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In murine models of Crohn's disease, rheumatoid arthritis and colon cancer, IL-6 (interleukin-6) signalling via the sIL-6R (soluble IL-6 receptor; termed IL-6 trans-signalling) has been shown to promote the pathology associated with these conditions. These detrimental activities can, however, be selectively blocked by soluble forms of the gp130 (glycoprotein 130) receptor. Although sgp130 (soluble gp130) therefore represents a viable therapeutic modality for the treatment of these conditions, the mass manufacture of such biologics is often expensive. The advent of molecular farming has, however, provided an extremely cost-effective strategy for the engineering of recombinant proteins. Here, we describe the expression and production of a biologically active sgp130 variant that is expressed in transgenic tobacco plants as an ELP (elastin-like peptide)-fusion protein (mini-gp130–ELP). Mini-gp130–ELP consists of the first three domains of gp130 (Ig-like domain and cytokine binding module) fused to 100 repeats of ELP. Expression of mini-gp130–ELP did not affect the growth rate or morphology of the transgenic plants, and purification was achieved using inverse transition cycling. This approach led to an overall yield of 141 μg of purified protein per g of fresh leaf weight. The purified mini-gp130–ELP specifically inhibited sIL-6R-mediated trans-signalling as measured by binding to the IL-6–sIL-6R complex and through its ability to block sIL-6R-mediated activation of STAT3 (signal transducer and activator of transcription 3) phosphorylation and proliferation in human hepatoma cells and murine pre-B-cells. Consequently, the present study validates the potential application of molecular farming in transgenic tobacco plants as a strategy for the expression and purification of therapeutically advantageous biologics such as sgp130.
- cytokine
- elastin-like peptide (ELP)
- glycoprotein 130 (gp130)
- inverse transition cycling
- interleukin-6 (IL-6)
- tobacco
Cytokines are a series of small soluble polypeptides, which control cellular functions such as proliferation, differentiation and viability. Dysregulation of cytokine activity is often associated with the progression of chronic disease, and selective targeting of these proteins in various disease states has proven to be clinically beneficial. Such an approach is exemplified by the use of anti-tumour necrosis factor α regimes in rheumatoid arthritis and Crohn's disease.
Cytokines of the gp130 (glycoprotein 130) family, including IL-6 (interleukin-6), IL-11, LIF (leukaemia inhibitory factor), OSM (oncostatin M), CNTF (ciliary neurotrophic factor), cardiotropin-1, CLC (cardiotrophin-like related cytokine)/NNT-1 (neurotrophin-1)/BSF-3 (B-cell stimulating factor 3), neuropoietin and IL-27, share the membrane glycoprotein gp130 as a common signal transducer subunit (reviewed in [1,2]).
On target cells, IL-6 first binds to the IL-6R (IL-6 receptor). The resulting complex of IL-6 and IL-6R then associates with the signal transducing membrane protein gp130, thereby initiating intracellular signalling [3]. The gp130 subunit is expressed by most, if not all, cells in the body, whereas IL-6R expression is mainly limited to hepatocytes, neutrophils, monocytes/macrophages and specific lymphocyte subsets. Moreover, a sIL-6R (soluble IL-6R) has been found in various body fluids [4–11]. sIL-6R forms an agonistic complex with IL-6 that stimulates cells that only express gp130 [12,13] – a process that we have termed trans-signalling [12,14].
Interestingly, a naturally occurring sgp130 (soluble gp130) possesses antagonistic properties on IL-6 acting in complex with the sIL-6R, but not on IL-6 acting via the membrane IL-6R. At 100-fold higher concentrations, sgp130 also inhibits LIF and OSM, but not CNTF and IL-27 [15–17]. Therefore we have concluded that sgp130 is the natural inhibitor of IL-6 trans-signalling. Moreover, in mouse models of human diseases, sgp130 has been shown to be a potent candidate for therapy of, e.g., Crohn's disease, rheumatoid arthritis and colon cancer [18–21].
Following the initial report on expression of complete antibodies in transgenic plants [22], several alternative expression strategies have been described for the high-yield production of recombinant proteins in transgenic plants [23,24]. However, for many proteins, increased ER (endoplasmic reticulum) retention leads to a trapping of the recombinant protein within the plant [25,43]. Moreover, we have demonstrated a 2-fold increase in spider silk protein accumulation in tobacco leaves and a 40-fold increase in single-chain antibody accumulation in tobacco seeds after fusion to 100 repeats of an ELP (elastin-like peptide), with one peptide unit made up of Val-Pro-Gly-Xaa-Gly (Xaa is any amino acid except proline; [26–28]). This amino acid stretch is derived from the characteristic repeat motif VPGVG found in the native sequence for mammalian elastin. ELPs and ELP-fusion proteins are soluble in water below their transition temperature and become reversibly insoluble when the temperature is raised above the transition temperature (inverse transition cycling) [27]. Since we have shown that inverse transition cycling can be used to purify heat-stable spider silk proteins from transgenic tobacco leaves [26], we have now asked whether this principle could be transferred to non-heat-stable proteins with therapeutic potential. We therefore generated transgenic tobacco plants, which express a sgp130–ELP (mini-gp130–ELP) fusion protein under the transcriptional control of the plant virus promoter CaMV (cauliflower mosaic virus) 35 S. Such an approach led to the accumulation of large amounts of mini-gp130–ELP in the ER, which could be purified as a functionally active variant using inverse transition cycling.
Cells and reagents
HepG2 cells (human hepatoma cells) were obtained from the American Type Culture Collection (Rockville, MD, U.S.A./Manassas, VA, U.S.A.). BA/F3 cells (murine pre-B-cells) stably transfected with human gp130 (Ba/F3-gp130 cells) or gp130 and IL-6R cDNAs (Ba/F3-gp130-IL-6R cells) have been previously described [29,30]. All cells were grown in DMEM (Dulbecco's modified Eagle's medium) High Glucose culture medium (PAA Laboratories, Pasching, Austria) supplemented with 10% (v/v) FCS (foetal calf serum), penicillin (60 mg/l) and streptomycin (100 mg/l) at 37 °C with 5% CO2 in a humidified atmosphere. For the cultivation of Ba/F3-gp130-IL-6R cells, the standard DMEM was supplemented with 10% FCS, penicillin (60 mg/l), streptomycin (100 mg/l) and 10 ng/ml IL-6 or 10 ng/ml Hyper-IL-6. Hyper-IL-6 is a fusion protein of IL-6 and sIL-6R connected by a flexible polypeptide linker. sgp130Fc is a fusion protein of the extracellular portion of gp130 and the Fc portion of IgG1 [16,29]. Both recombinant proteins were expressed and purified as previously described [16,29]. Hyper-IL-6–Fc is a fusion protein of the Hyper-IL-6 and the Fc portion of an IgG antibody, which was expressed in transfected EBNA (Epstein–Barr nuclear antigen)-HEK-293 cells (human embryonic kidney cells) and was directly used as conditioned supernatant. Mouse anti-STAT3 (signal transducer and activator of transcription 3) mAb (monoclonal antibody) was purchased from Cell Signaling Technology (Beverly, MA, U.S.A.), and rabbit anti-phospho-STAT3 mAb (Tyr705) was from Cell Signaling Technology. Mouse anti-human IL-6R mAb M91 was from Immunotech (Marseille, France). Mouse anti-c-Myc mAb 9E10 was from Abcam (Cambridge, U.K.). Sheep anti-mouse IgG–horseradish peroxidase and goat anti-rabbit IgG–horseradish peroxidase were from Amersham Biosciences (Little Chalfont, Bucks., U.K.). All restriction enzymes were obtained from MBI Fermentas (St. Leon-Rot, Germany). [3H]Thymidine was purchased from Amersham Biosciences (Freiburg, Germany).
Construction of the binary vector for mini-gp130–ELP under the control of the CaMV 35 S promoter for the transformation of tobacco plants
The cytokine-binding portion (D1–D3) of the cDNA coding for sgp130Fc-opt (see below) was amplified by PCR using the sense-strand oligonucleotide primer gp130-Δsignal (5′-GACGTCACATGTTTAGCAGAGCTGCTGGATCCTTGC-3′) and the gp130-NaeI (5′-CAGTGCCGGCTCTGTCCTCGTAGGTGATGC-3′) anti-sense oligonucleotide primer. The plasmid pCR-Script-sgp130Fc-opt served as a template (GeneArt, Munich, Germany). Here, the codon usage of sgp130Fc was adapted to the codon bias for Cricetulus griseus (Chinese hamster) genes (for sequence information of the cDNA coding for mini-gp130–ELP and the protein sequence of mini-gp130–ELP, see Supplementary Figures 1A and 1B at http://www.BiochemJ.org/bj/398/bj3980577add.htm). The purified PCR product was digested with AflIII and NaeI and cloned into the plasmid pRTRA7/3-35S-anti-KRES-c-myc-100xELP [27], which was linearized with AflIII and NaeI. The generation of the 100xELP-fusion protein was described previously [26]. The resulting plasmid pRTRA7/3-35S-mini-gp130-ELP was digested with HindIII, and the fragment containing the expression cassette [35 S promoter/LeB4 signal peptide/mini-gp130/c-Myc tag/ELP/KDEL (ER-retention signal)/CaMV 35 S terminator] was cloned into the binary plasmid pCB301-Kan. The vector pCB301-Kan is based on the vector pCB301 [31] and was produced by the transfer of a BglII–BamHI T-DNA fragment containing the kanamycin resistance gene of the pBIN19 vector [32].
Transformation of tobacco
The mini-gp130–ELP encoding construct was transferred into Agrobacterium tumefaciens C58C1 (pGV2260) by electroporation. Tobacco (Nicotiana tabacum cv. SNN) leaf discs were transformed as described elsewhere [33]. Regenerated transgenic plants were grown in vitro on Murashige–Skoog medium containing 100 mg/l kanamycin. Regenerated plants were grown to maturity in the greenhouse and were screened for high expression by Western-blot analysis using the anti-c-Myc mAb 9E10.
Purification of recombinant mini-gp130–ELP
Green leaves from transgenic tobacco plants were ground in a mortar under liquid nitrogen. PBS (50 ml) was added to 10 g of ground leaves and the suspension was stirred for 5 min at room temperature (21 °C). The extract was cleared by centrifugation at 8000 g for 30 min. The supernatant was filtered [Syringe Filter 0.22 μm pore diameter (Roth, Karlsruhe, Germany)] and sodium chloride was added to a final concentration of 2 M. The solution was incubated in a water bath at 40 °C for 30 min to allow for aggregation of the ELP-fusion proteins. The aggregates were precipitated by centrifugation at 8000 g for 30 min at 40 °C. The precipitate was dissolved on a vertical shaker at 200 rev./min in 50 ml of PBS for 15 min at 20 °C. Insoluble material was removed by centrifugation at 8000 g for 15 min at 20 °C. The supernatant was supplemented with sodium chloride to a final concentration of 2 M. The solution was again incubated in a water bath at 40 °C for 30 min. The precipitate was removed by centrifugation at 8000 g for 30 min at 40 °C. The precipitate was dissolved on a vertical shaker at 200 rev./min in 2 ml of PBS for 15 min at 20 °C. Insoluble material was removed by centrifugation at 8000 g for 15 min at 20 °C.
Size-exclusion chromatography
The mini-gp130–ELP protein was further purified on a calibrated HiPrep 26/60 Sephacryl S-300 High Resolution column (Amersham Biosciences, Germany) using PBS as the mobile phase with a constant flow rate of 1.0 ml/min. For calibration, the high-molecular-mass standards (Amersham Biosciences, Germany) were used (see Supplementary Figures 4A and 4B at http://www.BiochemJ.org/bj/398/bj3980577add.htm). Fractions of 2.5 ml were collected, analysed by SDS/PAGE, pooled and concentrated.
Protein concentration
The method of Waxman et al. [34] was used to determine the protein concentration. The molar absorption coefficient (ϵ) of mini-gp130–ELP at 280 nm was calculated to be 64890 litres·mol−1·cm−1. The absorption spectra were recorded in the range of 240–320 nm (Supplementary Figure 5A at http://www.BiochemJ.org/bj/398/bj3980577add.htm).
Edman sequencing
The purified mini-sgp130–ELP (10 μg) dissolved in 10 μl of PBS was sent for N-terminal sequence determination by automated Edman degradation to Sequence Laboratories Göttingen (Seqlab, Göttingen, Germany).
Purified mini-gp130–ELP protein was incubated with Hyper-IL-6–Fc in DMEM containing 10% FCS overnight at 4 °C followed by addition of 50 μl of Protein A–Sepharose (50% slurry; CL-4B from Amersham Biosciences, Germany) for at least 1 h at 4 °C. Immunoprecipitates were washed twice with washing buffer (10 mM Tris, pH 7.6, 150 mM NaCl, 0.2% Tween 20 and 2 mM EDTA) and once with PBS prior to addition of Laemmli sample buffer [2×Laemmli: 100 mM Tris, pH 6.8, 4% (w/v) SDS, 200 mM dithiothreitol, 20% (v/v) glycerol and 0.1% Bromophenol Blue] and boiling at 95 °C for 5 min.
Microtitre plates (Greiner Microlon, Solingen, Germany) were coated with purified mini-gp130–ELP (5 μg/ml) in PBS. The plate was incubated overnight at room temperature. After blocking with 3% (w/v) BSA in PBS for 2 h, 100 μl aliquots of Hyper IL-6 (0.1, 0.5 and 1 μg/ml) [with or without antagonistic sgp130Fc (5 μg/ml)] were added. The plate was incubated for 1 h at room temperature. Hyper-IL-6 bound to the plate was detected by the non-neutralizing mAb M91 (working concentration 0.1 μg/ml) followed by anti-mouse IgG–horseradish peroxidase (1:1000) diluted in PBS/3% BSA. The enzymatic reaction was performed with soluble peroxidase substrate (BM Blue POD from Roche, Mannheim, Germany) at 37 °C for 30 min, and the absorbance was measured at 450 nm on an SLT Rainbow plate reader from Tecan (Maennedorf, Switzerland).
Immunoblotting and ECL (enhanced chemiluminescence) detection
For Western blotting, proteins separated by SDS/PAGE were transferred on to PVDF membranes (Hybond-P from Amersham Biosciences, Germany) by a semi-dry electroblotting procedure. Membranes were blocked in a solution of TBS (Tris-buffered saline; 10 mM Tris, pH 8, and 150 mM NaCl) supplemented with 0.02% Tween 20 and 5% (w/v) skimmed milk powder, and probed overnight with the indicated antibodies at 4 °C, followed by incubation at 21 °C for 1 h with horseradish peroxidase-conjugated secondary antibody. Immunoreactive proteins were detected using a chemiluminescence kit (ECL® plus Western Blotting Detection system; Amersham Biosciences, Germany) following the manufacturer's instructions. Band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.).
Proliferation assays
Ba/F3-gp130-IL-6R cells were washed three times with sterile PBS and resuspended in DMEM containing 10% FCS at 5×103 cells/well of a 96-well plate. The cells were cultured in a final volume of 100 μl with additional cytokines as indicated in the legend of Figure 4(C) for 72 h and subsequently pulse-labelled with 0.25 μCi of [3H]thymidine for 4 h. The specific radioactivity of [3H]thymidine was 25 Ci/mmol. Cells were harvested on a glass fibre filter (Filtermat A from Wallac, Turku, Finland) and the filter was microwave-baked for 2 min. Subsequently, the filter was soaked in 4.5 ml of liquid-scintillation cocktail (Betaplate Scint from Wallac). [3H]Thymidine incorporated into cellular DNA was determined by scintillation counting using the MicroBeta TriLux counter from PerkinElmer (Wellesley, MA, U.S.A.). Bioassays were performed with each value being determined in triplicate.
The mini-gp130–ELP-fusion protein is expressed in high amounts in leaves of transgenic tobacco plants and can be purified by inverse transition cycling
A cDNA coding for the cytokine-binding domains D1–D3 of gp130 was fused to a cDNA encoding 100 repeats of the ELP and placed under the control of the plant virus promoter CaMV 35 S (Figure 1A). For sequence information, see Supplementary Figures 1(A) and 1(B).
We used ELP units that were made up of the pentapeptide Val-Pro-Gly-Xaa-Gly, where Xaa is valine, glycine or alanine residue [26]. The LeB4 signal peptide and the KDEL signal at the C-terminus caused ER retention. KDEL-mediated ER retention was chosen because maintenance of single-chain Fv fragments and spider silk proteins as ELP-fusion proteins in the ER resulted in high-level accumulation in transgenic tobacco seeds and leaves respectively [26,27]. The expression of mini-gp130–ELP without an ER-retention signal in transgenic tobacco plants was not examined. A c-Myc tag was used for detection of mini-gp130–ELP in Western blotting. Transgenic tobacco plants were generated by leaf disc transformation [33], and leaves were harvested and tested by Western blotting (results not shown). High-producers were chosen (12 out of 25 for mini-gp130–ELP; Supplementary Figure 3A at http://www.BiochemJ.org/bj/398/bj3980577add.htm) and proteins were extracted from leaves with PBS. Protein expression of recombinant mini-gp130–ELP could already be detected after SDS/PAGE separation and Coomassie Blue staining of raw leaf extracts (Figure 2A, lanes 1 and 2). Recombinant mini-gp130–ELP was purified by two rounds of inverse transition cycling as described in the Materials and methods section (Figures 1B and 2A, lanes 3–8).
Finally, 2 ml of pure mini-gp130–ELP protein from 10 g of green leaves was further purified by size-exclusion chromatography on a calibrated HiPrep 26/60 Sephacryl S-300 High Resolution column (for calibration curve, see Supplementary Figure 4). We determined the molecular mass of monomeric mini-gp130–ELP by SDS/PAGE analysis to be 100 kDa. The difference from the calculated molecular mass of mini-gp130–ELP (77.4 kDa) is likely due to glycosylation within the gp130 portion of the mini-gp130–ELP-fusion protein. Since most of the mini-gp130–ELP behaved on the column like a 240 kDa protein as estimated by comparison with a calibration curve of molecular mass standards, it is likely that most of the mini-gp130–ELP formed at least dimers (large peak in Figure 2B). A smaller fraction formed even higher ordered oligomers (650 kDa), whereas monomeric mini-gp130–ELP could not be detected. On the other hand, it could not be excluded that the presence of the ELP region resulted in an elongated protein that was eluted in the position expected for a globular protein of higher molecular mass.
Mini-gp130–ELP from the major peak was collected and the concentration was quantified by UV spectroscopy using the method of Waxman et al. [34] (see Supplementary Figure 5A). Therefore the final yield of purified mini-gp130–ELP was 141 μg of purified protein per g of leaf fresh weight (Supplementary Figure 5B).
The correct cleavage of the LeB4 signal peptide is important, since the N-terminus of gp130 is needed for efficient binding to the IL-6–IL-6R complex [36,37]. The four N-terminal amino acids of the mature mini-gp130–ELP protein were determined by automated Edman sequencing (performed by Seqlab) to be ELLD, which corresponds to the wild-type sequence of mature gp130. Purified mini-gp130–ELP protein was routinely stored at −20 °C but was also stable at 4 °C and room temperature without detectable loss of soluble recombinant protein integrity (Supplementary Figures 2A and 2B at http://www.BiochemJ.org/bj/398/bj3980577add.htm).
Mini-gp130–ELP is biologically active and inhibits exclusively IL-6 trans-signalling, but not classical IL-6 signalling
Purified mini-gp130–ELP (4 μg) was incubated at 21 °C for 1 h with a molar excess of Hyper-IL-6–Fc and precipitated by Protein A–Sepharose. Hyper-IL-6 is a fusion protein of IL-6 and the sIL-6R connected by a flexible linker [29], which can directly bind to gp130. Here, Hyper-IL-6 was additionally fused to the Fc portion of an IgG antibody to allow for direct precipitation with Protein A–Sepharose. Hyper-IL-6–Fc was produced in EBNA-HEK-293 cells and was directly used as conditioned supernatant. As depicted in Figure 3(A), mini-gp130–ELP was precipitated by Hyper-IL-6–Fc (lane 2), whereas no mini-gp130–ELP remained in the cleared supernatant after immunoprecipitation with Hyper-IL-6–Fc (lane 1). Moreover, mini-gp130–ELP could not be precipitated by Protein A–Sepharose alone (lane 3: supernatant; lane 4: immunoprecipitated protein). Since it was very likely that mini-gp130–ELP formed dimers at higher protein concentrations (Figure 2B), we cannot exclude that these dimers also existed at the lower mini-gp130–ELP concentrations used for the immunoprecipitation and for the following experiments. Therefore it is conceivable that the interaction of one out of two sgp130 molecules of the mini-gp130–ELP dimer with Hyper-IL-6 is necessary for complete immunoprecipitation of mini-gp130–ELP. Since there was no detectable mini-gp130–ELP remaining in the supernatant after immunoprecipitation with Hyper-IL-6–Fc, we conclude that at least 50% of the mini-gp130–ELP molecules are biologically active in terms of binding to the complex of IL-6 and sIL-6R.
An ELISA was developed to detect the binding of mini-gp130–ELP to the IL-6–sIL-6R-complex. Mini-gp130–ELP protein was surface-adsorbed and increasing amounts of Hyper IL-6 were added, followed by detection via the anti-IL-6R antibody M91 (Figure 3B). The mAb M91 directed against the human IL-6R was chosen for the immunochemical detection of Hyper IL-6. M91 recognizes the Ig-like domain of IL-6R and therefore does not affect binding of Hyper-IL-6 to mini-gp130–ELP. Minigp130–ELP alone did not cross-react with the detection antibodies. Binding of Hyper-IL-6 to mini-gp130–ELP could be competed by a molar excess of sgp130Fc. sgp130Fc consists of the complete extracellular part of gp130 (domains 1–6) fused to a dimerization domain (Fc part of a human IgG-antibody) [16]. These experiments clearly show that mini-gp130–ELP binds to Hyper-IL-6, which can be competed with the sgp130Fc protein.
Next, we asked whether mini-gp130–ELP could inhibit IL-6- or Hyper-IL-6-induced STAT3 phosphorylation. Human hepatoma cells (HepG2) were stimulated with 50 ng/ml of either IL-6 or Hyper-IL-6. We have shown previously that sgp130Fc exclusively inhibits the IL-6–sIL-6R complex without affecting the activity of IL-6 [16]. As shown in Figure 3(C), increasing amounts of mini-gp130–ELP led to a dose-dependent inhibition of Hyper-IL-6-induced STAT3 phosphorylation. On the other hand, mini-gp130–ELP was not able to inhibit STAT-3 phosphorylation when the cells were stimulated with IL-6 alone. These results indicate that mini-gp130–ELP specifically inhibits IL-6 signals that were mediated by the sIL-6R but not by the membrane-bound IL-6R.
The IL-3-dependent murine pro-B-cell line Ba/F3 is the only mammalian cell line known that completely lacks gp130 expression [38]. However, if these cells are transfected with gp130 (Ba/F3-gp130 cells), their reliance on IL-3 can be circumvented by stimulation with Hyper-IL-6 [29,39–41]. Ba/F3-gp130 cells were stimulated with a fixed concentration of Hyper-IL-6 in the presence of increasing amounts of mini-gp130–ELP. Minigp130–ELP inhibited Hyper-IL-6-induced STAT3 phosphorylation of Ba/F3-gp130 cells (Figure 4A). To prove that mini-gp130–ELP selectively blocked sIL-6R-mediated responses, an identical experiment was performed in Ba/F3 cells transfected with cDNA for both gp130 and the IL-6R (Ba/F3-gp130-IL-6R cells). These cells respond to either IL-6 or Hyper-IL-6; as shown in Figure 4(B), mini-gp130–ELP selectively blocked Hyper-IL-6-induced activation of STAT3, but not the IL-6-mediated phosphorylation of STAT3 in these cells. The activity of sgp130Fc was used as a comparative control protein. Moreover, the proliferation of Ba/F3-gp130-IL-6R cells stimulated by Hyper-IL-6 could be blocked by mini-gp130–ELP in a dose-dependent manner (Figure 4C). As expected, mini-gp130–ELP had no inhibitory effect on the proliferation of Ba/F3-gp130-IL-6R cells treated with IL-6 alone. Thus mini-gp130–ELP specifically inhibits IL-6 trans-signalling, but not classical IL-6-signalling.
Plant expression systems have the potential to be used for the production of safe and biologically active recombinant proteins. It has been estimated that proteins can be produced 10–50 times cheaper in plants than in Escherichia coli [42]. Therefore the combination of safe and low-cost protein production with a protein-intrinsic purification strategy would be very desirable. So far, there have been examples of high-yield production of recombinant proteins in transgenic plants, mainly in the field of molecular medicine such as antibodies, enzymes and vaccines [42,44,45]. Moreover, health risks that may arise from contamination of recombinant proteins with potential human pathogens or toxins are minimized by molecular farming [46]. Recently, an improvement of the production of recombinant single-chain antibodies and spider silk proteins respectively in seeds and leaves has been achieved by ER retention of ELP-fusion constructs [26,27]. Furthermore, inverse transition cycling of a spider silk ELP-fusion protein has been used for purification from leaf extracts [26]. Spider silk proteins are heat-stable and therefore represent an ideal model for inverse transition cycling, since the temporary heat treatment did not lead to protein denaturation. Initially, we generated transgenic tobacco plants accumulating sgp130Fc or sgp130Fc–ELP in the ER of leaves. It turned out that these proteins of approx. 800 and 1300 amino acids respectively were produced very poorly (<0.01 and 0.05% of total soluble protein respectively; Supplementary Figure 3A). Therefore we decided to generate transgenic plants, which accumulate a minimal mini-gp130–ELP-fusion protein in the ER of leaves. Mini-gp130–ELP only contains the cytokine-binding portion of gp130 (domains 1–3303 amino acids) fused to 100 repeats of ELP and was found to be produced in high amounts in transgenic tobacco leaves by ER retention (compare Supplementary Figures 3A and 3B). So far, it has not been examined whether ELP-fusion proteins, which are expressed in the cytoplasm or in plastids, will also result in increased protein accumulation compared with the non-fusion proteins.
Inverse transition cycling was applied to purify the mini-gp130–ELP to homogeneity. It has previously been shown that for sgp130Fc, a 2–5-fold molar excess was sufficient to inhibit IL-6/sIL-6R-mediated BA/F3-gp130 cell proliferation by 50% [16]. In sgp130Fc, the complete extracellular part of gp130 (domains 1–6) was fused to a dimerization domain (Fc part of a human IgG antibody). Thereby sgp130Fc mimics the receptor signal complex on the membrane, which also exists as a preformed dimer [47]. Interestingly, the preformed sgp130Fc dimer has a 10-fold higher efficiency to inhibit IL-6/sIL-6R-mediated responses than the sgp130 monomer [16]. For mini-gp130–ELP, an 80-fold molar excess inhibited IL-6/sIL-6R-mediated BA/F3-gp130 cell proliferation by 50%. Therefore we conclude that plant-produced mini-gp130–ELP antagonized the activity of Hyper-IL-6 less efficiently than the preformed sgp130Fc dimer but with efficiency comparable with a sgp130 monomer purified from mammalian cell-culture supernatants [16]. In this respect, mini-gp130–ELP resembles the functional properties of the highly truncated spliced variant of sgp130 known as gp130-RAPS (splicing variant of the gp130 mRNA; soluble gp130 receptor), which consists of D1–D3 of gp130 plus a unique C-terminal amino acid sequence [48,49]. Interestingly, mini-gp130–ELP very likely formed dimers, which in contrast with the preformed sgp130Fc dimer are non-directional, since mini-gp130–ELP exhibited antagonistic properties comparable with the monomeric sgp130 and not with dimeric sgp130Fc.
In summary, we have generated transgenic tobacco plants accumulating high amounts of biologically active mini-gp130–ELP in the ER, which could be purified to homogeneity by inverse transition cycling. The results reported in the present study are encouraging and will stimulate the generation of constructs with a cleavable ELP portion, which can then be dimerized artificially and in a directed fashion, e.g. by free accessible C-terminal cysteine residues. This strategy might lead to plant-produced mini-gp130-cytokine antagonists with even higher activity.
We thank Christine Helmold and Stefanie Schnell for excellent technical help and Simon Jones (Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, Wales, U.K.) for discussions and the help with the preparation of this paper. The work in the laboratory of S.R.-J. and J.S. was supported by grants from the Deutsche Forschungsgemeinschaft (Bonn, Germany). U.C. was supported by the Pharma Planta Project supported by the European Community.
Abbreviations: CaMV, cauliflower mosaic virus; CNTF, ciliary neurotrophic factor; DMEM, Dulbecco's modified Eagle's medium; EBNA, Epstein–Barr nuclear antigen; ECL, enhanced chemiluminescence; ELP, elastin-like peptide; ER, endoplasmic reticulum; FCS, foetal calf serum; gp130, glycoprotein 130; HEK-293, cell, human embryonic kidney cell; IL, interleukin; LIF, leukaemia inhibitory factor; mAb, monoclonal antibody; OSM, oncostatin M; sgp130, soluble gp130; IL-6R, IL-6 receptor; sIL-6R, soluble IL-6R; STAT, signal transducer and activator of transcription
- The Biochemical Society, London


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NewswireToday - /newswire/ -
Alexandria, VA, United States, 2013/03/06 - MorphoIDent Quickly Searches Both State and FBI Databases - MorphoTrak.com / Safran-Group.com. PAR: SAF | SAFRAN
MorphoIDent, a handheld mobile fingerprint identification device provided by MorphoTrak, a US subsidiary of Morpho (Safran), now allows patrol officers in Missouri to identify wanted or dangerous persons in the field without transporting them to a patrol station. With an interface to the FBIs Repository for Individuals of Special Concern (RISC), officers are even able to tell if the person is wanted in another state. The national RISC database includes approximately 2 million records of wanted persons, sex offender registry subjects, and known or suspected terrorists. The Missouri State Highway Patrol (MSHP) can now simultaneously search the state and the RISC fingerprint databases.
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In addition to determining true identity when a person gives a false name or has no ID, MorphoIDent can clear someone with the same name as another person, or confirm identification of incapacitated or deceased individuals. If there is no match, the print is discarded and the officer sends the individual on their way.
MorphoTrak, Inc. (morphotrak.com), a subsidiary of Morpho (Safran), provides biometric and identity management solutions to a broad array of markets in the U.S. including law enforcement, border control, driver licenses, civil identification, and facility/IT security. Reporting to Morpho, the Safrans security division, MorphoTrak is part of the worlds largest biometric company. Morpho is a leading innovator in large fingerprint identification systems, facial and iris recognition, as well as rapid DNA identification and secure credentials. With over 35 years experience, Morpho has captured more than 3 billion fingerprints and Morpho products are used by more than 450 government agencies in over 100 countries. MorphoTrak is headquartered near Washington D.C., with major corporate facilities in Anaheim, CA Concord, CA and Federal Way, WA.
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Monday, March 11, 2013 | 7:30 p.m.
The driver of a vehicle that hit a light pole Saturday night, killing a 15-year-old passenger, allegedly admitted to drinking several beers and smoking marijuana before the crash, according to an arrest report released Monday.
The fatal accident happened at 8:13 p.m. in the northbound lanes of Rainbow Boulevard, near Lake Mead Boulevard. The Clark County Coroner's Office identified the victim as Las Vegas resident Robert Lee Brown.
Police arrested Arturo Castillo, 23, in connection with felony driving under the influence of alcohol and drugs resulting in death. He was booked into the Clark County Detention Center, where he remains without bail.
Witnesses told police a Chevrolet Blazer was speeding — and appeared to be on two wheels — when the driver apparently lost control and struck a light pole, according to the arrest report.
Brown, who was in the backseat, was pronounced dead on the scene, police said.
Castillo allegedly failed field sobriety tests and a preliminary breath test, the report states. Officers also noted that he seemed confused and lethargic.
"Castillo could not grasp the severity of his actions and that a passenger who was riding in his (Castillo's) vehicle was deceased," police wrote in the report. "Castillo seemed very relaxed, calm and care-free."
Castillo and a front passenger suffered minor injuries, police said.


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By Godfrey Ojore
The leading youth television in Uganda, Urban Television, spent Monday in Soroti town meeting with youth and interacting with communities in the four divisions.
A crew of Urban TV led by the station manager, Michael Owor, combed the streets of Soroti town amidst ululations from the youth.
Excited fans celebrating the arrival of Urban Tv in Teso
"This is the only station that has given the youth a voice. I love it because all its programming's are pro youth," an excited Mary Atim said.
Business in Soroti town came to a standstill on Monday morning as boda boda riders clad in Urban Tv t-shirts rode along the streets celebrating the arrival of Urban Tv crew in Soroti.
Excited youth doning Urban Tv t-shirts in Soroti town
The crew visited Eastern, Northern, Western, and Central divisions and nearby sub-counties in Soroti districts like Arapai, Gweri and Asuret.
"We want to interact with our viewers. Two months since Urban TV went on air in Teso we have been receiving feedback and that is the reason we are here today," said Owor.
Fill in your Name and Email Address to receive a Free Newsletter
Excitement as Urban TV crew tours Soroti town


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Andrew Giuliani, the controversial son of Rudolph, himself the controversial former mayor of New York and presidential candidate, has made a solid start to his professional golf career. Taking some time off from suing his alma mater Duke University over being dismissed from the golf team, Giuliani notched his first pro victory in the Met Open on August 27, an event that is hotly contested among New York-area pros.
The win earned him $27,500. Then just two weeks ago he pre-qualified for PGA Tour Q-School in Dade City, Fla. He begins the long battle through Q School in pursuit of a PGA Tour card later this month.
But this weekend Giuliani learns that -- to paraphrase Tip O'Neill -- all golf is local. On Oct. 10 he tees it up in the Champions Cup against some of the best amateurs from New Jersey's most storied clubs. Though billed as a friendly matchplay event, the tournament comes with a PGA Tour subplot.
The Champions Cup lineup includes Ridgewood Country Club (which hosted The Barclays, the first leg of the FedEx Cup playoffs, in 2008 and will do so again in 2010); Plainfield Country Club (which will host in 2011); and Essex County Country Club, an underrated gem that bears the design imprints of A.W. Tillinghast, Seth Raynor and Charles Banks. Essex County CC recently underwent major improvements with an eye to snagging The Barclays in the future.
Noticeably absent from the lineup is Liberty National, the upscale private club that hosted this year's Barclay's, and which was almost universally panned by the pros.
Giuliani's opponents are Mike Stamberger (a two-time club champion at Plainfield), David Rahal (last year's champ at Ridgewood) and Greg Stebbins (the reigning Essex County club champion).


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- Reports
- Business Case for Emotional Intelligence
- Do Emotional Intelligence Programs Work?
- Emotional Competence Framework
- Emotional Intelligence: What it is and Why it Matters
- Executives' Emotional Intelligence (mis) Perceptions
- Guidelines for Best Practice
- Guidelines for Securing Organizational Support For EI
- Johnson & Johnson Leadership Study
- Ontario Principals' Council Leadership Study
- Technical Report on Developing Emotional Intelligence
- Measures
- Emotional Intelligence Quotient (EQ-i)
- Emotional & Social Competence Inventory 360 (ESCI)
- Emotional & Social Competence Inventory-University (ESCI-U)
- Genos Emotional Intelligence Inventory (Genos EI)
- Group Emotional Competence Inventory (GEC)
- Mayer Salovey Caruso Emotional Intelligence Test (MSCEIT)
- Profile of Emotional Competence (PEC)
- Schutte Self-Report Inventory (SSRI)
- Trait Emotional Intelligence Questionnaire (TEIQue)
- Wong's Emotional Intelligence Scale
- Work Group Emotional Intelligence Profile (WEIP)
- References
- Model Programs
- Achievement Motivation Training
- Care Giver Support Program
- Competency-Based Selection
- Emotional Competence Training - Financial Advisors
- Executive Coaching
- Human Relations Training
- Interaction Management
- Interpersonal Conflict Management - Law Enforcement
- Interpersonal Effectiveness Training - Medical Students
- JOBS Program
- LeaderLAB
- Self-Management Training to Increase Job Attendance
- Stress Management Training
- Weatherhead MBA Program
- Williams' Lifeskills Program
- Article Reprints
- Members
- Events
- Bookstore
Emotional Intelligence - Theoretical and Cultural Perspectives
By: Robert J. Emmerling, Vinod K. Shanwal, Manas K. Mandal (Eds.)
This book is designed to meet the growing need among researchers, graduate students, and professionals to look into the existing theoretical models as well as developing theories related to emotional intelligence. The primary aim of the book is to help readers get a view of current conceptualizations of emotional intelligence, while providing an opportunity to see how emotional intelligence has been interpreted and applied throughout the world. Psychological processes are expected to vary according to cultural meaning and practices.
Recent studies indicate that emotional intelligence influences behavior in a wide range of domains including school, community, and the workplace. At the individual level, it has been said to relate to academic achievement, work performance, our ability to communicate effectively, solve everyday problems, build meaningful interpersonal relationships, and even our ability to make moral decisions. Given that emotional intelligence has the potential to increase our understanding of how individuals behave and adapt to their social environment, it is an important topic for study.
Click here to order from Amazon.com
Peter Salovey
Robert J. Emmerling, Vinod K. Shanwal, Manas K. Mandal
Chapter 1 Emotions and the Ability Model of Emotional Intelligence
David R. Caruso
Chapter 2 A Comprehensive Framework for Emotional Intelligence
Benjamin R. Palmer, Gilles Gignac, Gina Ekermans and Con Stough
Chapter 3 The Cascading Impact of Culture: Group Emotional Competence (GEC) as a Cultural Resource
Elizabeth Stubbs Koman, Steven Wolff and Anita Howard
Chapter 4 Emotional Intelligence of Managers in India: Creating an Understanding
Gopa Bhardwaj
Chapter 5 Toward an Applied Science of Emotional Intelligence in the Global Workplace: Key Issues and Challenges
Robert J. Emmerling
Chapter 6 Assessing Emotional Intelligence Competencies in Two Global Context
Joan Manuel Batista-Foguet, Richard E. Boyatzis, Laura Guillen and Ricard Serlavos
Chapter 7 Emotional Intelligence- A Universal or a Culture-Specific Construct?
Mugdha Gangopadhyay and Manas K. Mandal
Chapter 8 Research on Emotional Intelligence: The Indian Experience
Ashok K. Srivastava, Anjum Sibia and Girishwar Misra
Chapter 9 Emotional Intelligence in Education: Applications & Implications
Vinod K. Shanwal and Gurpreet Kaur
Chapter 10 Cross-Cultural Competencies in a Major Multinational Industrial Firm
Lyle Spencer, Geoff Ryan and Urs Bernhard
Chapter 11 Creating an Emotionally Intelligent Classroom Culture
Janet Pickard Kremenitzer, Justyna K. Mojsa and Marc A. Brackett
News and Events
Check out our new EVENTS section to find out about the latest conferences and training opportunities involving members of the EI Consortium.
New Video Series From Dr. Daniel Goleman
Dr. Daniel Goleman has released a new video series outlining the 12 competencies of emotional intelligence in Crucial Competence: Building Emotional and Social Leadership. The series features in-depth video interviews with Daniel Goleman, George Kohlrieser, Richard Davidson, Richard Boyatzis, and Vanessa Druskat about how to foster emotionally intelligent leadership skills from an individual and organizational standpoint. It includes research-based evidence to show how EI competences differentiate performance, and specific methods to integrate these findings for effective leadership.
Listen to Consortium member Chuck Wolfe interview some of the thought leaders in emotional intelligence.
Interview with Dr. Maurice Elias
The show is about the Joys and Oys of Parenting, a book written by a respected colleague, Dr. Maurice Elias, an expert in parenting and emotional and social intelligence. Dr. Elias wrote a book tying Judaism and emotional intelligence together to help parents with the challenging, compelling task of raising emotionally healthy children. And while there are fascinating links to Judaism the book is really for everybody. Click here to listen to the interview.
Interview with Geetu Bharwaney
Challenges abound and life is stressful for many. So how do we cope? Chuck Wolfe interviews Geetu Bharwaney about her book, Emotional Resilience. Geetu offers research, insights, and most importantly practical tips for helping people bounce back from adversity. Click here to listen to the interview.
Interview with Dr. Helen Riess
Changes in healthcare have led to pressures on providers to spend less time with patients resulting in less time for questions, empathy and compassion. Helen Riess, M.D., a Harvard Medical Associate Clinical Professor of Psychiatry, and Chief Scientist and Chairman of Empathetics, is one person working hard to reverse this trend. Dr. Riess's company teaches empathy to doctors and other healthcare professionals often leading to very positive outcomes. Click here to listen to the interview.
Interview with Dr. Daniel Goleman
Listen to an interview by with Dr. Goleman on his new book Focus: The Hidden Driver of Excellence. In the book Dan helps readers to understand the importance and power of the ability to focus one's attention, will power, and cognitive control in creating life success. Click here to listen to the interview.
Interview with Dr. John Mayer
How Personal Intelligence Shapes Our Lives: A Conversation with John D. Mayer. From picking a life partner, to choosing a career, Jack explains how personal intelligence has a major impact on our ability to make successful decisions. Click here to listen to the interview.
Interview with Dr. Cary Cherniss
Click HERE to listen to an interview with Dr. Cary Cherniss co-chair of the EI Consortium. Dr. Cherniss discusses the issue of emotional intelligence and workplace burnout.
Interview with Dr. Marc Brackett
Click HERE to listen to an interview with Dr. Marc Brackett, the newly appointed leader of the Center of Emotional Intelligence which will begin operation at Yale University in April, 2013. In this interview Dr. Brackett shares his vision for the new center.
Special Issue of Cross Cultural Management: An International Journal.
Guest Editors:
Robert Emmerling
Richard Boyatzis
Emotional and Social Intelligence Competencies: Cross Cultural Implications
Continued research on the assessment and development of emotional and social intelligence competencies represents an opportunity to further both theoretical and applied applications of behavioral science to the management of human capital. While the field has continued to expand over the preceding decades, research has often trailed application, especially as it relates to cross-cultural validity. The purpose of this special issue of Cross Cultural Management - An International Journal serves to focus on cultural issues related to applied use of emotional and social intelligence competencies in diverse cultures. Articles in the special issue include data from various countries including India, Peru, China, Italy, Australia, and the United States. Click here to read more.


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A film exploring social media in the lives of consumers has gained new followers in the fresh produce industry.
Irving, Texas-based DMA Solutions Inc. and several produce companies have provided money and other resources to back a documentary film inspired by the popular Twitter hashtag "#followfriday."
The crew and cast of "Follow Friday the Film" set out June 26 to make a documentary in what the film's website described as an 8,000 mile, 38-day cross-country journey "to meet 140 strangers whose online recommendations shape our daily lives." The film's Twitter address is @ff_thefilm.
In a news release, Erin Faulk said her film will seek out experts in social media across the country and get their views on how platforms like Twitter and Facebook are changing the way business and consumers speak to each other.
The filmmakers raised funds for the production of the project with the help of the Kickstarter website, which allows interested parties to invest in creative projects. The film had a Kickstarter goal of $15,000, and received $22,000 with the help of produce companies, including California Giant Berry Farms, Duda Farm Fresh Foods, Mann Packing, Potandon Produce/Klondike Brands, Crunch Pak, DMA Solutions, Gills Onions, Mastronardi/Sunset Produce, Moonlit Farms, Onion Crunch and Stemilt Growers.
DMA president Dan'l Mackey Almy heard about the film and helped connect the project with produce companies.
"We are all involved with Twitter and we all see the value in social media," Mackey Almy said.
Mackey Almy said she met with the movie crew in Los Angeles in early June and discussed how produce companies may be a part of the effort.
Mackey Almy said she anticipates the film will have footage of fruit and vegetable growing regions, with scheduled visits with Potandon Produce, California Giant, Mann Packing and Duda Farm Fresh Foods,
Mackey Almy said she will also be tweeting about the progress of the film crew on her Twitter account, @whynotfresh, and the DMA Twitter account, @thecoreblog.


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Forum Replies Created
January 19, 2014 at 12:57 pm in reply to: Site tagline not showing #85926
it doesn't look like the site description has been set in WordPress. can you set that and post back?
January 19, 2014 at 12:50 pm in reply to: Using html5 citation tag in Agency Pro #85924
the <cite> tag is mainly for the title of the publication you're citing. the volume, section and page shouldn't be included in there.
January 19, 2014 at 12:40 pm in reply to: What are att. tags & categories? #85921
Reading this might help some: http://en.support.wordpress.com/posts/categories-vs-tags/
January 19, 2014 at 12:31 pm in reply to: Remove Header Entry Title from Only Pages #85919
something like what i have in the linked gist should work. https://gist.github.com/ozzyrod/a7e89a822f882776e987
January 19, 2014 at 12:14 pm in reply to: test site, backup and transfer to new server #85914
I think what you're looking for is http://serverpress.com/products/desktopserver/
I use WP Migrate DB Pro and it's awesome.
December 21, 2013 at 9:35 am in reply to: Reposition Post Meta under Post Info #80618
yup, there were some hook changes in HTML5. all set now?
December 21, 2013 at 9:28 am in reply to: Reposition Post Meta under Post Info #80615
it depends if you're using HTML5 or not.
for non-HTML5 sites:
for HTML5 sites:
December 19, 2013 at 11:48 am in reply to: Modern Portfolio [non-HTML5] Remove Filed Under #80248
Glad to be of assistance!
December 19, 2013 at 11:42 am in reply to: Modern Portfolio [non-HTML5] Remove Filed Under #80246
oh, you're using the featured posts widget. it's not a function you change, but a setting in the widget.
under Show Post Info, change what you have to [post_categories before=""]
that should do the trick.
December 19, 2013 at 11:30 am in reply to: Modern Portfolio [non-HTML5] Remove Filed Under #80241
try this: https://gist.github.com/ozzyrod/8043041
it works as i tried it.
December 19, 2013 at 11:10 am in reply to: Modern Portfolio [non-HTML5] Remove Filed Under #80236
something like this in the functions file should work:
add_filter( 'genesis_post_meta', 'anitac_post_meta_filter' );
function anitcac_post_meta_filter($post_meta) {
if ( is_home() ) {
$post_meta = '[post_categories before=""]';
return $post_meta;
December 3, 2013 at 12:09 am in reply to: How to Style Responsive Slider? #76769
i'm not seeing the issues your describing. were you able to fix them?
August 12, 2013 at 1:05 pm in reply to: Creating Pages #55838
yes and then use the Custom Menu Widget under Appearance > Widgets
August 12, 2013 at 12:19 pm in reply to: Upgrade now stops custom wrap #55831
if you didn't activate HTML5 support, then the what you had before should work just fine.
it doesn't look like you have activated it, so the #wrap declaration should still work.
August 12, 2013 at 4:20 am in reply to: Move Archive Text Intro #55753
i'd try moving it to the genesis_after_content hook


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Ask Experts & Get Answers to Your Questions - ASAP
Good morning,
According to Florida Administrative Code, Chapter 474.215, you do not have to be a dvm to own a clinic. Any individual or corporation may do so, however, each establishment must have a licensed veterinarian responsible for the management of the establishment.
Here is the website for the Florida Department of Business and Professional Regulations that refers to Veterinary Medicine. http://www.myflorida.com/dbpr/pro/vetm/index.html
Thank you for choosing JustAnswer.


text:
Trump Threatens to 'Spill The Beans' About Cruz's Wife After Ad Shows Naked Photo of Melania
An open war has broken out between Donald Trump and Ted Cruz over their wives.
A Super PAC supporting Cruz circulated a nude photo of Melania Trump with the caption: "Meet Melania Trump. Your next first lady. Or you could support Ted Cruz on Tuesday."
A furious Trump fired back on Twitter:
Lyin' Ted Cruz just used a picture of Melania from a G.Q. shoot in his ad. Be careful, Lyin' Ted, or I will spill the beans on your wife!— Donald J. Trump (@realDonaldTrump) March 23, 2016
Cruz claims he never approved the offending ad which was aimed at conservative Mormon voters in Utah and may have helped Cruz to win with over 69 percent of the vote there on Tuesday night. Trump decisively won the GOP primary in Arizona.
Cruz tweeted:
Pic of your wife not from us. Donald, if you try to attack Heidi, you're more of a coward than I thought. #classless https://t.co/0QpKSnjgnE— Ted Cruz (@tedcruz) March 23, 2016
Cruz also appeared on the Today show on Wednesday and said: "Donald is a bully. I told him last night: 'Don't you try and attack my wife.'"
He added: "Donald's trying to threaten my wife speaks volumes about character."
On Wednesday morning, Heidi spoke at a press conference in Waukesha, Wisconsin and addressed Trump's 'beans' comment.
"I'm sure you're aware by now that most of the things that Donald Trump says have no basis in reality," she said.
"The beans" Trump has been threatening to spill on Heidi Cruz may refer to her bout with depression.
In 2005, police in Austin, Texas, responded to a 911 call about a woman in a pink shirt sitting next to an expressway. It was there they found Heidi Cruz sitting ten feet from speeding traffic with her head in her hands.
Mrs. Cruz rebounded from her depression and told The New York Times earlier this year: "I want to use it to strengthen people around me and to recognize that we all have rough patches."
Jeb Bush re-entered the fray on Wednesday and endorsed Cruz.
"For the sake of our party and country, we must overcome the divisiveness and vulgarity Donald Trump has brought into the political arena," he said
He also posted the endorsement on social media:
I am endorsing @TedCruz. Ted is a consistent, principled conservative who has shown he can unite the party. https://t.co/znDl9GrNZC— Jeb Bush (@JebBush) March 23, 2016


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It may not be something at the top of your mind, but Judy Guido says if you aren't communicating your sustainable practices and services to the public, then you are missing an opportunity. If you've ever planted a perennial, incorporated ground cover on a project or used a GPS program to size a property instead of driving there, you've taken sustainable steps.
Guido, owner of Guido & Associates, a marketing and PR consulting firm gave the keynote address at the second annual Hologanix Bionutrional Summit.
Guido discussed research she did where she surveyed/spoke with 2,000 distributors, contractors, homeowners – the green industry marketplace about sustainability.
Here are some highlights of her speech:
• Guido said the myth that sustainability is a fad is not true. She backed it up with numbers that showed profits were 61 percent greater when top management was involved with sustainability within a company, while it's only about 40 percent with no top management involvement.
• She said the myth that green products are more expensive is also not true. Guido found green products were 6-8 percent less expensive.
• When Guido asks companies why they don't have a sustainability program, they say that their customers haven't asked. That's not the way to operate, Guido said. "Followers wait to be asked, leaders introduce innovation," she said. "We have a lot of followers in our industry. Thank God we have a lot of leaders."
• When trying to introduce sustainable services to customers, never ask them how it fits into their budget. In fact, never use the word budget in any question because that makes you an adversary to the customer. Instead, ask what they are willing to invest. "In investments, there is a return. (The customer) gets something back. We went from adversaries to advocates," she said.
Marketing and recruiting.
Chris Heiler, founder and president of Landscape Leadership, spoke on the topic of "How to use inbound marketing to convert website traffic into qualified leads and customers."
The main focus of Heiler's talk was how companies can be more purposeful with their marketing strategies, especially when it comes to online marketing. Heiler said that today, companies need to use inbound marketing, not 'outbound' marketing, because people are turned off when they're bombarded with advertisements. Inbound marketing is all about attracting potential customers to you; putting your business in a position to be found when potential customers are looking.
Another tip Heiler highlighted was how to make your company website more appealing. He said 75 to 90 percent of people visiting your website are not ready to buy. They're in fact-finding mode, meaning they are not ready to pick up the phone and call, or fill out a consultation form.
Most people make the mistake of only appealing to the people ready to buy, and their websites end up being a turnoff. If someone comes to your site and feels to pressured to speak to someone, they'll leave the site and when they do decide to look more seriously, there's a slim chance they'll come back to look at your company again.
Heiler also said it is important for companies to put their URLs everywhere: shirts, vans, invoices, etc, in order to make their company more visible to potential clients.
Recruiting. Bill Arman and Ed Laflamme of the Harvest Group delved into the deep end of the recruiting process, highlighting the 10 steps to recruiting successfully: look into retention versus recruiting, build and shape your culture, take stock and good care of what you have, identify the needs, identify the target, get your recruitment tools ready, practice the recruiting "best practices," go to the source, screening and interviewing, and hiring and on boarding.
Halfway through the talk, the audience was asked how many were currently looking for employees. Around half of the group raised their hands, with some saying they knew they would be looking soon. Arman and Laflamme said you always need to be on the lookout for potential hires, whether it be in an elevator or by looking up local colleges that offer courses for students with English as a second language. Instead of expecting potential employees to come to you, reach out and find them on your own.


text:
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Leadership accountability is becoming a more important issue with every pastor, politician, and leader in the limelight who has a moral failure. Personal accountability has always been important, but now people are beginning to realize it.
Good intentions aren't enough. You need prayer, worship, and a steady diet of Scripture to stay focused. And for good measure, I also recommend using accountability questions and Internet accountability software.
15 Accountability Questions
Having an accountability partner is only successful if you consistently meet, are brutally honest, and ask the right questions. Ed Stetzer has compiled a list of 48 accountability questions used by leaders such as John Wesley, Chuck Swindoll, Neil Cole, and Church Multiplication Associates. For my own use, I've adapted these questions into my top 15 personal accountability questions.
- #1 – Did the Bible live in you this week?
- #2 – Do you give it time to speak to you everyday?
- #3 – Are you enjoying prayer?
- #4 – Do you trust God?
- #5 – When did you last speak to someone about your faith?
- #6 – Have you been honoring, understanding, and generous to your family and important relationships?
- #7 – Have you damaged another person by your words, either behind their back or face-to-face?
Lust of the Eyes (Generosity)
- #8 – Have you been materialistic or too focused on having something?
- #9 – Have you been generous?
Pride of Life (Humility)
- #10 – Have you been proud or too focused on being something?
- #11 – Are you giving God the glory?
Lust of the Flesh (Integrity)
- #12 – Is God honored in the way you eat and drink?
- #13 – Are you improving your health through nutrition and exercise?
- #14 – Have you been exposed to sexually alluring material or allowed your mind to entertain inappropriate thoughts about someone who is not your spouse this week?
- #15 – Have you been with a woman anywhere this past week that might be seen as compromising?
These questions don't cover everything, but they are manageable and help keep a broad scope of lifestyle goals in the forefront of your mind throughout the week.
Internet Accountability Software
According to a March 2002 Pastors.com survey, 54% of pastors had viewed Internet pornography within the previous year, and 30% of those pastors had visited within the previous 30 days. Keep in mind that those statistics do not include any inappropriate content often found on news sites, online ads, and countless other non-ponographic sites.
Good Internet accountability software helps keep leaders above reproach. I recommend and use Covenant Eyes because of its highly thorough monitoring and customization. I tested a free Internet accountability program but was disappointed by its inability to report questionable non-pornographic content.
Covenant Eyes allows users to add accountability partners who receive regular reports detailing and labeling Internet usage in order of maturity – highly mature (porn), mature (smut), mature teen, and teen. It also charts the average Internet usage for each hour of the day.
The content labels and customized reports are convenient, but the real value is in its detailed tracking. Covenant Eyes analyzes the URLs of all photos, stories, ads, and more on each Web page that is viewed. If there is any potentially questionable content on a page, Covenant Eyes usually catches it.
Good Internet accountability software is not just about porn. It tracks sexual content, violence, crude humor, and vulgarity. That scope makes it a great tool for quick and easy discussions between accountability partners about what types of media they let themselves consume on a regular basis.
Special thanks to Covenant Eyes for a complimentary review subscription of their Internet accountability software.


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GOLDEN POND, KY: Land Between The Lakes National Recreation Area hosts the annual Hummingbird Festival, Saturday and Sunday, August 2 and 3, from 10 a.m.-4 p.m. at Woodlands Nature Station. Admission to this special event is $6 ages 13 and up, $4 ages 5-12, and free for children 4 and under.
Ruby-throated Hummingbird migration in this area peaks in August. Highlights include hummingbird banding demonstrations, a wildlife art show, a wildlife sketching workshop, a native plant sale, live animal encounters, local wildlife experts, and activities for kids. Food, prepared by Land Between The Lakes Brandon Spring Group Center cooks, will be available for purchase.
Nature Station Interpreter, Brooke Gilley, explains "a large number of hummingbirds stop here on their journey south to Mexico and Central America. During this time, up to 200 hummingbirds might visit our feeders in a single day. The festival is an opportunity for people to witness these powerful birds, about the size of an adult thumb, in a fun, learning atmosphere."
Weekend events at Nature Station include:
Saturday, August 3
- 11 a.m. Hummingbirds 101
- 12 p.m. Gardening for Hummingbirds and Butterflies
- 1 p.m. Hummingbirds 101
- 2 p.m. The Art of Photography, Presented by Teresa Gemeinhardt
- 3 p.m. Hummingbirds 101
Sunday, August 4
- 11 a.m. Hummingbirds 101
- 12 p.m. Stories of the Hummingbird, Presented by Sonya Wood Mahler
- 1 p.m. Hummingbirds 101
- 2 p.m. Sketching Workshop: Find the Wildlife Artist in You, Presented by Lennie Fottrell
- 3 p.m. Hummingbirds 101
Land Between The Lakes National Recreation Area manages more than 170,000 acres in western Kentucky and Tennessee as part of the U.S. Forest Service and the Department of Agriculture's public lands program. Land Between The Lakes offers outdoor recreation, lake access, land management, and environmental education to over 1.4 million visitors annually. The Friends of Land Between The Lakes works in partnership with the Forest Service providing program services, funds development, and volunteer support.
To find more information about Land Between The Lakes, log on to the official website at www.landbetweenthelakes.us or call toll-free at 1-800-LBL-7077 or local at 270-924-2000. For more information on the Friends of Land Between The Lakes, log on to www.friendsoflbl.org. To subscribe to the official electronic newsletter, send an email to firstname.lastname@example.com. Land Between The Lakes official Twitter accounts include @LBLScreechOwl, @LBLNature, @LBLHomeplace, and @LandBtwnLakes.


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One sure sign that spring is just around the corner: I again missed the arrival of this year's Sports Illustrated swimsuit edition. I shouldn't be surprised.
The sight of lithesome young women writhing on the beach while wearing what appear to be three Post-It notes held in place by dental floss stopped titillating me around 1960. That was "The Year the Mystery Died." The year I began "borrowing" my brother's Playboy magazines.
What drew my attention to the current swimsuit edition was the furor over the appearance of Barbie® on an "ad overwrap" that makes her seem to be the cover girl. The overwrap appeared on only 1,000 of the nearly 6 million copies the issue is projected to sell.
But it was savvy marketing by Mattel, Barbie's manufacturer, which is offering a Sports Illustrated Swimsuit Edition Barbie that retails for $29.95. Little girls are not SI's target audience, but Mattel likely anticipated the press coverage the cover garnered.
"So what?" you say. SI's swimsuit issue is already filled with plastic women. Why get all huffy over the addition of one more? But to rational beings of both sexes, Barbie and her impossibly sculpted body perpetuate stereotypes. This is not a new observation.
Complaints of this sort began almost immediately after Barbie debuted in 1959. Even in that "Mad Men" era of rampant female objectification, women quickly pointed out that there was something wrong with Barbie's vital statistics.
Barbie stands 11.5 inches tall and measures 5.5-2.75-5.25. Using the Secret Barbie Multiplier of 6.55, I have determined that in the real world, these figures translate to 5 feet, 9 inches tall and 36-18-34.
Never mind how I calculated this. Let's just say that the image of a grown man hunched over a Barbie doll, a cloth tape measure in hand, is not one I want to appear on my Facebook page.
These are not unrealistic numbers, but women seldom achieve them in real life. Disclaimer: I once dated a Barbie-like young woman who had an 18-inch waist. Yes, she was slim and blonde, had closetsful of clothes and walked on tiptoes. Yes, she left me for a guy named Ken. Need I say more?
Despite my experience, and Mattel's attempts over the years to make Barbie a role model for girls by introducing Astronaut Barbie, Veterinarian Barbie and even Episcopal Priest Barbie, women remain upset. I understand. Because I hate Ken.
Barbie's boyfriend appeared in 1961 yet is impervious to the ravages of time. When did you last run across a real 53-year-old man who looks like Ken, dressed in the latest fashions, disgustingly ripped and being foisted upon young boys as a role model?
Mattel, it's time to get real. Ken 2014 should reflect the true state of most 53-year-old males.
Introducing Beer Gut Ken. An understated "Git–R-Done" baseball cap first draws our attention and leads the eyes slowly downward past an eight-day growth of beard to a red-and-black plaid flannel shirt, tucked into and spilling over the elasticized waistband of faded denim boot-cut jeans, which break over the top of immaculate white New Balance cross trainers. Accessories include "Just for Ken" hair coloring, a tiny tube of testosterone-boosting cream and, in the Dream House medicine chest, an itty-bitty bottle of Barbie-turates to help him cope with the success of his plastic paramour. The bottle's safety cap is missing because Ken's nonarticulated fingers never did work so well.
I envision a Ken-based magazine ad-overwrap campaign next spring targeting female readers.
A Guns & Ammo swimsuit edition, maybe.
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Today marks the 67th birthday of one Betty Mabry, muse and second wife of Miles Davis and funk-rock innovator in her own right. I'm reminded that Right To Rock author and now NYU professor Maureen Mahon scored a rare interview with the reclusive Davis that was posted on the now-defunct EbonyJet.com (hat tip to Duke University's Mark Anthony Neal for excerpting a party of it). In it, she wrote:
A former model and one-time Jet Beauty, Davis wore an enormous Afro and an electric smile, but she was more than another pretty face. She was the rare female recording artist who wrote, performed, and produced her own music. Davis did the arrangements for Betty Davis and produced her subsequent releases, They Say I'm Different, Nasty Gal (1975), and Crashin' From Passion (1979). "I was just interested in keeping the sound pure," she explained. She is matter-of-fact about these accomplishments. "I've been writing music since I was 12 years old," she said. "I always thought of myself as a songwriter more so than an artist." In fact, some of her earliest material was performed by other acts. In 1967, the black rock 'n' roll group The Chambers Brothers recorded her composition "Uptown" for their The Time Has Come album, and in the early 1970s, she wrote material for the Commodores, helping the fledgling band secure a deal at Motown. But it was when she began to write for herself that her artistic vision came into full flower.
Here's "Your Man My Man" from her debut album Betty Davis:
NYC will be celebrating Betty's birthday this Sunday at DROM, when Nucomme presents Betty's Story: A Multimedia Tribute. Still time to get advanced tix.
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Single Father Graduates from Providence House
When Sean finally hit bottom due to drug abuse, he decided to get help for him and his son, Austin, at Providence House. Sean earned his GED through the Providence House Working-to-Learn program and is now enrolled at LSUS. Sean continues to make the future better and brighter for his family.


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By Tamara L. Waters
Meow! Cats inspire more than just cartoons and humor, they can inspire crafts too. If you are a cat fan or know someone who is, try out a few homemade projects to decorate your home or give as gifts. If you have kids, they will also enjoy these kitty cat crafts on a rainy day or to bust some boredom.
Printable Cat Crafts for Kids
For young children, printable paper crafts are fun and easy to do. Whether you are looking for templates to create toilet paper tube kitties, cross stitch patterns or other simple cat crafts, this website has lots of them. This is my favorite go-to site for simple crafts for the kids.
Kitty Treat Jar
Save a plastic peanut butter jar with lid to create a cute kitty treat container. Wash the jar out thoroughly. Use felt to cut out cat eyes, nose, mouth and ears. Glue them onto the jar to create a cat face. Be sure you don't glue anything in such a way as to obstruct the lid. Store your kitty treats in the jar.
Assorted Cat Crafts
I found a great website that offers a variety of cute crafts: daniellesplace.com has cat-inspired crafts that are fun to make and appeal to all ages. From a decorated tuna can food bowl to placemats for feeding your kitty on, to simple paper plate crafts and more, you can find it here. There are step-by-step instructions and links, and it is definitely a craft site worth checking out. About.com also has a listing of assorted cat crafts and links to websites featuring cat crafts.
Cat Face Luminary
For this craft you will need craft paint, slick puff paints, clear spray paint (to seal the paints), a glass jar and a candle (a battery operated tea light will be safest).
Start by making sure the jar is clean and dry. Use the puff paints to create a cat's face on one side of the jar. Leave parts of the face open (the mouth, the eyes) to allow the candle to shine through. Paint and decorate the rest of the jar so it looks like a cat. Use a fine-tipped paint brush and black paint to create whiskers. After all of the paint has dried, take the jar outside and spray the outside with clear spray paint to seal it. Allow the jar to dry thoroughly, add a candle and you've got a unique decoration!
Sock Mouse Toy
This craft is more for your kitty than for you, but you will benefit from the fun of watching your kitty play. Cut the toe end off of a sock – any color will do. Put a jingle bell and a bit of crinkly cellophane (or use a piece of a grocery bag – you know, the noisy kind) in the sock piece. If you have catnip, add some of it. Sew up the sock piece then sew a length of yarn for a tail onto one end. Tie another jingle bell onto the end of the tail. For a variation, use a pipe cleaner instead of yarn (you might have to attach it by pushing it through the sock material then twisting it back onto itself). My cats seem to adore pipe cleaners. Don't forget the jingle bell on the "tail."
There are lots of books that offer instructions for fun cat crafts. I checked Amazon.com and found "Cat Crafts (Kids Can Do It)" by Linda Hembry, "Cat Crafts: More than 50 Purrrfect Projects" by Dawn Cusick, and "Sassy Cats: Purr-fect Craft Projects" by Sheila Haynes Rauen. Browse through your local bookstore or library to see what is available, and enjoy your crafting fun!
Read more articles by Tamara L. Waters
The personal opinions and/or use of trade, corporate or brand names, is for information and convenience only. Such use does not constitute an endorsement by CANIDAE® All Natural Pet Foods of any product or service. Opinions are those of the individual authors and not necessarily of CANIDAE® All Natural Pet Foods.


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Carolyn A. Staton
Carolyn A. Staton undertook her Ph.D. studies in the role of fibrinogen and its related fragments on the process of tumor angiogenesis at the University of Sheffield, during which she discovered a number of novel antiangiogenic agents which were patented by the University of Sheffield and led to the funding of her postdoctoral research position to investigate these further. These studies led to her being given the AACR–Novartis Scholar In Training Award (2000) and the BACR Translational Research Award (2005)—both in open competition. She was appointed a Lectureship within the Microcirculation Research Group in the Academic Unit of Surgical Oncology at the University of Sheffield in 2004, and her research focus is on the regulation of angiogenesis, the development of new blood vessels from the preexisting vasculature, in wound healing and tumor development and progression. Angiogenesis and hemostasis, the coagulation cascade leading to clot formation, are among the most consistent host responses associated with cancer. Many haemostatic proteins stimulate angiogenesis by upregulating the expression of vascular endothelial growth factor (VEGF) and its receptors on endothelial cells. Other haemostatic proteins act directly on endothelial cells to inhibit or stimulate angiogenesis. Moreover, recently proteins originally identified as important in neuronal guidance are now suspected to be involved in regulating angiogenesis, both positively and negatively. Thus, her research aims to establish a comprehensive understanding of the relationships between these important processes, both in physiological conditions and the manner in which this changes during cancer development as this has implications for cancer therapy. Staton also has a strong interest in the development and refinement of assays used to study angiogenesis and as such was an Executive Editor on a book entitled Angiogenesis Assays: a Critical Appraisal of Current Techniques published by John Wiley & Sons.
Biography Updated on 7 September 2011


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INFANT OBSERVATION -VOLUNTEERING OPPORTUNITY(1 Post)
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Math made fun
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Kidsday reporters James Rose, Isabel Ryan, Faith McQuiston and Joyce Kim, all from Locust Valley Middle School, hit the Math Museum in Manhattan. The museum features hands-on exhibits for children of all ages. Videojournalist: Patrick Mullooly (Dec. 13, 2012)
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Carbs to muscle: seven ways to enhance your muscle glycogen stores without increasing fat.
Great article...
We constantly tell bodybuilders about the muscle-building benefits of protein. It plays a direct role in muscle development by providing the body with amino acids. However, looking at the larger picture, muscle growth is not solely dependent on protein consumption. Carbohydrate consumption also plays an active role. In fact, the amount of carbohydrates stored inside muscles--called muscle glycogen--can determine whether or not muscles remain in an anabolic, or muscle-building, state. How vital are well-stocked glycogen stores? Protein intake above and beyond what reputable nutritionists say is "enough" won't boost muscle mass if glycogen stores are too low. On the other hand, if glycogen stores are full, chances of faster recovery and improved growth markedly increase.
So, why not simply load up on carbs in hopes of getting huge? Because consuming too many carbs creates the potential of increasing bodyfat. Taking steps to ensure that carbs are stored in muscles rather than as fat is half the battle in building mass without turning into the Pillsbury Doughboy. This month, I'll explain how to build glycogen stores without increasing bodyfat stores.
1 | Eat six meals a day Eating multiple daily meals leads to greater glycogen storage with less fat storage. For example, if you eat 450 grams (g) of carbohydrates daily divided among three meals, your body will digest those carbs in 150 g increments. Some will head toward muscles to make muscle glycogen, and some will be stored as fat. Splitting the same daily amount evenly among six meals (75 g per meal) will take away from their ability to uptick fat storage, leaving more for muscle glycogen. The result of splitting the same number of carbs among six meals a day is greater glycogen storage for better growth and fewer carbs stored as bodyfat.
2 | Honor your training Carbohydrates eaten before you train help power your workout and spare the breakdown of muscle tissue. Therefore, pretraining carbs have a job to do: fuel your training. Consequently, relatively few are stored as bodyfat. Carbohydrates eaten after training refill muscles with glycogen before having any ability to increase bodyfat storage. Carbs consumed before and after training protect your body against muscle breakdown and support glycogen levels, ultimately helping your body to grow. When carbs are performing an anabolic role--supporting growth--they are not making you fat.
3 | Determine your insulin sensitivity No, you don't have to take a blood test. Insulin sensitivity is a fancy description for a body's ability to handle carbohydrates. If carbs make you tired or tend to quickly smooth out your physique, you're likely to be more "insulin resistant" than an average person. For our purposes, this means that you likely pump out more insulin than someone who gets a lot of energy from carbs or does not gain bodyfat quickly by eating them.
If you are insulin resistant, you should stay away from sugar, juices, refined carbs (such as rice cakes), cold cereals, mashed potatoes and white rice. Instead, choose slower-burning carbs, such as red potatoes, yams, brown rice, pasta and buckwheat noodles. Their slower-burning character tends to facilitate the storage of glycogen instead of the storage of bodyfat by keeping insulin release at moderate levels.
4 | Alter your carb intake When you eat fewer carbs, your body undergoes all kinds of changes. Interestingly, your muscles start to "crave" carbohydrates. With fewer carbs, the ability of your muscles to utilize them--rather than store them as bodyfat--actually increases. When you return to eating more carbs, virtually all of them are stored in your muscles, making your physique look fuller and more impressive. This increase in glycogen stores triggers and supports protein synthesis, meaning that your muscles grow. So, pulling back on your carb intake for two or three days can actually help you grow. Just be sure to keep your protein intake a little higher during a carb cutback to protect against potential muscle breakdown, which is sometimes associated with a decrease in carbohydrate intake.
5 | Supplement with alpha-lipoic acid Alpha-lipoic acid (ALA) is an antioxidant supplement that also improves muscle glucose uptake by mimicking the action of insulin on muscle cells. The benefit is that carbs are more readily removed from blood and stored in muscles as glycogen. Greater glycogen storage translates into more energy and muscle growth, and it also staves off bodyfat storage. Try taking 100-200 milligrams two or three times a day with higher carb meals.
6 | Try acetic acid Acetic acid is found in vinegar. Yes, what you use on salads and vegetables. Touted in folk remedies, vinegar actually does have some benefits. Through the ages, vinegar has been used to increase appetite, offset fatigue and boost mineral absorption. Vinegar increases the absorption of calcium and, in animal studies, vinegar has been shown to augment glycogen formation--therein lies "offsets fatigue." Vinegar may help prevent fatigue by improving the body's ability to load carbohydrates into muscles (rather than allowing the carbs to flow into fat-storing pathways, which contributes to a lack of energy and vitality). Try adding three or four tablespoons of vinegar to your pre- and posttraining meals.
7 | Use omega-3s Omega-3 fatty acids are the fats in fish oil and flaxseed oil that help muscles become more receptive to the anabolic effects of insulin. Your body releases insulin when you consume carbohydrates, and insulin has an anabolic effect, helping to push carbohydrates into muscles. At the same time, insulin also has a bodyfat-storing effect. It can push those same carbohydrates toward fat storage and increase enzymes that help manufacture bodyfat.
Omega-3 fatty acids can help create an anabolic effect by allowing muscles to "pull" carbs in--with the help of insulin. This not only increases glycogen storage, but it also minimizes the ability of insulin to store carbs as bodyfat. Take 5-6 g of omega-3s per day.
Carbohydrates present bodybuilders with their biggest dietary dilemma. It's virtually impossible to get muscles to grow without carbs, but it's hard to keep bodyfat stores from swelling with them. Bodybuilders sometimes need a little help to coax their bodies to use carbs for muscle building rather than for bodyfat. If you follow the seven suggestions presented here, you'll be able to make much more muscle--and far less bodyfat--out of the carbs you're consuming on your bodybuilding diet.
Chris Aceto "Carbs to muscle: seven ways to enhance your muscle glycogen stores without increasing bodyfat". Flex. FindArticles.com. 25 Nov, 2009. http://findarticles.com/p/articles/m.../ai_n13790098/
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A New Sign Marks a Grand Old House in Almond
September 22, 2000
The Almond Historical Society's Hagadorn House, built in the early 1830s, sporting a new sign made by Almond residents Bill Banker and Dick Baker.
By TAMMY JAYCOX - Staff Writer
ALMOND - In 1971, Kenneth Wetherby Hagadorn, third generation Hagadorn to reside here, bequeathed his house on Main Street to the Almond Historical Society. This Victorian dream land is open to the public as a historical museum and visitor's attraction.
The Hagadorn House is located at 7 Main St., showing its new addition. Bill Banker and Dick Baker of Almond recently made the new sign that overlooks the front lawn. Baker's family are long time natives of Almond, and his wife, Kitty, is the president of the village's historical society. Banker is an Alstom worker by day and a blacksmith by night.
The house was built in the early 1830s, according to Kitty Baker, by Jesse Angell, a prosperous merchant whose general store was located across the street from the house. In 1867, the house was owned by the John Wetherby family. His daughter, Cornelia Wetherby, married Dr. William Hagadorn in 1869. The young couple lived with her mother until her parents, the Wetherbys, moved to their farm and house. It then became the home of the young Hagadorns.
There were several generations of Hagadorns who lived in the house. The second generation to live in the home was William and Alice Simmons Hagadorn. The couple owned a hardware store across the road from the home. The third generation was Kenneth and Marie Dodds Hagadorn. Marie was a nurse and Ken was a postmaster in Almond. The couple never had children.
As one enters the front parlor, the Hagadorn's furniture is still intact, sitting, telling its history to all those who walk past. Kenneth's postmaster's desk sits by the front window, a memorial of his years of postal service. A mahogany chair, bought in 1910 sits aside a pile of early 1900s books and magazines as one looks at the Victorian hat rack with an umbrella stand and a Pier mirror sitting in the hall containing a cherry wood stair case.
Cornelia's diary mentions the addition of the kitchen area in her 1868 entry. The two parlors are believed to have been built around the same time Dr. Hagadorn built his doctor's office, which today is a gallery of Victorian garb, jewelry and accessories.
Passing through the second parlor doorway, one is mesmerized by the enormous cooking fireplace exposed on the back wall over looking the original Hagadorn center table and oriental rugs. The fireplace was uncovered almost intact in 1942 by Kenneth and Marie Hagadorn. The two were curious as to what was behind an abnormally thick wall, tearing it down to reveal the fireplace that had been concealed for a hundred years.
The Historical Society found that the former owner, many years ago, finding more efficient ways of cooking and heating than fireplaces, sealed up the cooking fireplace, with the kettles, cranes, and other cooking utensils. The Society believes that all the fireplaces that are now seen by visitors were at one time covered by walls. The fireplace lays amid the portraits and china collections donated by local families, including a painted grain cupboard and a desk from 1840.
From the front door, one is led up the staircase to where they are met by a small children's room, restored with an 1870s wicker baby carriage holding a small doll and other original Hagadorn belongings. The next door is the entrance into "The Roosevelt Room," named due to a claim that young Teddy Roosevelt slept there for one night. The room is now used as a workroom for society members containing resource material and general printed histories and photographs.
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Where Can I Find Updates on the U.S. Money Supply?
Question: The money supply of the United States is such a key economic indicator, why don't we get a reading of it on a daily basis? It seems like it would be a lot more valuable gauge of the economy and the future than anything like the S&P500, Nasdaq100, Russell—and so on.
Paul Solman: The Fed reports the money supply weekly, Bill. Here's the latest report.
You can begin to decipher it by reading about the various money supply measures on the Internet.
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Nature Photographer
I have been taking nature photos all my life and have sold some as well. I love being somewhere that no one else has gone and taking a photo and offering it to the world that way the people who can not truly experience those stunning places can enjoy them in print. This is truly one of the joys of my life. I don't know how to live without a camera. But recently my camera was stolen from my car and I am not financially able to replace it. I feel lost and adrift without this part of me that felt like an extension of myself.
NoWhereToGoButUp 36-40, F 1 Response 1 Jan 10, 2010


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When I put out my own call for stories from Sallie Mae borrowers about its collection practices, my inbox flooded instantly with angst-ridden messages. Many of the messages spoke of being called several times a day at all hours, every day of the week whether at home or work. More than one person said they set up a separate phone number just for Sallie Mae, and several said they filed Chapter 7 bankruptcy just to stop the calls. (Filing can stop the calls, but does not usually discharge the debt.)
"My interest-only payments [from Sallie Mae] were going to be about $1,200 a month. If I were to pay that amount I would have had to either be homeless, not pay any of my credit card bills and not buy food," said Greg, a 37-year-old media specialist from Virginia. "The people I talked to on the phone ... treated me like I was some kind of delinquent because I tried to ask for some type of assistance on my huge payment. They even threatened to sue me and garnish my wages."
Another borrower messaged me that she had her tax refund garnished for several years by Sallie Mae, but those garnishments weren't applied to her debt.
"I have a receipt from the IRS showing [my refund] went to Sallie Mae. Yet Sallie Mae has no record of the thousands garnished from my tax refunds," said Carole, whose original debt of $38,000 has ballooned to nearly $90,000, mostly due to interest and fees.
Carole was not the only person who reported payments or garnishments later discounted or lost by Sallie Mae, and others sent stories about being misinformed about paperwork or deadlines over the phone and later slapped with late fees and penalties that drove up their balance -- despite following closely the instructions offered by Sallie Mae representatives.
Sallie Mae sees it differently.
"Sallie Mae provides financial literacy tools, proactive outreach and guidance to help customers experience success. For those who need additional assistance, we work with them to identify a payment plan scaled to their financial circumstances," says Patricia Nash Christel, vice president of corporate communications at Sallie Mae. "Helping customers successfully repay their loans is our top priority. "
Christel also said that at the end of 2013, nearly 85% of customers in repayment were up to date with their payments and that federal loan borrowers serviced by Sallie Mae had a default rate below 30% of the national average.
But all of the responses I got from Sallie Mae borrowers expressed not only frustration, but an underlying desperation, with many arguing that they felt they had no recourse.
"There are few options for private loan borrowers because there are no comprehensive laws requiring private student lenders to offer any type of relief," said Deanne Loonin, an attorney with the NCLC and author of the Sallie Mae report. "As for bankruptcy, [the NCLC] supports restoring full rights in bankruptcy for both federal and private loan borrowers, but it's no secret that this is a difficult time to get protections and relief for consumers passed through Congress."
Besides extending bankruptcy to include student loan debt, the NCLC policy recommendations include increased legal and federal oversight and accountability of financial lenders such as Sallie Mae, as opposed to the bailouts and incentives that have mostly been lavished on them up to this point.
For those former students who feel they suffer from harassment or deceptive practices by Sallie Mae, Loonin suggests they visit the NCLC's Student Loan Borrow Assistance Project or consult with a lawyer to find out more about their rights and other resources available to them.
For many, though, there is another answer.
"Sallie Mae needs to go out of business," said borrower Andie, 46, of Scottsdale, Ariz. "They are feeding off of a national crisis in the economy and made a choice to not work with people. Their treatment is absurd, inhumane, unethical, greedy and frankly blatantly illegal."


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nate:chevrolux: I just swapped my parents to VoIP as Dad wanted cheaper tolls. Mum wouldn't have any of this 'internet phone nonsense' though. So we did it on the down low.
Good work on the sly!
With 2talk, its very important to realise that 111 may not work as expected. The call might not be connected, and even if it is - the location information is not transmitted to 111 services.
From 2talk FAQ
Q. Can I make 111 calls on 2talk?
A. 2talk do not support emergency calling. However if you do make a 111 call it will connect if possible. The operator will not know your physical location so you will need to provide this information to them.
It might not seem like a big deal but if people are not aware of this - it could be extremely dangerous as emergency services may not know where to go. I've seen quite a few posts on geekzone where people have swapped out their folks or friends POTS lines to 2talk - it is critical that they understand this limitation and have backups (such as cellphones or using their neighbours phones).
Other VoIP providers such as WorldxChange do provide this service. For me it was a big driver in shutting down my 2talk connection as it was better safe than sorry. I now use my POTS line for my voice and while it could suffer outages (power outages, cable cuts, exchange outages etc), if the 111 call connects - at least they will know where I am.


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Andrew Wiggins wants to open up his recruitment a little bit and see what other schools have to offer other than just Kentucky and Florida State.
Many have thought for some time that Kentucky will be where Wiggins will play college basketball, but the current 2014 recruit wants to do some window shopping to see what other schools have to offer.
Schools have also gained interest because of the commitment of James Young to Kentucky.
Because Young and Wiggins are both wing players, schools like North Carolina, Kansas, Ohio State and Syracuse may now have added motivation to recruit the player most everyone agrees is the best non-NBA player in North America.
Still, Fulford said Wiggins is so unselfish, competitive and confident in his own abilities, that he wouldn't necessarily rule out Kentucky simply because they have added other talented wing players.
"No, Andrew's an extremely unselfish palyer so I don't know that that would have a determining factor on his school choice, as to what guys are there or what guys aren't there," Fulford said.
Calipari was at Huntington to see Wiggins the day the Harrisons committed. And Fulford reiterated that Wiggins still could potentially play with the Harrisons and Young, in what one NBA Director of Scouting told SNY.tv would be the greatest recruiting class ever.
"Obviously, it's not like they all can't play together," Fulford said. "I think some of the other schools maybe feel like they have a window now."
He added: "It takes certain personalities to be able to coexist like that. Obviously, Andrew is one of those guys who can do that because he's fairly unselfish."
Kentucky fans shouldn't get too excited about this, I would still bet the farm that Andrew Wiggins ends up in Lexington, no matter what class he decides to commit.