown previously [17], the active transcription complex on the genes appeared to contain IκB-ζ and both p50 and p65 subunits of NF-κB. It is noteworthy that the NF-κB-binding sites in the hBD-2 and NGAL promoters are not canonical, as revealed by the fact that their respective p65 subunit-mediated inductions are much weaker than that of the E-selectin promoter or the promoter in pNF-κB-Luc (Figures 1, 2D and 4C, see induction in the absence of IκB-ζ). IκB-ζ binding to NF-κB might alter the preferred DNA-binding sequences of the p65/p50 heterodimer from the canonical NF-κB-binding sequences to non-canonical sequences flanked by a C/EBP-binding site.
Although the recruitment of IκB-ζ and the NF-κB subunits was shown by the ChIP analyses, we could not detect the binding of the IκB-ζ-containing complex to the DNA fragment of the hBD-2 promoter by EMSA (electrophoretic mobility-shift assay) using lysate from cells transfected with IκB-ζ and NF-κB (results not shown). This suggests that the complex is very fragile or that it requires another protein(s) for DNA binding. The C/EBP family of proteins could be such candidates, since NF-κB and the C/EBP family of proteins associate directly via their Rel homology domain and the basic leucine-zipper domain [32–34]. In fact, the IκB-ζ-mediated activation of the NGAL promoter was severely impaired in C/EBPβ-depleted HEK-293 cells (Figure 8). However, in vivo studies using C/EBP-deficient mice have provided results contrary to this hypothesis. The expression of IL-6, an IκB-ζ target gene, following LPS stimulation is unchanged in macrophages from wild-type and C/EBPβ- [35] or C/EBPϵ- [36] knockout mice. In addition, no defect in cytokine production has been detected in macrophages from C/EBPδ-knockout mice [37]. Our preliminary studies indicated that the IκB-ζ-mediated activation of the hBD-2 and NGAL promoter in A549 cells was not significantly affected by depletion of either C/EBPβ or C/EBPδ. Thus other proteins could be involved, in other cell types, in the transcriptionally active complex consisting of IκB-ζ and NF-κB that binds to the promoters harbouring the NF-κB- and C/EBP-binding sites.
The NGAL promoter with the multiple C/EBP-binding sites was induced by transfection of C/EBPβ and C/EBPδ alone (results not shown). As C/EBPδ is one of the IκB-ζ target genes [19], the induction of NGAL could be sustained by C/EBPδ in a late phase. Although the C/EBPδ-mediated induction was completely abolished by the triple mutations of C/EBP-binding sites 1, 2 and 3 in the NGAL promoter (results not shown), marginal levels of IκB-ζ-mediated induction was observed with the mutant (Figures 4 and 5), suggesting that the recognition sites of the IκB-ζcontaining complex and C/EBPs overlap, but are not identical.
Despite intensive studies on the molecular mechanisms of IκB-ζ-mediated transcription, its physiological roles in vivo remain to be analysed. In addition to several cytokines, the present studies indicated that the hBD-2 and NGAL genes are targets for IκB-ζ-mediated transcription. Consistent with the induction spectrum of IκB-ζ, both the hBD-2 and NGAL genes are induced preferentially by IL-1β, but not by TNF-α in A549 human lung adenocarcinoma cells, keratinocytes and other cells [25–30]. A recent report has also shown critical roles for IκB-ζ in IL-1β-dependent induction of the genes [38]. Since both genes encode inducible antimicrobial proteins expressed in various epithelial tissues [39,40], one of the functions of IκB-ζ is likely to be to activate genes that play roles in elimination of infected bacteria. This concept is consistent with the observation that IκB-ζ is induced by microbial ligands of various TLRs, but not by double-stranded RNA, a ligand for TLR3, which is an intracellular receptor for RNA viruses [41].
There is accumulating evidence that both NF-κB- and C/EBP-binding sites are important for regulation of many immune responses and acute-phase response genes such as IL-1β [42], IL-6 [32] and granulocyte CSF [43], in addition to hBD-2 and NGAL. Recent studies also showed that NF-κB- and C/EBP-binding sites are required for induction of IL-6 and 24p3, a mouse homologue of NGAL, on co-stimulation with IL-17 and TNF-α [44,45]. The induction probably involves IκB-ζ, because co-stimulation with IL-17 and TNF-α induces expression of IκB-ζ [20,46]. Nonetheless, not all promoters harbouring both NF-κB- and C/EBP-binding sites are targets for IκB-ζ. For example, the IL-8 promoter contains both sites and is activated synergistically by overexpression of NF-κB and C/EBPβ [32]. IκB-ζ, however, is not recruited to the IL-8 promoter and acts negatively on expression, as revealed by the ChIP assay (Figure 6) and mRNA quantification (results not shown). Probably, the order, distance and the intervening sequence between the NF-κB- and C/EBP-binding sites, as well as the sequence of each binding site and/or chromatin structures, would affect transcriptional activation by IκB-ζ. Comprehensive analyses of genes whose transcription is regulated by IκB-ζ should provide more information on the common promoter structures of these genes. Furthermore, identification of the transcription complex containing IκB-ζ and biochemical characterization of the complex would be invaluable towards understanding the sophisticated mechanisms for regulation of a series of inflammatory reactions.
This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to T.M., K.T. and S.Y.), and grants from the Naito Foundation (to T.M.), the Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T.M.), the Kaibara Foundation (to T.M.) and Japan Foundation of Applied Enzymology (to T.M.).
Abbreviations: AP-1, activator protein 1; C/EBP, CCAAT/enhancer-binding protein; ChIP, chromatin immunoprecipitation; CSF, colony-stimulating factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; hBD-2, human β-defensin 2; HEK-293, human embryonic kidney-293; IκB, inhibitor of nuclear factor κB; IκB-ζ(L), long-form IκB-ζ; IκB-ζ(S), short-form IκB-ζ; IL, interleukin; LPS, lipopolysaccharide; MBP, maltose-binding protein; NF-κB, nuclear factor-κB; NGAL, neutrophil gelatinase-associated lipocalin; RNAi, RNA interference; RT, reverse transcription; siRNA, small interfering RNA; TLR, Toll-like receptor; TNF, tumour necrosis factor
- © 2007 Biochemical Society


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Reading the success stories of people seeking Arthur's help will give you an idea about how he solves different kinds of problems, including anxiety and phobias.
Below you can read some messages that people have sent several weeks or months after their last session to update and thank Arthur:
"After seeing you my life changed. I do not devour my meals anymore. Now I only eat when I want to and need to. I lost about 20 pounds."
— Jose Manuel
"I enjoyed eating chocolate very much but now I can control it. I don't have cravings anymore. I want to thank you because you helped me a lot."
— Pat
"After three months of quitting, I can't believe it. It feels unreal that I don't need to smoke and I can be happy (or happier) without cigarettes. I have the feeling that I never smoked in my life."
— Natalia
"As promised, I'm writing to you after several weeks to thank you because thanks to your help my sleep quality has improved. I will recommend you to all my friends."
— Joe
I don't know if you remember me. I'm Julie and I did a session with you for my non-stop coffee drinking. I'm still coffee free! Thank you very much Arthur."
— Julie
"About three months ago I came to see you for my fear of public speaking. First of all, I apologize for not writing you sooner, but I want to thank you for your help. I presented my novel in several bookstores, had radio and television interviews. All went well. I have to work on my diction but I don't get paralyzed with fear anymore. Thanks for everything."
— Anthony
"Hi Arthur, I was in your office last September. We agreed that I would write you back to tell you how things are going. The truth is that I'm very happy. Only on one night (Monday) did I wake up to eat at night — and that was because I skipped dinner and only an had apple. Thank you very much."
— Agnes
"I write to you to confirm that now several months after doing therapy with you for my anxiety, I feel fantastic. I'm still able to do all those things that I couldn't before — my fears have been reduced to a very small thing. I cannot deny that this still seems to me VERY CURIOUS, but I'm delighted and thank you."
— Ramona
"As agreed I'm writing to you three months after doing therapy with you, to let you know the results. I went 'full steam ahead!' I'm free from my fear of the ocean and boats. Infinite thanks to you and I will always remember you for your help."
— Mary
"The truth is that I feel great. Before, whenever I dieted, I never felt satisfied nor happy like I do now. I don't have any anxiety. I'm not obsessed with my physical look or with my weight. I used to weight myself twice a day. Now I don't have this obsession. I learned to eat well and lose weight. Thank you."
— Rose
"Hi Arthur, I'm Robert, you must remember me. I'm fine now. I'm social and have gotten to know many new friends. I recently met a girl and we are dating. I don't think about my ex anymore. Thanks for everything,"
— Robert
"I must admit that I was very skeptical when I came to see you. But I'm finally free from anxiety. Thank you."
— Maria

subset: commoncrawl


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What is the Mission of United Methodism?
The mission of the United Methodist Church is to make disciples of Jesus Christ for the transformation of the world. We, as a local church, help to provide the most significant arena for disciple making to occur.
What do United Methodists Believe?
We, along with millions of Christians through the ages, affirm that God is a Trinity: Father, Son, and Holy Spirit. We believe that God became human in Jesus of Nazareth, and that his life, death, and resurrection demonstrate God's love for us. We believe that the Holy Spirit is God's present activity in our lives, providing God's leading, challenge, support and comfort.
We celebrate two Holy Sacraments that Christ himself participated in: Baptism and the Lord's Supper.
Learn more about the Basics of United Methodist Belief.
We are a Connectional Church
We are part of the United Methodist Denomination. We are a connectional church, meaning that we are connected to other United Methodist churches on many levels: locally, as a conference (think state, roughly), nationally, and internationally.
Locally, we are part of the Missouri River District. This district spans across south eastern Nebraska, with the district office in Omaha.
The Missouri River District is part of the Great Plains Conference, newly formed in 2014, which covers Nebraska and Kansas.
The South Central Jurisdiction covers the Southern Central part of the US, including the Great Plains Conference.
And finally, on a global level, we are part of the United Methodist General Conference: all United Methodist Churches are part of this world-wide body of believers.


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Why Is Betting On Horse Racing Events So Popular?
One of the oldest forms of sports is horse racing and there are horse racing events in every country. They are very thrilling and exciting and invite interest from people all over the country and also from all over the world. Local horse racing events attracts thousands of people who come to watch the race and enjoy money through betting. International horse racing events are e thrilling events where a lot of money can be put into various racing outcomes. There are so many smaller racing events held that gives the opportunity to place more bets and gain money from betting.
Types Of Betting Opportunities Offered In Horse Racing
Betting on horse racing events is quite exciting because of the many types of betting opportunities that you can avail here. Here is a look at the different types of bets that you can place in such events:
· Daily double – In this type of bet, you will be placing bets on the outcome of two races happening one after another
· Bridge Jumper - In this bet type, you will put a large sum of money onto a single horse. This type of horse bet is risky a you stand to lose a lot of money of your bet does not win
· Quinells – Here you will be facing your bet on two horses to finish the race.
· On the nose - Betting that a horse will only win the race
· Pick six – in this bet, you pick the winner of several raving events
These are some of types of bets that can be placed for horse racing events. There are many more and you should learn about them in detail, to understand how to use them for your betting requirements.
Betting Through Online Sports Betting Sites
Now horse racing can be easily done from the comfort of your home through online booking site. In order to initiate horse racing in such sites like sbobet or sbo, it is essential to open an account. Once you have an account and verified it, you can proceed to managing your account by depositing some money in it for betting. After this, you can proceed to placing the money on various racing outcomes by looking into the list of racing events that are going to happen. Check out local racing events where you can place bets as well as international s racing events where the thrill and excitement is more.
Use the different kinds of betting opportunities to place exciting bets and win money quickly and easily. These online portals like sbo222 offer an easy system for placing bets and withdrawing the money after winning back to your account. They are safe to use because they offer good security features which ensures that money transactions and use of personal information is kept confidential. They also provide detailed information on various odds, handicaps and betting types that can be used to place attractive bets and enjoy the opportunity of making money quickly and easily.


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|Professor Goong Chen, Texas A&M University, Texas, USA
Goong Chen is Professor of Mathematics at Texas A & M University. He received his Ph.D. in Mathematics from the University of Wisconsin at Madison in 1977 and BSc in Mathematics from the National Tsing Hua University in Hsinchu, Taiwan in 1972. Before joining Texas A & M University in 1987, he had taught at the Southern Illinois University at Carbondale (1977–1978) and the Pennsylvania State University at University Park (1978–1987).
He has research interests in many areas of applied and computational mathematics: control theory for partial differential equations (PDEs), boundary element methods and numerical solutions of PDEs, engineering mechanics, computational fluid dynamics, chaotic dynamics, quantum computation, chemical physics and quantum mechanics.
He has written over one hundred and fifty papers, seven advanced texts/monographs, and co-edited four books. He is Editor-in-Chief of the Journal of Mathematical Analysis and Applications and Academic Press's Mathematics in Science and Engineering Book Series, and has served as the Editor-in-Chief of the Chapman & Hall/CRC Press Applied Mathematics and Nonlinear Science Series(2002-2011), and as Associate Editor for several other editorial boards, including the SIAM Journal on Control and Optimization, the International Journal on Quantum Information, Physica Scripta, and the Electronic Journal of Differential Equations.
He is also a co-holder of a U.S. Patent on certain quantum circuit design for quantum computing.
|Professor Ilias Kotsireas, Wilfrid Laurier University, Waterloo, Canada
Ilias S. Kotsireas is a Professor of Computer Science at Wilfrid Laurier University. He also serves as the director of Computer Algebra Research Group of Wilfrid Laurier University http://www.cargo.wlu.ca/ . He received his Ph.D. in Computer Science from the University of Paris 6 in 1998, MSc in Computer Science from the University of Paris 6 in 1995, BSc in Computer Science from the University of Paris 6 in 1994, and BSc in Mathematics from the University of Athens in 1990. Before joining Wilfrid Laurier University in 2001, he was a postdoctoral fellow at the Ontario Research Center for Computer Algebra (1999-2001). He has taught at the University of Paris 6 and worked as laboratory assistant at Lycee Saint-Louis and University of Versailles Saint-Quentin-en-Yvelines.
He has research interests in symbolic computation, combinatorial designs, theoretical and algorithmic aspects of polynomial system solving. Kotsireas uses algorithmic, meta-heuristic and high-performance computing techniques to study existence questions for combinatorial designs.
He has written more than 100 papers and serves as the Editor of ACM Communications of Computer Algebra for 10 years. He serves on the Editorial Board of 7 international research journals including: Journal of Combinatorial Designs, Mathematics in Computer Science, Journal of Computational Science, and Optimization Letters. He has served as a referee for 34 journals especially including Journal of Symbolic Computation, Mathematics of Computation, Discrete Mathematics, Theoretical Computer Science, Journal of Combinatorial Designs, Numerical Algorithms, SIAM Journal on Scientific Computing, etc. He was the chair of Applications of Computer Algebra Conference and had organized over 60 conferences. He has supervised 20 students including graduate and postgraduates.
His research has been funded by NSERC for 15 years and he currently serves as the Chair of ACM SIGSAM http://www.sigsam.org/
|Professor James Nagy, Emory University, Georgia, USA
James Nagy is a Professor of Mathematics and Computer Science at Emory University. He received his Ph.D. in Applied Mathematics from North Carolina State University in 1991. Before joining Emory University in 1999 he had postdoctoral research fellowships with the IMA at the University of Minnesota, with the NSF at the University of Maryland, and was on the faculty at Southern Methodist University.
Nagy has published 75 research articles, a book on image deblurring, and has produced a variety of freely available software and educational material. His work has made a great impact in the field of numerical linear algebra, which has been recognized through numerous invited plenary talks, grants from the U.S. National Science Foundation and Air Force Office of Scientific Research. He has supervised 10 PhD students and 10 undergraduate Honors students.
He was Vice Chair of the Society of Industrial and Applied Mathematics (SIAM) Linear Algebra Activity Group from 2013-2016, and from 2016 is serving as Chair. He is on the editorial boards of SIAM Journal on Scientific Computing (SISC), SIAM Journal on Matrix Analysis and Applications (SIMAX), Numerical Algorithms, and the Electronic Journal of Linear Algebra (ELA). His research interests include numerical linear algebra, structured matrix computations, and numerical solutions of inverse problems in image processing
|Dr. Sivaguru S. Sritharan, Air Force Institute of Technology, Ohio, USA
Sivaguru S. Sritharan serves as the Provost and Vice Chancellor for the Air Force Institute of Technology (AFIT). He received his BSc in Mechanical Engineering from University of Sri Lanka, Peradeniya Campus in 1977 with first class honors. He went to USA for postgraduate studies in 1978 and received a MS in Aeronautics and Astronautics from University of Washington, Seattle in 1979 and then a PhD in Applied Mathematics from University of Arizona in 1982. In the past he has been a faculty member in University of Sri Lanka (Peradeniya), University of Southern California and University of Wyoming. He has also served as the Head of the Mathematics Department at University of Wyoming and the Dean of Engineering and Applied Sciences at the Naval Postgraduate School at Monterey, California.
He has widely published in mathematical fluid dynamics, magneto-hydrodynamics, control theory and stochastic analysis and has given over three hundred lectures and invited colloquia in academic and research institutions world-wide
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g-Bar Canted-4-Bar Suspension System
Total Controls second generation g-Bar and g-Link systems represent the current state-of-the-art in canted-4-bar suspension design. The g-Bar system consists of three different combinations of upper and lower link bars with your choice of coil-over or air-spring shocks. These options create six different variations to better suit your particular performance application. Each can be used with our vehicle-specific bolt-in FAB9 housing or the vehicles existing housing.
Adjustable Geometry
Adjustable Shock Mounts
System Options: VariShock coil-over or air-spring shocks;single- or double-adjustable valving; direct-fit FAB9 housing or bolt-on axle brackets (for use with existing housing); urethane, pivot-ball, or billet pivot-ball suspension links; splined-end or sliding-link anti-roll bar.
For detailed product information and specific pricing download the g-Bar technical data sheet or contact our sales staff directly.
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Includes: g-Bar cradle, axle brackets, upper and lower links, VariShock single-adjustable coil-overs, springs, and mounting hardware
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Includes: g-Bar cradle, axle brackets, upper and lower links, single-adjustable air-spring shocks, and mounting hardware
System Options
Coil-Over Shocks
Accurately corner-balance the vehicle by varying spring preload. Choose from a selection of shock types and spring rates to match your vehicle's performance.
SensiSet (SS) — Factory-preset valving for street-performance applications.
QuickSet 1 (Q1) — Single-adjustable; 16-position knob adjusts bump and rebound simultaneously.
QuickSet 2 (Q2) — Double-adjustable; 16-position knobs adjust bump and rebound independently.
QuickSet 4 (Q4R) — 4-way-adjustable with remote reservoir; two pairs of 16-position knobs adjust bump and rebound independently at high- and low-piston speeds.
Air-Spring Shocks
Air spring equipped systems offer instant ride-height and ride-quality adjustment, with the VariShock air spring, in single- or double-adjustable versions. When fully deflated, the vehicle rests 5-6 inches below stock ride height and can be raised to driving height at the push of a button. A compressor system available separately) is required for operation.
Sliding-Link Adjustable Anti-Roll Bar
Splined-End Tubular Anti-Roll Bar
|Note: Anti-roll-bar mounts are integrated into the g-Bar brackets system and must be selected at time of g-Bar purchase. Download the technical data sheet for specific information.
g-Bar Direct-Fit FAB9 Housings
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Since she could not delete the image of a hot stallion from her mind the babe decided to use it while fingering her cunt.
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A new update for The Division coming out on June 28 together with the Underground expansion will introduce some "significant" changes to weapon balance.Weapon balance changes are coming "very soon."
The Division's 1.3 update will also add some other things like unspecified bug fixes and gameplay changes, as well as new weapons and new gear sets.
The Division's Underground expansion will be available for Xbox One and PC players on June 28 but for PS4 it's coming on August 2, players on all platforms will receive update 1.3 on that day.
Which weapons will need balance changes in The Division? Let us know in the comments below.//TODO: Rijesiti rating i odkomentarisati ovo kad rijesim ?>


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Some of the world's biggest miners are running Australian mines at a loss because the fixed costs of their rail contracts mean it would cost more to close them.
Several coalmines in Queensland are understood to be operating under such a scenario, because of long-term ''take-or-pay'' rail access contracts that miners struck when commodity prices were high.
Several coking coalmines run by global miner Anglo American are understood to be affected in this way and one source said he was confident that Anglo was not ''Robinson Crusoe'' in that regard.
Australia's coal sector is already facing multi-year lows for both thermal coal and coking coal prices, and the fixed-rail contracts highlight the multitude of challenges facing the local industry.
When asked to confirm the situation in Queensland an Anglo spokeswoman said the company would not comment on ''matters of this nature''. But Anglo is known to have take-or-pay contracts in place with Aurizon for rail access, and Anglo chief executive Mark Cutifani referred to the problems it was causing during the company's annual results in February.
''Unfortunately in the industry there is still a lot of sticky production with take-or-pay contracts on the rail lines in both the US and Australia. Some of that capacity is shaking out, but it might be more than a year before we see the sorts of numbers that are going to be needed to improve the pricing focus,'' he said.
''If we have to make some tough decisions on reducing our capacity we will go there, but certainly I think the improvement programs are making a big difference.''
Unfortunately for Anglo, some of those contracts were struck during the absolute peak of coal prices around February 2011, when Queensland coking coal was fetching more than $US300 a tonne.
The bulk commodity was circling $US110 a tonne this week, having folded under the dramatic rise in exports from producers in North America and Queensland.
The likes of Glencore Xstrata, BHP Billiton, Peabody Energy and Rio Tinto also have rail contracts with Aurizon for their Queensland mines, while take-or-pay contracts are also used by rival rail providers Asciano and Pacific National.
Many of the miners are also tied to long-term take-or-pay contracts for port capacity.
An Aurizon spokesman defended the rail providers, saying they had to invest significant amounts in rail infrastructure to provide a transport solution. ''Given the scale, capital intensity and 25-year-plus life of investments, contracts seek commitments from customers to long-term use of the assets,'' he said.


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Medical Student
Getting into Medical School: A Guide for Older Nontraditional Students
|FAQ - Frequently Asked Questions
Am I too old to get into medical school?
Do medical schools discriminate against older applicants?
How can I get my family to support my decision to go to medical school?
How can I get my spouse to support my decision to go to medical school?
Will I have time for my family or to have children?
Can I get into medical school with a GPA below 3.0?
Will it really take 2 or 3 years to complete the prerequisites before I can start medical school?
Are there any medical schools that do not require the MCAT or the standard premedical courses?
Should I take the MCAT in April or August?
If I don't get in the first time, can I reapply to medical school?
How can I afford medical school if I have a family to support?
Will problems in my past prevent me from getting into medical school (eg, academic problems, bankruptcy, drug use, criminal record)?
Should I consider Osteopathic or Caribbean medical schools?
Do you have any regrets about changing careers later in life?


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Donegal bus for Dublin protest
Donegal Action Against Austerity (DAAA) are calling for the public to join them in Dublin on Wednesday, February 29, for a march against the government's austerity programme.
DAAA, which brings together several prominent Donegal campaigns, has organised buses to take people from Donegal to Dublin for the march.
The protest is scheduled to begin around 12 noon, at the Spire on O'Connell Street, and march to the Dáil at about 1.30pm.
Convoy haulier John McLaughlin of the JML Diesel Protest, a founding member of DAAA, said the group changed their plans to protest in Dublin earlier this month when they heard from groups in other counties who wanted to coordinate demonstrations in the capital.
"Now's our chance for everybody to get their points across and stand up and be counted," Mr McLaughlin said. "We're not standing for these austerity measures any longer."
Betty Holmes of Donegal Action for Cancer Care is also a founding member of the group.
Betty said that she hoped politicians would see from the Donegal response to the protest, "that the people of Donegal are in a place that they probably haven't been in a long time.
"I haven't see as much upset or annoyance," she said. "Normally this would have happened in kitchens and pubs in Donegal, but people now are prepared to stand up and be counted, and be proactive.
"That's a new experience for Donegal," Mrs Holmes said.
Buses are leaving Donegal for the protest at 7am on February 29. Anyone interested in booking a space on a bus should text their name and town to (086) 3383090 or send an e-mail to email@example.com.


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PARIS (Reuters) - Airbus (AIR.PA) Chief Executive Tom Enders, who is stepping down in 2019, urged employees on Friday to allow the board to pick his successor without being distracted by speculation about who would replace him.
"You may be wondering who will replace me in 2019," Enders said in a letter seen by Reuters shortly after a management shake-up. "This is a matter for the board to decide in due course, behind closed doors and in the sole interest of Airbus, its employees and shareholders."
"We have much to achieve together between now and then, so my suggestion is to let the board do their work and we'll focus on ours."
Enders acknowledged past differences with his no.2 and chief operating officer, Fabrice Bregier, and said he was relying on Bregier's team to welcome his successor Guillaume Faury into the role "with open arms and ease him into the job".
Bregier is leaving in February 2018, having told the board he would not seek the CEO position in 2019, Airbus said earlier.
Several people close to the company said tensions between the two top executives had contributed to Bregier's departure, though aides to Enders said it was solely related to the board-supervised succession process.
"I am personally very grateful to Fabrice for everything he has done for Airbus," Enders said.
Faury will be replaced as head of Airbus Helicopters in coming weeks, he added.
In his own letter, Bregier thanked staff and called for one last push to meet a "difficult" challenge of reaching a record target of more than 700 jet deliveries in 2017.
"It will require a huge effort, but I am confident that with your support and full dedication, we will be successful again."
Enders called for "all hands on deck" to meet the same goal, which is being closely watched by aerospace investors.
Reporting by Tim Hepher; Editing by Richard Lough


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Sunday, April 13, 2014
Simple Goodness
So. Don't be wearing your white gloves over here, okay?
But I glanced out the window when I was cleaning the library, Book of Thrones clasped to my ears, and I saw a guy in a white shirt and black jacket get out of a car.
"Oh Lord," I thought. Some church dude. Than I looked at the woman who got out of the car with him and she was so pretty and I knew her immediately.
It was Juancho and his bride, stopping by on their way back from an anniversary celebration in Monticello. I was just pleased as I could be to see them. Some people bring joy in the door with them and these two surely do. It's odd how some folks make me feel worried about how I'm dressed or how filthy my house is or how stinky my dogs are and some people- well, it's like they're family. The good kind.
I gave them the tour (Melissa looked around my kitchen and I could tell she wanted to ask me the very same thing Owen asked me the other day which was, "Where did you GET all this stuff?") and introduced them to the chickens and demonstrated how Elvis tid-bits for the ladies with grapes which is VERY hard for Elvis to do because he loves grapes more than anything on this earth. Tid-bitting, for those of you who may not know is what they call it when a rooster finds food and instead of eating it himself, he drops it to the ground and calls his hens with a deep, throaty call. The ladies love it.
I read in that article in Scientific American a few weeks ago about chickens that if there is an alpha rooster in the flock, other roosters, his "inferiors," shall we say, may tid-bit as well, but they do it on the sly. They do the food-dropping and the head toss that goes with it but they don't make the call which would alert the alpha rooster that someone is trying to make time with his hens. But that the hens remember and are more open to the beta males' advances.
What I have mostly learned from observing chickens is that they have far more in common with human beings than you would imagine.
Anyway, it was a sweet visit from sweet people and I've gotten a lot of stuff done, none of it important, and Mr. Moon has worked his ass off, mowing and now he's weed-whacking. God, that man. Where does he find the energy? I'm sitting here yawning myself and I slept with Owen and that child doesn't move all night whereas Gibson kicks and tosses all night and he slept with Mr. Moon so I know who did and did not get a good night's sleep.
I'm going to make something with leftover steak. Out of the four steaks the fellows grilled last night, the four of us didn't finish two of them. Oh well. If you're going to grill, you might as well grill, or as Mr. Moon says they say in Tennessee, "If you're gonna come on, then COME ON!"
I think that may refer more to fighting than to grilling but whatever. It's all manly.
And my heart is light and the house is a little tidier in a few select areas and some of the pine pollen has been swept and dusted away and the yard is pretty and I got some good hugs from some of the very best people and I repotted a rose bush and the cardinals are cracking seeds in their yellow beaks and it's just been a damn good day. I've washed and put away sweaters and now I'm doing the same with jackets. I feel certain that warm weather is here upon us surely and truly and the pecan leaves are coming out, bright green and feathery and I'm just one incredibly grateful lady, surprised and amazed to be at this place in my life.
10 comments:
Sounds like a wonderful day! We had 80 degrees and LOTS of sunshine yesterday, so did lots of outside stuff. The plan was to get some inside cleaning done today....then the storms came. So I sat on the deck and watched the storms roll through and got very little accomplished. I've been cleaning on the 1.5 level (or lower) for a while now, but I need to kick it up because I've got 20 people coming for Easter dinner. And two grands who insist they still believe and REALLY want to hung Easter eggs. Tell me, do you know of many 10 and 11-year olds who still believe? I'm such a pushover!ReplyDelete
...oh, and I forgot one teensy thing. The hippy-dippy weatherman tells us that we can expect sloppy wet, but trace amounts of sticking snow tomorrow afternoon. That ain't right!!!ReplyDelete
I don't like homes that can pass the white glove test, they make me nervous. Give me a home where you can put your feet up in the coffee table and just commune.ReplyDelete
Love that about COME ON! :) It is indeed all manly :)ReplyDelete
I sooo wish I could just stop by your house for a visit whenever I wanted! Other than this home-spot, that is :) If you put on a bra for me, I'd be insulted.ReplyDelete
Oh Elvis - he really is the best rooster, isn't he? Sounds like you had one of those days in a good way.ReplyDelete
- AnonymousApril 14, 2014 at 7:50 AM
If I know someone is coming over, I'm s clean freak. Pop in on me, and you get what you get. I prefer it when people see me at my worst and still hang around, then I can relax.ReplyDelete
The cats' sitting on me, palette should be itchy in 5, 4, 3...
These chicken tid bits never fail to amaze me.ReplyDelete
catrina- You ARE a pushover, but when it comes to our grandchilden, who the hell isn't?ReplyDelete
Watching the rain is a holy rite in my opinion.
Birdie- You would be most comfortable in my house. I promise you.
Jo- I love that you liked that. It is such a plain and honest thing. Well then, COME ON!
SJ- I would never put on a bra for you. I promise you that.
Jill- Elvis may be the world's best rooster. There has to be one somewhere, right?
heartinhand- And you have cats...why?
Ms. Vesuvius- Maybe someday you will have chickens. If you do, I am certain you will enjoy them.
I have dropped tidbits in front of ladies before. I think that the woman who became my wife appreciated them the most. LOL.ReplyDelete


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San Francisco Ballet – Shostakovich Trilogy – San Francisco
★★★✰✰ The choreography in Shostakovich Trilogy, which was co-commissioned by SFB and ABT and premiered in 2014, is as frustratingly capricious as the music.
San Francisco Ballet – Unbound Festival Program D: works by Edwaard Liang, Dwight Rhoden, Arthur Pita – San Francisco
★★★★★ A hundred monkeys might eventually type out Hamlet, but only a herd of unicorns on Ecstasy could come anywhere near Arthur Pita's Björk Ballet.
San Francisco Ballet – Serenade, The Chairman Dances, Rodeo – San Francisco
★★★✰✰ Not all, but many pas de deuxs have a romantic subtext, whether intentional or not. But in Rodeo Peck has crafted a pas de deux celebrating introductions and the process of getting to know another person.
New York City Ballet – Spring Gala with Odessa, Ratmansky premiere – New York
★★★✰✰ With only four works, no intermissions and a run time of less than two hours, New York City Ballet's Spring Gala was short and to the point.
San Francisco Ballet – Stravinsky Violin Concerto, Prodigal Son, Diamonds – San Francisco
★★★✰✰ The soloists and principals of San Francisco Ballet brought unique interpretations to George Balanchine classics for the company's fourth program of the season, a varied survey of Balanchine's oeuvre.
San Francisco Ballet – Fearful Symmetries (premiere), Rubies, Drink to Me Only with Thine Eyes – San Francisco
★★★✰✰ "But despite his high concept, Scarlett seemed to lose the thread halfway through, resorting to lascivious theatrics to complete the work…"
Dores André – Principal, San Francisco Ballet
If it was up to me, I would pay more [salary] to the corps than to the principals. Half the ballet depends on them, and it is very hard to be in a good mood and trying your hardest every day when you are not getting the attention and the development that we get as principals and soloists.
San Francisco Ballet – Caprice, The Four Temperaments, Swimmer – San Francisco
Swimmer is one man's journey from being the stereotypical breadwinner… to his own self-realisation in a kind of isolated freedom.
San Francisco Ballet – Dances at a Gathering, Hummingbird – San Francisco
San Francisco Ballet's Program 4 is a double bill coupling a welcome return of Robbins' Dances at a Gathering with a Liam Scarlett's Hummingbird.
San Francisco Ballet – Program 3 including Myles Thatcher premiere – San Francisco
Program 3 features Hans van Manen's Variations for Two Couples, William Forsythe's The Vertiginous Thrill of Exactitude, Manifesto by Myles Thatcher and "The Kingdom of the Shades" from La Bayadère.
San Francisco Ballet – Prog 1: Serenade, RAkU, Lambarena – San Francisco
America's oldest ballet company, San Francisco Ballet, opened its eighty-second season with a triple bill that encapsulates the uniquely varied repertory developed by artistic director Helgi Tomasson…
Fall For Dance Festival – Program 1: Black Grace, SF Ballet, Russell Maliphant, Mark Morris – New York
New York City Center's Fall For Dance Festival opened its 11th season with a very fresh program…
San Francisco Ballet – Caprice, Hummingbird, Symphonic Dances – Paris
Audience applause during the evening recognised that Scarlett's Hummingbird was the starry centrepiece of the three Paris premieres.
San Francisco Ballet – Giselle – San Francisco
Though Tan dances with a wonderful lightness and an elegance not quite suited to a naïve peasant girl, she never really emotionally engages with the ardent Karapetyan, who possesses nuanced acting and strong technique, and deserves to have a partner who can match his level of commitment to crafting a role.
Gallery – UK National Dance Awards 2013
Gallery by Dave Morgan…
2013 National Dance Awards – Winners Announced
The Critics' Circle National Dance Awards for 2013 took place today in London at The Place's Robin Howard Dance Theatre.


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Wallstreetscope.com 26.06.2015
VimpelCom Ltd. was up 0.39% to trade at USD5.18 on June 25, at a volume of 121,939 shares. VimpelCom Ltd. has a weekly performance of – 6.860% and profit margin of -2.60%. Its monthly performance stands at -17.970%, with an analyst rating of 3.1 and dividend yield of 0.97%.
Cameron International Corporation ( CAM ) of the Basic Materials sector is down -1.06% (change from open -1.00%) this morning trading at a volume of 123,862 shares and price of $53.42. Cameron International Corporation (CAM)'s weekly performance is 0.73% and compared to their 52 week low, they are up 35.17%. Cameron International Corporation (CAM)'s monthly performance stand at 4.23% with a profit margin of 8.30% and has an analyst rating of 2.3 Juno Therapeutics Inc. ( JUNO ) of the Healthcare sector (Biotechnology) is down -0.94% already this morning, a change from open of -1.42%. Juno Therapeutics Inc. (JUNO) is trading at $48.55 at a volume of 123,858 shares. Juno Therapeutics Inc. (JUNO) has a weekly performance of – 5.730% and EPS growth this year of -457.40%. Juno Therapeutics Inc. (JUNO)'S monthly performance stands at 4.630% and has an analyst rating of 2.3 UPDATED: Today's Top 50 Trending Stocks New Gold, Inc. ( NGD ) of the Basic Materials is up 0.35% this morning at $2.89 and 123753 shares. The return on investment for New Gold, Inc. (NGD) is currently -13.10% and weekly performance of – 7.690%. New Gold, Inc. (NGD)'S monthly performance stands at – 6.800% with an analyst rating of 2.6 Unilever plc ( UL ) of the Consumer Goods sector is up 0.67% trading at a volume of 122894 shares. Unilever plc (UL) is trading at $44.810, with a weekly performance of 1.070%. Unilever plc (UL)'S monthly performance stands at – 0.020% with an analyst rating of 1 and dividend yield of 2.88% VimpelCom Ltd. ( VIP ) of the Technology sector is up 0.39% so far today trading at $5.18 at a volume of 121939 shares. VimpelCom Ltd. (VIP) has a weekly performance of – 6.860% and profit margin of -2.60%. VimpelCom Ltd. (VIP)'S monthly performance stands at -17.970% with an analyst rating of 3.1and dividend yield of 0.97%


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Copper Creek Canyon is a fantasy destination. It's an ever changing designer haven where you generally find more than you're looking for. Although we carry over 300 lines of furniture and accessories, we know where another 300 or so are hiding, and we're happy to find them for you – because that's what we do. We put you in touch with designs that make your home your home.
Interior Outfitters began in 1997 in Lafayette, Indiana as Southwest Territory, Inc. In 2001 we opened a second location in Indianapolis under our new name, Copper Creek Canyon Interior Outfitters.
Today, Copper Creek Canyon carries over 300 lines of Furniture and Accessories. We find our items everywhere…from the best Furniture and Western Design Markets in the country to small time craftsmen and artisans, whom we know personally, from out west. Specializing in heirloom-quality case goods, upholstered and leather pieces, we also show home accessories, art, jewelry, handbags and clothing. We sell right off the floor, through our warehousing location, and by drop shipping.
At Copper Creek Canyon, we stand behind our Service and Merchandise. Our furniture is made through the best construction methods devised, be it eight-way-hand-tied upholstery, or dovetailed joints… and many pieces carry a lifetime warranty. We also do in-home consultations. If you don't see exactly what you are looking for here, contact us… we can usually find it!
Thanks for Visiting!


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Thursday, August 28, 2014
Monday, August 18, 2014
One Month Old!
Our Sophie turned one month old on the 14th! We celebrated with a trip to the zoo, complete with a ride on the carousel. We also managed to fit in a trip to Bagels Forever!
Breakfast sandwiches for Mommy and Daddy!
Of course we chose the badger for her first ride!
Chilling out with the penguins… Sophie is really into it.
Hanging out at home with Red Dog and Purple Hippopotamus (we took this same picture when she was just a few days old)
We ended the day with a fashion show/dance party featuring the onesies made by Lia and Elsworth for the baby shower. I also made up a joke:
What song do they play at baby Halloween parties?
Boo, Baby Baby!
Posted by Amanda at 12:26 PM 4 comments:
Monday, August 11, 2014
Announcing Your Pregnancy, Library-Style
1. Gather some basic materials:
Plain notecards (the kind that don't open) and envelopes
Book pockets and date due slips (we ordered ours from Demco); do yourself a favor and order more book pockets than you think you'll need for this project. You'll likely find lots of uses for them! These are the same pockets we've used for Advent calendars.
An ink pad and date stamp (also from Demco)
Heart-shaped printable stickers
2. Stick the book pockets to the notecards.
5. Print the call number for books about babies (there are several; just choose one and know that no one is going to correct you, at least not out loud) on the heart-shaped stickers, and seal the envelopes with the stickers.
6. Pop the cards in the mail and wait for a delighted response from friends and family!
Sunday, August 10, 2014
Ten on Ten: "I Only Take Pictures of My Baby" Edition
More about Ten on Ten here!
Posted by Amanda at 7:04 PM 2 comments:
Labels: Babies, Family, Ten on Ten
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The community is invited to nominate sites or attractions in Columbus for the National Geographic Society Gulf States geotourism mapguide. Some local sites, like the Tennessee Williams Home Welcome Center pictured, have earned recognition on previous mapguides and are eligible to be nominated again. Photo by: Courtesy photo
June 22, 2013 6:52:37 PM
Is there something special about Columbus you think everyone should know about? Well, this is your opportunity to tell someone.
The U.S. Gulf States Geotourism Council is working with the National Geographic Society to create a U.S. Gulf States Geotourism MapGuide, interactive website and mobile app -- and you're invited to nominate sites for potential inclusion by July 15.
Residents, visitors, community organizations, tourism stakeholders and local businesses are asked to visit usgulfcoaststatesgeotourism.com to fill out a nomination form. To nominate a site offline, fill out a printed nomination form and submit it to the contact listed on the form.
"It really is an advantage for us to have sites included in the geotourism guide," said Nancy Carpenter, executive director of the Columbus-Lowndes Convention and Visitors Bureau. "We wanted to open this up to the public and offer everyone a chance to nominate a site."
This isn't the first time the Friendly City has been included in the guide. The Tennessee Williams Home, for example, earned a previous listing.
"Just think how many places here are eligible, like the Tennessee Williams Home and Friendship Cemetery, where two Mississippi governors are buried as well as a soldier from every war from the War of 1812 to the Bosnian conflict," Carpenter said.
What is geotourism?
Geotourism is tourism that sustains or enhances the geographical character of a place -- its environment, heritage, aesthetics, culture and the well-being of its residents, according to the National Geographic Society. It encompasses a range of travel including culture and heritage, history, food, nature, the outdoors, water, music, trails, natural landmarks, artisans and storytellers, as well as local legends that capture the region's unique character.
The U.S. Gulf States Geotourism Program wants to map the best of the Gulf States -- Alabama, Florida, Louisiana and Mississippi -- by capturing and promoting the essence of the region through the voices and stories of the people that live here.
Using the public's recommendations, the National Geographic Society will produce a geotourism website, app and print mapguide to promote travel to the Gulf States.
"The guidelines for making nominations are very simple," said Carpenter.
Ask yourself
A test for inclusion on the mapguide would be: is the spot, attraction, business, activity or experience distinctive to our town, area or region, and is it authentic to the Gulf States?
A few examples given by the National Geographic Society include:
Nominators are asked to provide a 20-70 word description of what makes their recommendation unique or interesting.
The National Geographic Society will work closely with the U.S. Gulf States Geotourism Council to select the sites and themes that will appear on the map. The Society will make the final determination on working and site selection.
"We hope everyone will take time to go by the site and make a nomination by the July 15 deadline," urged Carpenter.
On the Web:
■ usgulfstatesgeotourism.com
Jan Swoope is the Lifestyles Editor for The Commercial Dispatch.
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Photo by Shari AltmanLast year, I lost all of my tomato plants to the plant disease late blight. It was such a devastating experience, especially since my husband and I had nurtured them from seed.
One day we had healthy tomatoes about to set fruit and the next day we walked into the garden and found irregular brown spots on the lower leaves. We had heard about this disease on the radio and knew we had to pull up the plants, bag them, and send them to the trash. It was sad, but we didn't want to be irresponsible and aid in the spread of the disease.
Experts don't think late blight will be as much as a problem in the Northeast and Mid-Atlantic this year, but I think it's better to err on the side of caution.
Here are few things you can do to help prevent late blight:
- Sow your own tomato seeds or buy transplants from a local source.
- Grow varieties that show some resistance to late blight. We're trying the Matt's Wild Cherry tomato variety this year.
- Buy certified seed potatoes if you're planting potatoes this year.
- If you grew potatoes last year, be on the lookout for volunteers and destroy them as they come up. Late blight can overwinter on potatoes that were accidentally left in the soil.
- Learn the symptoms and signs of late blight. Cornell University has a wonderful website with a photo gallery showing the symptoms of late blight. You'll also find answers to FAQ about the disease and a pdf that clears up some late blight myths.
- Be vigilant about inspection. Look early and often for signs of the disease
Will you be trying a resistant variety of tomato or potato in your garden this year?


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Want to know more about our business and the services we offer? Use our website to get detailed information about us and meet our dedicated staff members. We're looking forward to working with you.
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I found these vintage treasures this weekend!
1. Green atomic clock by General Electric
2. Heart Arabia Finel enamel bowl
3. Framed deer paint by number
4. Flocked jackalope figurine
5. Tin toy toaster with cute decals
I have never seen a clock like this...I love the overall lines of the clock, shape of the clock face, and the atomic style burst in the center!
This jackalope was way too cute/weird not to bring home and this tin toaster will go perfectly with the pink Wolverine vintage toy kitchen that I picked up this past fall.
I am so fortunate to live in northern MN where people have room to store so many great pieces from the past in various out buildings, barns, garages etc... It makes attending yard sales and auctions extra exciting because you never know what you will stumble upon next!
Have you found anything good lately?


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John H. Manny–Founder of our Fortune
Hello Folks,
Today I am going to explore the foundations of the Manny, Dorr, and Tinker fortunes. John H. Manny (who sometimes gets confused with John P. Manny) was the inventor of the Manny Reaper, which was an early version of a horse powered combine. John was born in 1825 in Amsterdam, NY and later moved to Illinois in the early 1850's. He brought his young wife, Mary Dorr Manny with him. The reaper had already been invented by a man named Cyrus McCormick who patented the reaper in 1834. Later, McCormick sued Manny for the sum of $400,000 in 1855. Abraham Lincoln and Edwin Stanton (his future Sec. of War–although they did not get along at first) were two of the lawyers that worked for Manny during the trial. The trial was also the reason that Lincoln came to Rockford in 1855, and the only documented case of Lincoln being in Rockford after the town was established (Lincoln had been through the area during the Black Hawk War of 1832). Anyway, Manny won the case and stayed out of the poor house, which was good for the whole family. Unfortunately, Mr. Manny passed away of an illness just after the case was settled and Mary Dorr Manny became a young widow. She later married our Robert Tinker in 1870 as he had known her from his employment at the Manny Reaper Works, after her husbands death. So, Mary became Mrs. Mary Dorr Manny Tinker, and lived on our historic site until her death. John Manny was the first husband of Mary Dorr, and the cornerstone that statred the future financial success of the three families. Thank you for your interest!
This Week in the Tinker Time Line:
1861-Confederate constitution was adopted by the Confederate States of America.
1868-Senate hears the impeachment charges (second attempt) against President Andrew Johnson because he replaced Edwin Stanton with Gen. Lorenzo Thomas, and a vast number of other complaints. The impeachment effort failed.
1918-First cases reported in the deadly influenza epidemic that killed thousands.
One thought on "John H. Manny–Founder of our Fortune"
How are John P. Manny and John H. Manny related?


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The best way to find unusual or unique homes for sale is to follow popular online real estate websites such as Zillow, Realtor.com and Trulia. These sites often feature unique and unusual property listings.
Zillow has a Unique Homes section on its blog that highlights unusual houses for sale in the United States. Users can sign up to receive email notifications whenever new articles are published under the Unique Homes topic.
Realtor.com also has a Unique Homes section on its site that showcases unusual real estate listings. On Realtor.com, the Unique Homes topic is a sub-category of the News & Advice section.
Trulia does not have a website section dedicated specifically to unique properties for sale, but the Trulia blog often features articles about interesting property listings. For instance, the site has published articles about dome-shaped homes and homes with water slides.
Zillow, Realtor.com and Trulia are active on social media, so users who are interested in unique real estate listings can follow these sites on Facebook, Twitter and Pinterest to make sure they never miss an update about an unusual home for sale in the United States. Zillow and Trulia also have Instagram accounts that often feature images from interesting listings on their sites.


text: Hello everybody. I just inherited a magical gift today - I was given (on a long-term loan basis ) a beautiful 6 string by a dear friend. It belonged to a man named David Trevis, who was a phenomenal player and whose life was tragically cut short when he committed suicide a few years ago. It has been sitting in a studio for these last few years and today the inheritor of the instrument told me that he would rather see it be played and loved than for it to hang on a wall for the forseeable future. I have been playing for a little over 12 years, but the 6 string is a new beast for me. I don't know much about them, so I feel like a new player all over again (which is a good thing, always). One nagging thing, however, is identifying this bass. I can't figure out what it is. I'll attach two pictures of it, in hopes that one of the experts here can give me a hand in identifying it. Thanks in advance for any help that anybody can give me.


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MOUNT KISCO, N.Y. -- Financial author Jane Bryant Quinn offered several points of retirement advice to a large crowd that gathered Saturday at the Mount Kisco Public Library.
Quinn was on hand to promote her best-selling new book, "How To Make Your Money Last." Following the talk, Quinn took questions from attendees and signed several book copies.
Although stock market bubbles and subsequent crashes tend to tug on people's emotions and grab major headlines, Quinn emphasized the need for retirees to invest in well-balanced portfolios for the long run.
Even people who are in their 60's, Quinn explained, are still long-term investors if they are planning for 20 to 30 years.
"You are a long-term investor, don't think like a short-term investor,"
Quinn told the crowd to consider putting a portion of their savings - it can range anywhere from 35 to 70 percent - into stock-based funds. Specifically, the author recommended low-cost funds that track a stock-market average, such as the S&P 500. Doing so, Quinn noted, is a better option than assembling an portfolio of individual stocks, where good-performing ones can be offsets by poor-performing counterparts, or in picking mutual funds that are managed by "hot-shot professionals."
"If you have these funds, you are paying your manager to lose and why would you want to do that?"
Additionally, Quinn suggested investing in U.S. government-bond funds, with one exposed to short-term bonds and another tied to Intermediate-term ones. Investing in government bonds offer a floor on losses when stocks go down, Quinn added, as they tend to rise during the same periods. By contrast, Quinn cautioned against investing in high-yield (or "junk") corporate-bond funds, which have securities that carry higher risks of default. High-yield bond funds, Quinn noted, also go down in value at the same time as the stock market.
Quinn also addressed the annuities market, suggesting either "immediate" ones, where an amount of money is given to a company in exchange for specific payments soon after, or a deferred fixed annuity, where such payments are made after a waiting period. Quinn cautioned against "black-hat annuities," such as the deferred variable kind. A deferred variable type, Quinn warned, come with commissions and higher fees, which can erode any payments advertised in addition to a basic amount.
Quinn, a Daily Voice founding editorial director, is a former resident of Chappaqua and North Salem. She currently resides in the Dutchess County community of Poughquag with husband and Daily Voice CEO Carll Tucker, who grew up in the Mount Kisco area and spoke to the audience.
Tucker once owned The Patent Trader, which was a prominent local newspaper in Northern Westchester. He also served on the board of directors for Northern Westchester Hospital.
A video interview with Quinn and Tucker is attached to this story.
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