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TAL (transaldolase) was originally described in the yeast as an enzyme of the PPP (pentose phosphate pathway). However, certain organisms and mammalian tissues lack TAL, and the overall reason for its existence is unclear. Recently, deletion of Ser171 (TALΔS171) was found in five patients causing inactivation, proteasome-mediated degradation and complete deficiency of TAL. In the present study, microarray and follow-up Western-blot, enzyme-activity and metabolic studies of TALΔS171 TD (TAL-deficient) lymphoblasts revealed co-ordinated changes in the expression of genes involved in the PPP, mitochondrial biogenesis, oxidative stress, and Ca2+ fluxing. Sedoheptulose 7-phosphate was accumulated, whereas G6P (glucose 6-phosphate) was depleted, indicating a failure to recycle G6P for the oxidative branch of the PPP. Nucleotide analysis showed depletion of NADPH and NAD+ and accumulation of ADP-ribose. TD cells have diminished Δψm (mitochondrial transmembrane potential) and increased mitochondrial mass associated with increased production of nitric oxide and ATP. TAL deficiency resulted in enhanced spontaneous and H2O2-induced apoptosis. TD lymphoblasts showed increased expression of CD38, which hydrolyses NAD+ into ADP-ribose, a trigger of Ca2+ release from the endoplasmic reticulum that, in turn, facilitated CD20-induced apoptosis. By contrast, TD cells were resistant to CD95/Fas-induced apoptosis, owing to a dependence of caspase activity on redox-sensitive cysteine residues. Normalization of TAL activity by adeno-associated-virus-mediated gene transfer reversed the elevated CD38 expression, ATP and Ca2+ levels, suppressed H2O2- and CD20-induced apoptosis and enhanced Fas-induced cell death. The present study identified the TAL deficiency as a modulator of mitochondrial homoeostasis, Ca2+ fluxing and apoptosis.
- apoptosis
- Ca2+
- mitochondrion
- pentose phosphate pathway
- transaldolase
Metabolism of glucose through the PPP (pentose phosphate pathway) fulfils two unique functions: formation of R5P (ribose 5-phosphate) for the synthesis of nucleotides, RNA and DNA, and generation of NADPH as a reducing equivalent for biosynthetic reactions and maintenance of a reducing environment [1]. ROI (reactive oxygen intermediates) have long been considered as toxic by-products of aerobic existence, but evidence is now accumulating that controlled levels of ROI modulate cellular function and are necessary for signal-transduction pathways, including those mediating PCD (programmed cell death) [2]. A normal reducing atmosphere, required for cellular integrity is provided by GSH, which protects cells from damage by ROI. Regeneration of GSH from its oxidized form, GSSG, is dependent on NADPH produced by the PPP [1].
Understanding regulation of the PPP has been complicated by the fact that the pathway is comprised of two separate, oxidative and non-oxidative, branches [1]. Reactions in the oxidative branch are irreversible, whereas all reactions of the non-oxidative phase are fully reversible. TAL (transaldolase) was, on the basis of metabolites and enzymatic activities detected in yeast [3], originally described as an enzyme of the non-oxidative branch of the PPP. However, certain organisms [4,5] and mammalian tissues do not express TAL [6] and the non-oxidative branch can function without this enzyme [7,8]. Hence the overall reason for the existence of TAL has not been clearly established. Mutations in TAL have been associated with liver cirrhosis in children from three families [9–11]. Deletion of Ser171 was noted in five out of six cases [9,11], and this was found to lead to inactivation, proteasome-mediated degradation and complete deficiency of TAL [12]. The pathogenesis of liver cirrhosis has been associated with increased cell death of hepatocytes and biliary epithelial cells [13]. To understand the consequences of TAL deficiency on cell-death-signal processing, B-cell LBs (lymphoblasts) available from the first TD (TAL-deficient) patient were investigated. Relative to control LBs, TD B-cells exhibit increased spontaneous, H2O2-, and CD20-induced apoptosis and reduced CD95/Fas-dependent apoptosis. Altered cell-death-signal processing is mediated by a unique pattern of metabolic changes affecting (1) the PPP (characterized by elevated S7P (sedoheptulose 7-phosphate) and diminished G6P (glucose 6-phosphate) levels, which reflect blocked recycling of G6P for the oxidative branch, (2) nucleotide metabolism (depletion of NADPH and elevation of ADP-ribose), (3) Ca2+ fluxing (increased cytoplasmic and mitochondrial Ca2+levels and elevated CD38 expresssion) and (4) decreased Δψm (mitochondrial transmembrane potential) and increased NO (nitric oxide) production, increased mitochondrial mass, increased ROI and ATP production. Normalization of TAL activity reversed the elevated CD38 expression and Ca2+ levels, the increased H2O2- and CD20-induced apoptosis and the diminished Fas-induced apoptosis. The results indicate that TAL regulates the PPP via G6P recycling and thus controls NADPH production, nucleotide metabolism, Ca2+ fluxing, the Δψm and pathway-specifically influences apoptosis signal processing of human B-cells.
Cell culture
Human EBV (Epstein–Barr virus)-transformed human B-cell LBs were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS (fetal bovine serum), 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B. EBV-transformed LBs from TD patients and human control donors [GM13416 (Coriel Cell Repositories, Camden, NJ, U.S.A.) and JAB (a B-cell line we established in our laboratory from a human donor)] were established as described previously [12]. Human FBs (fibroblasts) were maintained in Hams F-10 medium supplemented with 20% FBS, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B. FBs were isolated from a skin biopsy obtained from the lateral aspect of the left thigh from a TAL-deficient patient [9] and age-matched female controls (#1 and #2). All cell lines were maintained in a humidified atmosphere with 5% CO2 at 37 °C. Cell-culture products were purchased from Cellgro (Mediatech, Herndon, VA, U.S.A.).
Induction of apoptosis and cell viability assays
Apoptosis was induced with H2O2 (100 μM; Sigma, St Louis, MO, U.S.A.) and Fas Ab (antibody) CH-11 (1 μg/ml; Upstate Biotechnology, Saranac Lake, NY, U.S.A.) as previously described [14,15]. CD20-mediated apoptosis was induced by cross-linking with goat anti-human IgG. Tissue-culture wells were pretreated with 100 μg/ml goat anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, U.S.A.) at 37 °C for 2 h, washed twice with PBS, then treated with CD20 Ab (10, 100 or 1000 μg/ml rituximab; Genentech, South San Francisco, CA, U.S.A.), and washed again twice with PBS before the addition of cells at 106/ml. Apoptosis was quantified by staining with annexin V-Alexa 647 (Molecular Probes; excitation at 650 nm; emission at 665 nm recorded in FL-5) and exclusion of propidium iodide (excitation at 535 nm; emission at 617 nm recorded in FL-2).
Flow-cytometric analysis of Δψm, mitochondrial mass, ROI and NO production and cytoplasmic and mitochondrial Ca2+ level
Δψm was estimated by staining with 10 nM DiOC6 (3,3′-dihexyloxacarbocyanine iodide; Molecular Probes) [15] for 15 min at 37 °C in the dark before flow cytometry (excitation at 488 nm; emission at 525 nm recorded in FL-1). Δψm was also quantified using a potential-dependent J-aggregate-forming lipophilic cation, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide). JC-1 selectively incorporates into mitochondria, where it forms monomers (fluoresces green at 527 nm) or aggregates, at high transmembrane potentials (fluoresces red at 590 nm). Co-treatment with a protonophore, 5 μM mClCCP (carbonyl cyanide m-chlorophenylhydrazone; Sigma) for 15 min at 37 °C resulted in decreased DHR (dihydrorhodamine 123), DiOC6, and JC-1 fluorescence and served as a positive control for disruption of Δψm [15]. Δψm was also assessed by staining with 1 μM TMRM (tetramethylrhodamine methyl ester; excitation at 543 nm; emission at 567 nm recorded in FL-2; all from Molecular Probes). Mitochondrial mass was assessed by staining with potential-insensitive mitochondrial dyes 50 nM NAO (nonyl Acridine Orange; excitation at 490 nm; emission at 540 nm recorded in FL-1; Molecular Probes) or with 100 nM MTG (MitoTracker Green-FM; excitation at 490 nm; emission at 516 nm recorded in FL-1; Molecular Probes). Production of ROI was assessed using oxidation-sensitive fluorescent probes DCFH-DA (5,6-carboxy-2′,7′-dichlorofluoresceindiacetate), DHR and HE (hydroethidine; Molecular Probes) as previously described [14]. Although rhodamine 123, the fluorescent product of DHR oxidation, binds selectively to the mitochondrial inner membrane, ethidium and DCF (5,6-carboxy-2′,7′dichlorofluorescein) remain in the cytosol of living cells. DCF and HE (hydroethidine) preferentially detect H2O2 and superoxide (O2−) respectively [16]. Intracellular glutathione levels were assessed with 100 μM MCB (monochlorobimane; excitation at 380 nm; emission at 461 nm; FL-UV). Production of nitric oxide (NO) was assessed by using DAFFM (4-amino-5-methylamino-2′,7′-difluoroflourescein diacetate; Molecular Probes). Measurement of NO was calibrated by incubating cells with the NO donors NOC-18 {(Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium 1,2-diolate; 200 μM–1.8 mM} and sodium nitroprusside (400 μM–10 mM). C-PTIO (carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; 500 μM, 24 h), a specific NO chelator [17], was used to reduce NO levels and inhibit NO signalling. The [Ca2+]c (cytoplasmic Ca2+ concentration) was measured by loading the cells with 1 μM Fluo-3 acetoxymethyl ester (excitation at 506 nm, emission at 526 nm recorded in FL-1; Molecular Probes). The [Ca2+]m (mitochondrial Ca2+ concentration) was estimated by loading the cells with 4 μM Rhod-2 acetoxymethyl ester, which is compartmentalized into the mitochondria [18]. 2-APB (2-aminoethoxydiphenyl borane), a membrane-permeant antagonist of the IP3R [Ins(1,4,5)P3 receptor] and BAPTA-AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester); 5 μM] were used to inhibit Ca2+ signalling [17].
Western-blot analysis
Cytosolic lysates of LBs and FBs were prepared by harvesting cells in a mixture comprising 40 mM triethanolamine, pH 7.6, 10 mM EDTA, 1 mM sodium orthovanadate, 0.1 mM sodium molybdate, 10 mM sodium pyrophosphate and 50 mM NaF. Protein concentrations were determined by the Bradford method using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, U.S.A.). A 10 μg portion of protein lysates, unless otherwise indicated, were separated on an SDS/12%-(w/v)-polyacrylamide gel and electroblotted on to nitrocellulose. For whole-cell lysates, 2×105 cells were resuspended in 10 μl of sample buffer and boiled at 95 °C for 5 min prior to loading. Nitrocellulose strips were immunoblotted with anti-TAL Ab 170 and β-actin-specific mouse Ab 1501R (Chemicon, Temecula, CA, U.S.A.) that had been treated overnight with blocking reagents as previously described [19]. G6PD (G6P dehydrogenase) was detected with a rabbit Ab specific to G6PD (catalogue no. A300-404A; Bethyl Laboratories, Montgomery, TX, U.S.A.). Horseradish-peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories) was utilized as a secondary Ab for TAL, whereas biotin-conjugated goat anti-mouse IgG and streptavidin (Jackson ImmunoResearch Laboratories) were used as secondary and tertiary reagents respectively for the detection of β-actin. Protein bands were visualized by enhanced chemiluminescence using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Boston, MA, U.S.A.) using a Kodak Image Station 440CF and quantified with Kodak 1D Image Analysis Software (Eastman Kodak Company, Rochester, NY, U.S.A.).
Enzyme-activity assays
TAL, TK (transketolase) and G6PD activities were measured as described previously [14]. GPX (glutathione peroxidase) activity was measured in the presence of a mixture containing 150 mM KH2PO4, pH 7.0, 1 mM GSH, 0.25 mM NADPH, 3 units of glutathione reductase and 1 mM H2O2 [20]. Catalase was measured in the presence of 50 mM KH2PO4/Na2HPO4, pH 7.0, and 10 mM H2O2 at room temperature (20 °C) by continuous reading of A240 for 3 min [21]. SOD (superoxide dismutase) activity was measured in the presence of 0.05 M sodium pyrophosphate buffer, pH 8.3, 186 μM phenazine methosulphate, 300 μM Nitroblue Tetrazolium and 780 μM NADH [22].
Measurement of phosphorylated sugars by HPLC
A total of 107 cells were resuspended in 1 ml of 10% perchloric acid, and after three freeze–thaw cycles the precipitate was removed by centrifugation (15000 g, 10 min, 4 °C). The supernatant was neutralized with 120 μl of 10 M KOH. After centrifugation, the supernatant was filtered through a 0.2-μm-pore-size PVDF membrane, dried in a Speedvac apparatus and dissolved in 100 μl of water. The total protein content of each sample was determined using the Lowry assay [23]. A 25 μl portion was injected into the HPLC chromatograph equipped with a Dionex ED40 amperometric detector and the material analysed on a Carbopack PAI column (Dionex, Sunnyvale, CA, U.S.A.) using a gradient of 100 mM NaOH/1 M sodium acetate in 100 mM NaOH [24].
Measurement of nucleotides by HPLC
Pellets from 107 cells washed in PBS were resuspended in 50 μl of 0.16 M KCl/5 mM glucose at 4 °C. Then 100 μl of ice-cold 0.5 M KOH was added. The sample was mixed, diluted with 100 μl of ice-cold water and neutralized with 30 μl of cold 1 M KH2PO4. The extracts were centrifuged for 10 min at 15000 g at 4 °C, the supernatants were filtered through a 0.45-μm-pore-size polypropylene membrane, and 10–20 μl aliquots were injected into a 125 mm (length)×4.6 mm (diameter) Hypersil column packed with 3-μm-particle-size ODS-2 (Millipore Waters Chromatography, Milford, MA, U.S.A.) attached to a Waters Alliance HPLC chromatograph equipped with a Model 996 photodiode array detector set at 254 nm. The mobile phase was made with buffers A and B. Buffer A consisted of 0.1 M KH2PO4, pH 5.5, and 8 mM tetrabutylammonium hydrogen sulfate. Buffer B consisted of 70% (v/v) buffer A and 30% (v/v) methanol. Gradient elution was performed as follows: from 10% B to 75% B in 11 min; to 100% B at 12 min; back to 10% B at 21 min [25].
ATP measurement by chemiluminescence
Intracellular ATP levels were determined using the luciferin–luciferase method [26].
Electron microscopy
Cell pellets were fixed overnight in PBS with 2.5% (w/v) glutaraldehyde, post-fixed in 1% OsO4, dehydrated in a graded series of ethanols, infiltrated with propylene oxide and embedded in Araldite 502 epoxy resin. Ultrathin sections were stained with uranyl acetate and Reynold's lead citrate prior to examination using a Tecnai BioTWIN 12 transmission electron microscope (FEI, Hillsboro, OR, U.S.A.).
Microarray analysis of gene expression
For comparative gene-expression studies, RNA was extracted from TD and control FBs and LBs using the RNeasy kit from Qiagen. The mRNA fraction from approx. 5 μg of total RNA was amplified and labelled using the MessageAmp kit (Ambion). A 20 μg portion of biotinylated cRNA was hydrolysed randomly to 35–200 nucleotides in a fragmentation buffer solution (at 94 °C for 35 min). A 15 μg portion of the fragmented cRNA was then added to a hybridization buffer (100 mM Mes, 1 M Na+ (as NaCl), 20mM EDTA, 0.01% Tween-20, 0.1 mg/ml herring sperm DNA and 0.5 mg/ml acetylated BSA), containing known concentrations of positive control genes (50 pM Oligo B2, 1.5, 5, 25 and 100 pM Escherichia coli bioB, bioC, bioD and cre provided by Affymetrix, Santa Clara, CA, U.S.A.). The entire hybridization solution was heated at 99 °C for 5 min, equilibrated at 45 °C for 5 min, then centrifuged at 15000 g for 5 min before being injected into a U133A GeneChip (Affymetrix). After washing and staining, fluorescent images were scanned at 2 μm resolution using the Agilent G2500A Gene Array Scanner. The Microarray Analysis Suite, version 5.0 (Affymetrix), was employed to generate the comparative analysis presented in this study. Distinct algorithms of the software were used to determine the absolute call that distinguishes the presence (P) or absence of a transcript (A), the differential change in gene expression [increase (I), decrease (D), marginal increase (MI), marginal decrease (MD) and no change (NC)], and the magnitude (fold) change. Fold-change calculations were based on the average difference of each probe set, since this output is directly related to its expression level. After scanning the GeneChips, the Affymetrix software (MicroArray Suite 5.0) was used to calculate the intensity of the signal from each perfect match probe relative to the signal for the mismatch probe and to determine whether or not the gene was present in the sample [and a probability value (P<0.05) associated with this determination], as well as to measure the expression level of the gene. The overall chip intensities for each sample were scaled by linear adjustment to the same target value (1000). Pivot tables containing the scaled data from each experiment were generated and imported into GeneSpring (Silicon Genetics, Redwood City, CA, U.S.A.). In GeneSpring, the individual gene chip data were normalized by adjusting the median intensity of each array to a value of 1.0.
Flow cytometry of cell-surface antigen expression
TD and control FBs were freshly plated or 107 LBs/ml were resuspended in fresh medium 24 h before analysis. A total of 107 cells were stained in 200 μl of PBS with 1% (v/v) fetal-calf serum with primary antibodies specific for CD95 (ZB4 and IgG1; Upstate Biotechnology) or GGT (γ-glutamyl transferase; 3A8 and IgG2a [27]) for 30 min at 4 °C. IgG1 CD38 Ab was obtained from Caltag (Burlingame, CA, U.S.A.). ME0.5 IgG1 (Serotec, Oxford, U.K.) and OKT3 IgG2a antibodies (A.T.C.C., Manassas, VA, U.S.A.) were used as isotype controls. After washing them three times, cells were stained with phycoerythrin-conjugated goat anti-mouse or anti-human IgG for 30 min at 4 °C and analysed by flow cytometry.
Transduction of wild-type TAL cDNA by AAV (adeno-associated virus) vector (pAAV-IRES-hrGFP)
The full-length wild-type TAL cDNA [19] was inserted upstream of the IRES (internal ribosomal entry site) of the pAAV-IRES-hrGFP vector (Stratagene, La Jolla, CA, U.S.A.; where hrGFP refers to humanized Renilla green fluorescent protein). TAL-expressing AAV was produced by transfection of HEK-293 (human embryonic kidney 293) cells with TAL-containing pAAV-IRES-hrGFP, pAAV-RC (containing AAV replication and capsid genes) and pHelper plasmids, which supply the necessary exogenous gene products for virus production (Stratagene). At a 1:1 multiplicity of infection, >99% of cells infected with pAAV-TAL-IRES-hrGFP or pAAV-IRES-hrGFP control virus were GFP-positive. Maximal GFP expression was observed 24 h after virus infection. GFP and TAL expression were diminished by 50% after 48 h and became undetectable at 5 days post infection. For flow-cytometric analysis with FL-1 fluorochrome excited at 488 nm, changes elicited by the wild-type TAL-producing AVV were compared with those infected by AAV lacking TAL. In these viruses, GFP was replaced with the 792-bp neomycin phosphotransferase gene transferred from pUHD172.1neo [28]. Using Western-blot analysis, both TAL and NPT (neomycin phosphotransferase) were detected in pAAV-TAL-IRES-neo-transduced cells using rabbit Ab to NPT II (United States Biological, Swampscott, MA, U.S.A.; results not shown).
Results were analysed using the Student t test or Mann–Whitney rank sum test for non-parametric data. ANOVA was assessed with Graphpad (San Diego, CA, U.S.A.) software. Changes were considered significant at P<0.05.
Human experimentation
Our research was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association and had the approval of the ethical committees of our institutions.
Effect of TAL deficiency on metabolites and enzyme activities of the PPP and interconnected pathways
TAL catalyses the transfer of a three-carbon fragment, corresponding to dihydroxyacetone, to GA3P (D-glyceraldehyde 3-phosphate) and E4P (D-erythrose 4-phosphate) [1]. In the forward reaction of the PPP, TAL transfers dihydroxyacetone from S7P to GA3P, thus producing E4P and F6P (fructose 6-phosphate). The latter is converted by glucose-phosphate isomerase into G6P, which, in turn, becomes a substrate of G6PD. In the reverse reaction, E4P and G6P are converted into GA3P and S7P. To assess the impact of TAL deficiency on the PPP, unique substrates of the pathway were analysed by HPLC. As shown in Figure 1(A) and Supplementary Figure S1 at http://www.BiochemJ.org/bj/415/bj4150123add.htm, S7P was markedly accumulated both in TD FBs (P=0.012) and LBs (P=0.0049). By contrast, G6P levels were reduced in TD FBs (0.062±0.017 pmol/μg of protein) with respect to control FBs #1 (0.246±0.044 pmol/μg of protein; P=0.0132) or control FBs #2 (0.279±0.066 pmol/μg of protein; P=0.047). G6P levels were also diminished in TD LBs relative to the control LBs #1 (P=0.034) or control LBs #2 (P=0.039) (Figure 1A).
Accumulation of S7P and depletion of G6P indicate that TAL deficiency blocked the forward reaction, i.e. recycling of G6P for the oxidative branch of the PPP. The oxidative PPP generates NADPH [1]. NADPH, as well as NADP+, AMP, cAMP and NAD+ were diminished, whereas ADP-ribose levels were elevated in TD cells (Figure 1B and Supplementary Figure S2 at http://www.BiochemJ.org/bj/415/bj4150123add.htm). Following the alkaline extraction required to preserve the integrity of NADPH and NADH [14], ATP was degraded. Therefore, ATP was measured by the luciferase assay [26]. The ATP content of TD LBs was increased (2.8±0.3 pmol/μg of protein) in comparison with JAB (0.9±0.2 pmol/μg of protein; P=0.0002) and GM13416 control LBs (1.5±0.4 pmol/μg of protein; P=0.004).
Profound depletion of G6P and NADPH suggested diminished activity of the oxidative PPP. As previously reported [12], TAL protein and enzyme activity were undetectable in LB and FB of the TALΔS171 patient (Figure 2A). To assess the impact of TAL deficiency on the oxidative PPP, expression and activity of G6PD, the rate-limiting enzyme of NADPH production, was measured. Both G6PD protein (Figure 2A) and enzyme activity levels were markedly diminished in LBs and FBs of the TD patient relative to control cells (Figures 2B and 2C). Although activities of TK and GPX were not significantly affected, catalase and SOD were reduced in TD LBs and FBs (Figures 2B and 2C).
Loss of Δψm and increased mitochondrial biogenesis characterize mitochondrial dysfunction in TD cells
The oxidation–reduction equilibrium of pyridine nucleotides (NADH/NAD++NADPH/NADP+) regulates Δψm [29]. Since TAL deficiency may affect cell viability through controlling Δψm [14,15,30], annexin V-negative live cells were assessed for Δψm using potentiometric fluorescent dyes TMRM and JC-1 (Supplementary Figure S3 at http://www.BiochemJ.org/bj/415/bj4150123add.htm and Table 1). A significant decrease in Δψm was detected by both TMRM and JC-1 fluorescence. As altered incorporation of potentiometric dyes may represent changes in mitochondrial mass, the latter was assessed by staining with the potential-insensitive mitochondrial probes NAO and MTG. Surprisingly, the mitochondrial mass in TD cells was increased, as determined by both NAO (+104%, P=0.01) and MTG (+39%, P=0.0003) fluorescence. Along the same lines, electron microscopy showed increased numbers of mitochondria in TD LBs (29.2±5.2/cell) with respect to control LBs (+10.2±2.2/cell, P=0.0107). Mitochondria also appeared larger in TD cells (Figure 3), exhibiting features of megamitochondria [31]. Whereas intracytosolic and mitochondrial ROI production, monitored by HE (−15.0%, P=0.02) and DHR (−23.0%, P<0.01) fluorescence were reduced, DCF fluorescence, preferentially detecting H2O2 [16], was enhanced in TD LBs (+23.8%, P=0.0013). TD LBs also produced increased amounts of NO, as monitored by DAF-FM fluorescence (+61.3%, P=0.0081; Figure 3), which is a key trigger of mitochondrial biogenesis [32]. Since mitochondria store Ca2+ [18], we investigated the [Ca2+]c and [Ca2+]m levels. [Ca2+]c and [Ca2+]m of TD LBs were elevated with respect to control LB, as determined by Fluo-3 (+53.0%, P=0.023) and Rhod-2 fluorescence (+29.0%, P=0.001) respectively (Table 1 and Supplementary Figure S3).
Mitochondrial function was further tested by exposure of TAL-deficient and control LBs to a low dose (50 μM) of H2O2. Although H2O2 is freely diffusible, it is not, in itself, an ROI. Induction of apoptosis by H2O2, requires mitochondrial transformation into an ROI, e.g. OH−, through the Fenton reaction [33]. After exposure to H2O2, the potentiometric dyes showed mitochondrial hyperpolarization in accordance with previous findings [26,34]. Mitochondrial hyperpolarization is dependent on NO production [17]. TD cells exhibited a blunted response in H2O2-induced NO production and mitochondrial hyperpolarization (Supplementary Figure S3 and Table 1). Maintenance of GSH in a reduced state is dependent on the availability of NADPH [1]. Although NADPH was profoundly depleted, GSH levels were normal in TD cells, suggesting compensatory changes in GSH metabolism.
TAL deficiency elicits co-ordinated changes in expression of genes involved in the PPP, mitochondrial biogenesis, GSH metabolism and Ca2+ fluxing
The impact of TAL deficiency on the PPP and connected metabolic pathways was investigated in the context of global gene expression using the Human Genome U133A chip with probe sets of 22283 genes. The specificity of TAL-deficiency-induced changes on gene expression was validated by analysis of TD LBs transduced with an AAV vector expressing wild-type TAL. Expression of 18 genes was significantly and specifically altered by TAL deficiency, i.e., normalization of TAL expression by AAV-mediated gene transfer reversed the changes in expression of each of these genes (Figure 4). Reduced TAL transcription was detected in both TD FBs and LBs in comparison with control FBs and LBs, in agreement with earlier Northern-blot studies [12]. G6PD mRNA levels were reduced in a manner that correlated with diminished G6PD protein levels (Figure 2A). G6PD, TK, 6PGD (6-phosphogluconate dehydrogenase), PGI (phosphoglucose isomerase), PFKM (phosphofructokinase), TPI1 (triosephosphate isomerase 1), ALDC (aldolase C) and PGK1 (phosphoglycerate kinase 1) mRNA levels were all reduced in TD LBs and FBs, following the pattern of TAL expression. On the basis of ANOVA, expression of these enzymes was co-ordinately regulated in TD LBs and FBs (P=0.005). Interestingly, genes regulating mitochondrial biogenesis [PGC-1 (peroxisome proliferator-activated receptor γ-co-activator 1)', NRF-1 (nuclear respiratory factor 1), HMOX1 (haem oxygenase), NUDT1 (nucleotide diphosphatase type motif 1/8-oxo-7,8-dihydroguanosine triphosphatase), PTK2B (protein tyrosine kinase 2, Ca2+-regulated) and BNIP3L (bcl-2-like interacting protein 3-like mitochondrial protein)] were also co-ordinately altered in TD FBs and LBs. Expression of the Fas/Apo-1/CD95 cell death receptor was only increased in TD FBs but not in LBs (results not shown). Flow-cytometric staining with monoclonal Ab ZB4 confirmed increased surface expression of the Fas/Apo-1/CD95 on FBs but not on LBs of the TD patient (Figure 5A). Increased surface expression levels of GGT (Figure 5B) and CD38/NAD+ hydrolase were also confirmed on TD FBs and LBs by flow cytometry (Figure 5C). Overexpression of GGT may help maintain GSH levels in cells with diminished NADPH production. Indeed, incubation with GGT Ab for 48 h reduced GSH content of TAL-deficient LB measured by MCB fluorescence (results not shown).
Expression of wild-type TAL reverses changes in ATP, Ca2+ fluxing and apoptosis susceptibility of TD cells
The phenotype of homozygous TALΔS171/TD patient's cells may be influenced by mutations in genes other than TAL. Therefore, we normalized TAL activity by transduction of wild-type TAL cDNA using an AAV vector. At 1:1 multiplicity of infection, >99% of AAV-infected cells were GFP-positive (Figure 6A) and expressed functionally active TAL (Figures 6B and 6C). After AAV infection, TAL expression (Figure 6B) and activity levels reached ∼100% of those in control LB (Figure 6C). Normalization of TAL protein expression and activity resulted in reduced ATP (Figure 6D), increased GSH (Figure 6E) and reduced [Ca2+]c in TD LBs (Figure 6F).
B-cell homoeostasis is maintained through H2O2- [35], Fas/CD95- [36] or CD20-mediated apoptosis [37]. To investigate the impact of TAL deficiency on the processing of these cell-death signals, cells were exposed to 100 μM H2O2 or Ab to Fas/CD95 or CD20 12 h after AAV-mediated transduction of wild-type TAL and survival was measured by annexin-V staining 24 h later. H2O2-induced apoptosis was enhanced in TD LBs as compared with control LBs, a situation that was reversed after infection with wild-type TAL-producing AAV (Figure 6G). Although microarray analysis and cell-surface staining showed similar CD20 expression levels (results not shown), cross-linking of CD20 resulted in accelerated apoptosis of TD LBs (Figure 6H). Enhanced CD20-induced cell death was reversed by expression of wild-type TAL (Figure 6H). In accordance with its dependence on Ca2+ fluxing [37], increased CD20-induced apoptosis of TD cells was also reversed by pretreatment with 5 μM BAPTA-AM for 30 min (results not shown). In contrast, Fas-induced apoptosis was reduced in TD LB; which was also reversed by infection with TAL-producing AAV (Figure 6I). These results reveal that the apoptosis-susceptibility of B-cells is regulated by TAL in a signal-dependent manner.
TAL is an enzyme of the non-oxidative branch of the PPP which is the principal supplier (1) of NADPH for reductive biosynthesis and regeneration of glutathione from its oxidized form, and (2) of ribose 5-phosphate for nucleotide production. G6PD has been uniformly regarded as the rate-controlling enzyme for both of these functions of the PPP [1]. TAL deficiency has been found to be associated with liver cirrhosis in children from three families [9–11]. Utilizing LB and FB cells available from the first patient [12], the present study provides evidence that deficiency of TAL influences apoptosis signal processing in a death-pathway-specific manner. TD LBs exhibit increased spontaneous and H2O2-induced apoptosis. Fas-induced apoptosis was reduced in LBs of the TD patient. Fas-induced caspase activity is dependent on maintaining of active-site cysteine residues in a reduced state [38]. Co-ordinate up-regulation of GSH and Fas-dependent apoptosis by normalization of TAL expression suggests that oxidative stress limits activity of caspases in TD LBs. NADPH depletion may underlie oxidative stress. Increased H2O2 levels in TD cells is also consistent with deficiency of NADPH, which is required for conversion of H2O2 into water by catalase [21].
The mechanism of oxidative stress in TD cells was investigated at the level of PPP enzymes and metabolites. S7P, a substrate of the forward TAL reaction, was accumulated, whereas G6P was depleted. Accumulation of S7P is compatible with a failure to recycle R5P into G6P through the non-oxidative branch of the PPP, thus reducing NADPH production by the oxidative branch. ADP-ribose and ATP levels were clearly elevated, indicating that nucleotide synthesis was not limited, but rather stimulated, by the availability of R5P. The five-carbon sugars R5P, xylulose 5-phosphate and ribulose-5-phosphate are likely to accumulate and inhibit 6PGD [39]. Co-ordinate accumulation of ADP-ribose and depletion of NAD+ may be attributed to overexpression of CD38 [40,41]. CD38 or NAD+ hydrolase metabolizes NAD+ into ADP-ribose and cADP-ribose, which, in turn, stimulate the release of Ca2+ from the endoplasmic reticulum [40]. Normalization of TAL activity reduced CD38 expression and [Ca2+]c, observations that are consistent with the notion that oxidative stress is responsible for overexpression of CD38 and the resultant NAD+ depletion, ADP-ribose accumulation and increased [Ca2+]c. Moreover, mitochondria constitute major Ca2+ stores [42]; thus enhanced mitochondrial mass also favours higher [Ca2+]m levels. Increased mitochondrial mass allows for increased production of ATP. In turn, excess ATP could also inhibit G6PD and 6PGD [43].
Expression of genes associated with the PPP and mitochondrial biogenesis were co-ordinately regulated with TAL deficiency and normalized by AAV-mediated transduction of the wild-type TAL gene. Overexpression of Fas/CD95, both at the mRNA level and on the surface of TD FBs, has been associated with increased Fas-dependent apoptosis of these cells (results not shown). Fas expression was not enhanced, and Fas-dependent apoptosis was diminished, in TD LBs. On the basis of microarray and confirmatory flow-cytometric studies, CD38 was seen to be overexpressed on both TD LBs and FBs. The promoter region of CD38 harbours several redox-sensitive AP-1 (activator protein 1) motifs [44]. Thus oxidative stress may account for increased expression of CD38 in TD B-cells. Normalization of TAL activity reversed the elevated CD38 expression and Ca2+ levels, the increased H2O2- and CD20-induced apoptosis and the diminished Fas-induced apoptosis of B cells, clearly indicating that each of these changes resulted from TAL deficiency. Increased CD20-induced apoptosis was also reversed by the Ca2+ chelator BAPTA-AM, suggesting that the enhancement of this cell-death pathway is mediated by high Ca2+ levels. Although the role of B-cell apoptosis in human TAL deficiency is presently unclear, enhanced spontaneous and H2O2-induced apoptosis and inhibited CD95/Fas apoptosis could contribute to the pathogenesis of liver disease. Clinical manifestations of TAL deficiency are dominated by the consequences of liver cirrhosis [9–11], which results from increased cell death of hepatocytes and biliary epithelial cells [8,13]. Increased expression of CD38 [45], elevation of [Ca2+]c and [Ca2+]m, as well as depletion of NAD+ and NADPH, have been implicated in mitochondrial dysfunction, oxidative stress [46] and cirrhosis of the liver [47]. There is growing evidence for the involvement of the CD95/Fas cell-death-receptor-initiated apoptosis pathway in physiological regulation of hepatocyte turnover [48], and defective Fas signalling predisposes to hepatocarcinogenesis [49]. As oxidative stress leads to cirrhosis [47], whereas CD95/Fas resistance facilitates hepatocarcinogenesis [49], the present study identifying TAL as a signal-specific regulator of apoptosis is likely to be relevant for the pathogenesis of TAL deficiency.
We thank Dr David Fernandez (Department of Medicine, SUNY Upstate Medical University, Syracuse, NY 13210, U.S.A.) for helpful discussions and Dr Paul Phillips (Department of Medicine, SUNY Upstate Medical University, Syracuse, NY 13210, U.S.A.) for continued encouragement and support. This work was supported in part by grant DK 49221 from the National Institutes of Health, the Central New York Community Foundation and the Children's Miracle Network.
Abbreviations: AAV, adeno-associated virus; Ab, antibody; ALDC, aldolase C; annexin V-FITC, fluorescein-conjugated annexin V; annexin V-PE, phycoerythrin-conjugated annexin V; 2-APB, 2-aminoethoxydiphenyl borane; BAPTA-AM, 1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester); BNIP3L, bcl-2-like interacting protein 3-like mitochondrial protein; [Ca2+]c, cytoplasmic Ca2+ concentration; [Ca2+]m, mitochondrial Ca2+ concentration; C-PTIO, carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide; DAF-FM, 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate; DCF, 5,6-carboxy-2′,7′-dichlorofluorescein; DCFH-DA, 5,6-carboxy-2′,7′-dichlorofluorescein diacetate; DHR, dihydrorhodamine 123; DiOC6, 3,3′-dihexyloxacarbocyanine iodide; EBV, Epstein–Barr virus; E4P, D-erythrose 4-phosphate; FBS, fetal bovine serum; F6P, fructose 6-phosphate; FB, fibroblast; GGT, γ-glutamyltransferase; G6P, glucose 6-phosphate; G6PD, G6P dehydrogenase; GA3P, D-glyceraldehyde 3-phosphate; GLH, gluconolactone hydrolase; GPX, glutathione peroxidase; HE, hydroethidine; HMOX1, haem oxygenase; (hr)GFP, (humanized Renilla) green fluorescent protein; IP3R, Ins(1,4,5)P3 receptor; IRES, internal ribosomal entry site; JC-1, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide; LB, lymphoblast; MCB, monochlorobimane; mClCCP, carbonyl cyanide m-chlorophenylhydrazone; MTG, MitoTracker Green-FM; NAO, nonyl Acridine Orange; NPT, neomycin phosphotransferase; NRF-1, nuclear respiratory factor 1; NUDT1, nucleotide diphosphatase type motif 1/8-oxo-7,8-dihydroguanosine triphosphatase; PCD, programmed cell death; PFKM, phosphofructokinase; 6PG, 6-phosphogluconate; PGC-1, peroxisome proliferator-activated receptor γ-co-activator 1; 6PGD, 6-phosphogluconate dehydrogenase; PGK1, phosphoglycerate kinase 1; PGI, phosphoglucose isomerase; PPP, pentose phosphate pathway; PTK2B, protein tyrosine kinase 2; Ca2+-regulated, R5P, ribose 5-phosphate; ROI, reactive oxygen intermediates; SOD, superoxide dismutase; S7P, sedoheptulose 7-phosphate; TAL, transaldolase; TD, TAL-deficient; TK, transketolase; TMRM, tetramethylrhodamine methyl ester; Δψm, mitochondrial transmembrane potential
- © The Authors Journal compilation © 2008 Biochemical Society


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Even as the weather shifts from snowy to seasonable, colds and flu season isn't ready to say so long just yet.
Spring time often means the final swan song for feeling under the weather, even thought most of us aren't thinking about getting sick when March and April hits.
But inevitably either you or someone you know will get bitten by the flu or cold bug.
And when that happens, you likely want to be the first of many to wish a friend, family member or co worker a quick recovery that goes above and beyond a text message, simple phone call or even a card sent through the mail.
What about a gift basket?
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subset: commoncrawl


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Inspired by Cameddie, I also create my own iCarly fan character. But this is a "guest" character.
Travis Redfield is the creator of NeoGrounds. He "appears" in my fanfiction blog iFind NeoGrounds.
Relationships with other characters
Freddie Benson
Travis met Freddie one day in the AV Club on Ridgeway. They are very good friends.
Fanship: Fravis.
Carly Shay
Carly falls in love with Travis since she first sees him. They are friends.
When Travis had a good idea, Carly hugged him and Travis always asked nicely to let him go. That means, Travis wants to be just friends with Carly.
Fanship: Cravis.
Sam Puckett
It seems that Travis can deal with people like Sam. At first, Sam thought Travis was a "nerd techy". Travis frequently ignores Sam's mean comments and "gives her food" in change to not picking on him.
Travis considers Sam as a "frenemy", just like Freddie does.
Fansip: Savis.
Spencer Shay
Not much interaction, but they seem to be in good terms.
Spencer helps Travis and the iCarly gang to take payback to Nevel.
Fanship: Trencer.
Gibby Gibson
They are in good terms.
Gibby helps Travis and the iCarly gang to take payback to Nevel.
Fanship: Gravis.


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With the Inauguration of the 45th president, there's a lot to think about. WE ACT, a local organization (based right here in Harlem!) devoted to protecting our environment, is particularly concerned about what could happen under Scott Pruitt, who has been nominated to be the head of the Environmental Protection Agency.
Pruitt, under oath, claimed to not know important information about lead poisoning, asthma, and climate change. For those of us who are concerned about these crucial health issues, this is very worrying. WE ACT proposes four ways to resist the damaging environmental policies of the incoming administration.
If you're feeling worried, remember: you have power. There are many things you can do to make your voice heard, locally and nationally.


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Bolivia suspends airline after deadly crash in Colombia
Published 3:51 pm, Thursday, December 1, 2016
MEDELLIN, Colombia — Bolivia's Civil Aviation Authority on Thursday indefinitely suspended operations of the airline behind this week's air tragedy in Colombia, as relatives of the victims spoke out in anger, many saying the crash was avoidable.
The Bolivian agency said it was stopping all flights operated by LaMia airlines, amid reports its charter aircraft ran out of fuel moments before slamming into the Andes mountains on Monday, killing all but six of the 77 people on board.
Many of the victims were players and coaches from a small-town Brazilian soccer team that was headed to the finals of one of South America's most prestigious tournaments after a fairy-tale season that had captivated their soccer-crazed nation.
Latest news videos
Grieving relatives of the dead spoke out in disbelief and anger on Thursday after a recording of conversations between a pilot of the doomed flight and air traffic controllers, as well as the account of a surviving flight attendant indicated the plane ran out of fuel.
Williams Brasiliano, uncle of midfielder Arthur Maia, said the crash was avoidable if the team had chosen a commercial airline to travel to Colombia — not a charter.
"Look how complicated that flight was going to be even if it had arrived," Brasiliano said tearfully. "Even if they had arrived, it is clear that they would be tired from the trip to play a final. This can't be right. I doubt that a bigger club would have done the same."
Meanwhile, British aviation authorities said the flight data and cockpit voice recorders recovered from the accident site were being brought to Britain for study.
A recording of the exchange between the pilot and air traffic controllers, leaked to Colombian media on Wednesday, showed the pilot repeatedly requested permission to land because of "fuel problems," although he never made a formal distress call. He was told another plane had been diverted with mechanical problems and had priority, and was instructed to wait seven minutes.
Just before going silent, the pilot said he was flying at an altitude of 9,000 feet and made a final plea to land.
At a vigil late Wednesday, thousands of soccer fans jammed the stands of the 40,000-seat stadium where Brazil's Chapecoense team had been scheduled to play Medellin's Atletico Nacional in the Copa Sudamericana finals. With the words "Eternal Champions" blazing on a big screen, the Atletico fans urged the rival team be named the champion.


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Actors Ben Affleck and Matt Damon are set to reunite after 10 years for a biopic of New England's most notorious gangster, Whitley Bulger.
"Matt and I have been looking for something to do together for some time. We've heard about Whitley Bulger since we were kids, and we are excited by the prospect of putting it on screen," said Affleck.
Both the actors earlier teamed up for 1997 film "Good Will Hunting" for which they also won an Oscar.
While Affleck will direct the film, Damon will play the role of Bulger. Both of them will produce the movie under their banner, Pearl Street Films, reports aceshowbiz.com.


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We love the Internet. MakeUseOf readers would give up alcohol, caffeine and various limbs before giving up on this network. That's the value of the Internet. But what is it, exactly?
Let's ask Google, together.
Google's Suggest isn't just a useful feature: it's insight into the minds of millions. Start typing something into the search bar and Google will guess what you might be asking, based on what others have asked. This view into the collective can be disturbing, but I'm convinced the meaning of life is hidden somewhere in it.
Tina showed you interesting Google Suggest results and what they reveal 12 Interesting Google Suggest Results & What They Reveal 12 Interesting Google Suggest Results & What They Reveal Google Search is the modern world's confessional. Whatever it is that we need help with, we just type it into the patient white search box and hope for relevant results. Google even helps us complete... Read More . Today I'm going to use Google Suggest to find what exactly the Internet is. Here's what I found:
The findings are in – it's time for analysis. The Internet is…
…A Series Of Tubes
The top result shouldn't surprise anyone – "a series of tubes" is arguably one of the first memes to break into mainstream culture. But where did it come from?
The year was 2006, and the Senate was debating net neutrality. The late Senator Ted Stevens (R-Alaska), the man in charge of regulating the Internet, decided it would be helpful to explain just what the Internet is.
Turns out it's not a big truck.
Millions watched this speech, ironically using up a great deal of valuable tube space in the process. Still more spent their time making remixes, using up even more tubes.
Stevens was a respected statesman, beloved in Alaska, but this speech will always remind tech-savvy people just how ignorant government can be of the systems it tries to regulate. Since then the Internet's rallied to defend itself from politicians who don't understand it much really, something that will hopefully keep up in the future.
…Made Of Cats
Long, Nyan, ceiling or otherwise, The Internet loves cats. So much so, in fact, that people regularly use Google to find this song, which "explains" the feline origin of the Web.
This song conveys important scientific information, to be sure. But have you ever wondered why the Internet is so obsessed with cats? Mike Rugnetta, host of PBS Idea Channel on YouTube, certainly has. His conclusion: the Internet is cats.
Whoa. If that inspired you, check out Dave's list of cat websites 8 Purrfect Cat Websites For Feline Lovers 8 Purrfect Cat Websites For Feline Lovers Cats are everywhere online… as photos shared by friends on social networking sites, as cute pics on Reddit, on any of the cat-centric Tumblr sites, and even here on MakeUseOf. Oh, and then there's Nyan... Read More . You're welcome.
…A Playground
This result is actually about David Thorne's book "The Internet is a Playground". Thorne paid for a phony chiropractic service with a drawing of a spider. When he later tried to sell this drawing on eBay it reached $10,000, but the top bidder didn't pay. Thorne didn't mind: ""The internet is a playground," he said, "And I would not have it any other way."
Background aside, The Internet really can be seen as a playground. It's perfectly possible for everyone to hang out with whoever they want, yet people tend to stick to their cliques. Some people spend their time playing games, while others prefer to just hang out. And sure, cyberbullying 5 Websites That Help Parents & Children Deal With Bullying Or Cyberbullying 5 Websites That Help Parents & Children Deal With Bullying Or Cyberbullying For years bullying had been thought of as a necessary rite of passage to adulthood. If bullying is a devil's trait, then we as concerned citizens and parents can play protectors. Educating ourselves about how... Read More is a problem, but for the most part everyone here just wants to have a good time before they need to get back to work.
Anyway, while we're on the subject of David Thorne, you should really read one of the funniest things on the Internet. You'll like it.
…For Porn
The 2003 Broadway show Avenue Q is many things: a Sesame Street parody, a look at what it means to grow up, and a musical. It's perhaps most famous online, however, for a song about porn. Trekkie Monster, the show's not-so-subtle nod to Cookie Monster, very clearly prefers explicit images to baked goods.
Porn makes up a staggering percentage of Internet traffic, something that's rarely talked about. Porn addiction Pornography Addiction: The Hidden Struggle & How to Break Free [Feature] Pornography Addiction: The Hidden Struggle & How to Break Free [Feature] Anon22 discovered Internet porn when he was just 12 years old. For around 10 years, Anon22 has enjoyed pornography using his computer once or twice a day, a compulsion that he claims ruined his social... Read More is a hidden struggle for many. This song uses humor to get at this without it becoming weird, so it's no wonder people are constantly Googling it to this day.
There you have it: the sum total of what the Internet is, as described by the collective consciousness of millions. You cannot refute the logic of the masses, so don't even try. If you're feeling futile, however, let us know what you think the Internet is in the comments below. I can't wait to hear them.
Image Credit:Ezgisu Atacan


text: Armada Hoffler Properties, Inc. (NYSE: AHH), today announced that it will participate in REITWeek 2014: NAREIT's Investor Forum on Wednesday, June 4, 2014 in New York City. The live webcast of the Company's presentation, which begins on Wednesday, June 4 at 8:00 a.m. Eastern Time, can be accessed on the Investor Relations section of the Company's website at www.ArmadaHoffler.com. Supporting presentation materials will also be available on the Company's website prior to the webcast. A replay of the webcast and the supporting presentation materials will be available for 90 days and can be accessed at the same web address. About Armada Hoffler Properties, Inc. Armada Hoffler Properties, Inc. is a full service real estate company with extensive experience developing, building, owning and managing high-quality, institutional-grade office, retail and multifamily properties in attractive markets throughout the Mid-Atlantic United States. The Company has elected to be taxed as a real estate investment trust (REIT) for U.S. federal income tax purposes.


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I really don't understand this. I honestly think this is worse then Facebook.
Most Helpful Girl
Many guys confuse and reuse GAG for a dating site. Many times I have seen and heard this on here, dear. I, for one, just Strictly keep it platonic and business as usual when it comes to sticking to the book of... Helping people with problems only.
Nowadays, where there is Internet, there is a female face, and some guys jump on this, hoping for a hook up or even if it is a hit and miss, they find it a challenge and a chase to at Least try for A... Pretty girl's face, @PrettyRican.
Good luck. xx22|00|0
Thank you hun!!
- Show AllShow Less
Oh, thank you so much for your kind and sweet words.. Wise owl with diplomacy who flies free to lend a helping hand:)) xxoo
Beautiful profile, thank you for sharing.:)) xxoo


text: 10+ Cartoon Nails That Totally Pop Hello viewers, today we are going to share with you another nail design which is entitled "10+ Cartoon Nails That Totally Pop". All the designs are awesome and wonderful, when you apply each design you will get stunning look. loading... 11:04 AM Nail Art, Nail Designs Email ThisBlogThis!Share to TwitterShare to Facebook


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MIAMI TO CURACAO (caribbean island) FOR $272 R/T NS (winter-spring)
Amazing deal: Miami to Curacao flights roundtrip as low as $272.
Miami, USA
Curacao, Curacao
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Departures: January 15 – March 18
Return: January 15 – March 23
There are few blackout dates, but overall lots of tickets!
Insel Air
We do not sell travel products or services. We just provide you with information (how/where) about flight deals or links to websites, where you can read more about it.
Information is valid at time of publication. Due to a high amount of posts – it is hard to track down all expired offers, so sometimes you may not find the price mentioned in the post. It means that the deal was sold out or supplier (airlines/agency) has changed the conditions or removed the offer. To avoid these frustrating situations, you can subscribe to our newsletter, Facebook, Twitter, Tumbler.
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text:
Zeasla (Maori: Aotearoa) is a nation located in the southwestern Pacific Ocean. The country geographically comprises two main landmasses ‒ that of the several former English colonies‒ as well as numerous smaller islands. Zeasla is situated some 4500 km (900 mi) east of the Greater Khmer Confederacy across the Tasman Sea and roughly 1000 km (600 mi). Because of its remoteness, it was one of the last lands to be settled by humans.
On 1 January 1901, the six colonies federated, forming the Commonwealth of Zeaslans. Since Federation, Zeasla has maintained a stable liberal democratic political system which functions as a federal government. The federation comprises sixteen states and several territories. The population of 33.9 million is heavily concentrated in the Middle states and is highly urbanised.
A highly developed country, it holds as the country with the third largest economy and tenth largest messages.
Capital: Wellington
Official Languages: English, Maori,
Type of Government: Comersist Democracy
Doge: Alan Bollard
Population: 34 Million
Currency: Dollar
Religion: 85% Anglican, 15% Roman Catholic
Literacy: 98%


text:
Robbie Henshaw injury a blow: Lam
Connacht coach Pat Lam is confident others will step forward and fill the void as he comes to terms with the loss of Robbie Henshaw for the next six weeks.
The Irish international suffered a fracture to his left hand in the closing stages of Saturday's 18-12 win over Munster at Thomond Park.
Lam said that the injury to the 22-year-old was a big loss but pointed out that his side are on an eight-match winning run and Henshaw had only been available for three of those. He marked his return from World Cup action to score a try against Treviso and also featured against Brive in the Challenge Cup before setting up the clinching try for Bundee Aki against Munster.
"After Bundee's try the kick-off went to Robbie and he went up in the air and there was a collision," said Lam.
"When he came down, he held his hand. We finished the play, and then straight after the game, we could see that it was blown up."
The Connacht coach, who takes his side to Cardiff this Friday and who will have Challenge Cup games against Newcastle Falcons before Irish derbies at home to Ulster and away to Leinster over the festive period, will also have to do without prop Denis Buckley (ankle sprain) and No.8 Eoghan Masterson (thumb) for the next three weeks.
Belfast Telegraph


text: Sure, Joe Tereshinski wasn't the sexiest choice in the four-way battle to be Georgia's starting quarterback. But he was the safest choice. When the success or failure of a football season might hinge on a second-week showdown in South Carolina where the Bulldogs traditionally struggle, playing it safe is a good idea. Head coach Mark Richt knows better than anybody else what his team needs to do to win now, and throwing a true freshman like Matthew Stafford into the fire isn't the smart play. If he was clearly that much better than Tereshinski, he would be the starter in the opener. Clearly, he was not ... for now. Tereshinski is a loyal, third-generation Bulldog who bided his time and learned the system behind NFL-drafted quarterbacks David Greene and D.J. Shockley. Whether or not Richt owed him the opportunity is not the point. Tereshinski earned the opportunity. He's smart, steady and knows the system better than Joe Cox (his current backup), Blake Barnes or Stafford. Considering the skill he'll have around him, that's the kind of QB the Bulldogs need to keep the mistakes to a minimun and get the ball in the hands of the running backs and tight ends who will be the key to making the offense click in 2006. Whether or not Tereshinski will succeed in the starting role remains to be seen. But whether or not he was the right choice is certain. The job was always his to lose and he hasn't lost it.


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ITT Technical Institute-Greenville
About ITT Technical Institute-Greenville
ITT Technical Institute is a technology and career focused school granting associate's and bachelor's degrees. ITT Tech has a campus in Greenville, South Carolina. Greenville is within driving distance of Charlotte, North Carolina, Charleston, South Carolina, and Atlanta, Georgia and has a population of approximately 56,000. For art and culture, Greenville has the Greenville County Museum of Art and the Greenville County Youth Orchestra. The Carolina Ballet Theater performs often in Greenville and the Centre Stage is a professional theater troupe performing in Greenville.
ITT Technical Institute is run by ITT Educational Services, Inc, started in 1946 and headquartered in Carmel, Indiana since 1964. This company runs a system of private schools with over 100 campuses across the country. ITT Tech schools focus on technology education and training for careers. The six career paths the schools offer are information technology, drafting and design, electronics technology, business, criminal justice, and health sciences. The popular and ubiquitous chain of schools granted more electronics and drafting degrees than any other school in the U.S. in 2001.
ITT Tech offers an associate's degree in computer drafting and design. Students take courses in basic computing, computer-aided drafting, graphic communication, technical illustration, architectural drafting, engineering graphics, three-dimensional modeling, and project planning. The program teaches how to translate ideas and sketches into working plans. Students are prepared for entry-level careers in mechanical drafting, architectural drafting, interior design, and civil drafting. The Bensalem campus of ITT has a student affiliates chapter of the American Design Drafting Association to enhance professional development.
Students can earn an associate's degree in information technology with a focus on multimedia. The program prepares students to create and design multimedia communications and to write computer programs. Students are ready for entry-level careers in multimedia information technology.
Students interesting in gaming can study for a bachelor's degree in digital entertainment and game design. Coursework includes general education classes, gaming technology, game design, and animation. The program prepares students for entry-level careers in designing games or creating animations.
To earn any federal financial aid, students must complete the Free Application for Federal Student Aid (FAFSA). This will help determine eligibility for federal grants and loans. Students may also seek out state aid, private loans, or scholarships to fund their education.
ITT Technical Institute is accredited by the Accrediting Council for Independent Colleges and Schools. Because ITT Tech is not regionally accredited, other colleges and universities may not accept transfer credits.


text:
What is JotForm?
JotForm is a free online form builder which helps you create online forms without writing a single line of code. No sign-up required.
At JotForm, we want to make sure that you're getting the online form builder help that you need. Our friendly customer support team is available 24/7.
We believe that if one user has a question, there could be more users who may have the same question. This is why many of our support forum threads are public and available to be searched and viewed. If you'd like help immediately, feel free to search for a similar question, or submit your question or concern.
Can we export to PDF including the form text?Asked by thiningsoftware on May 21, 2015 at 03:54 AM
We're looking at JotForm as a replacement for Adobe Forms. We've imported our forms and everything works great except for a little PDF issue.
Once the person files out our form, we then used to save it as a PDF and email it to them as a confirmation. However, since our form includes some instructions, we need all of the content to be included on the PDF, not just the responses.
Is that possible?
JotForm Support
Hi thiningsoftware,
Thanks for contacting us.
Do you mean you want to export the submissions to PDF with the same format as the form itself?
If yes, currently JotForm does not have this feature but there is a workaround that you can try. See instructions below.
1. Add Edit Link to email notification.
2. When you receive the email notification, do the following:
If you need further assistance, please feel free to contact us again.
JotForm Support
I created a separate thread for your own issue here https://www.jotform.com/answers/1171157
We will attend to it shortly.


text:
I apologize if this has been previously discussed - I looked in past posts for an answer but didn't see anything related.
This question is largely directed toward hosts of whole-house rentals, but might apply to others who can imagine staying in a whole-house for a vacation, as well. Otherwise, if someone is hosting guests in a room in their home many of the potential issues would already be addressed being on-site, and likely not add up enough to need to do something like this.
Okay, my question:
When someone stays with us (because we don't normally greet them personally) I send out a rental package with the instructions on their stay…
The operational instructions include how to handle garbage. As (hopefully) the world becomes more eco-friendly, our area has strict rules about disposing of garbage. There is a centrally-located "manned" waste depot where everyone takes recycling and garbage. They check to make sure things are disposed of properly: plastics in one bin, paper in another, cardboard, metals, organics for composting, and anything else left over.
Our kitchen has built-in bins to separate the garbage accordingly. We also have a garbage compactor that will hold 1-2 weeks worth of garbage for our large family. Part of our Rental Policies (agreed to before we accept a rental) states that guests are expected to handle their own garbage to avoid extra fees.
This is the current case in point: the last group told me - after they had left - that they didn't have time to take their garbage away. And, they had actually arrived early while we were still prepping (that's another story!) so I took the time to explain it all to him in person - I know they were aware of how this all works.
They left 7 extra-large bags of garbage for us in our foyer (I mean, they had 2-3 standard-large bags in each!). None had been separated, except for one that was completely filled with cardboard. We spent a total of 3 hours dealing with their garbage, separating it so that it was acceptable to go to the dump, then of course had to take it in.
As well (also noted in our up-front policies) is that the group is to leave the bbq and kitchen as they found it. Some groups will eat in restaurants all the time so they don't have to cook on vacation, while others just love to cook in our kitchen that is ultra-well equipped… BUT, cleaning up their kitchen mess is not included in the cleaning fee (it nearly always needs additional cleaning to be up to snuff but people are expected to make the effort). In this case the kitchen needed SO much work.
Also, the heat was left at full-blast (my cost is about $30 a day to heat an empty house in winter); windows were left open; garbage/small items left all over the rooms - all things that are covered in the Check-out list.
I really should charge an additional fee to cover these items. I usually talk myself out of it, unless there is something so blatant that I cannot justify it. I don't want the hassle, or appearing to "nickle and dime". I usually end up just feeling crappy about it all, making everything normal again and eating the cost if it is less than about $200.
But that got me to thinking… perhaps I should have a list of charges / fees that I leave at the house and let the guest fill it in. I suppose such a thing would look like a mini-bar in a hotel suite where you mark down the items you want and expect to be charged for it at the end of your stay.
It would look something like this:
$50 - kitchen cleanup
$10 - remove food (alcohol gladly accepted for free)
$20 - bbq cleanup
$30 - first bag of garbage
$10 - each additional garbage bag
$10 - broken dishes
$75 - each unregistered guest (it is only $15 if they register ahead)
Rather than looking punitive, it may offer another option to guests in case they don't want to handle any of those items and are willing to pay for someone else to handle it for them. Even if they don't fill in the sheet this will tell them in advance how much they should expect to get a bill for so that I can recoup those extras that I keep absorbing.
This is getting rather lengthy, so I'll just leave it here and look forward to hearing from a host and potential guest perspective on whether this is a good solution.
Good idea. Are you going to send them a charge for excess cleaning? Did you take pictures? Obviously they will get a negative review.
Hi @Louise! I thought you'd appreciate this because your posts indicate that you're also eco-friendly.
This is one of those cases where I've almost talked myself out of charging them extra. They really were a nice group overall. They are wealthy people from a country where labour is cheap so they probably have house staff to normally do these things for them.
My ultimate reason for "letting it go" was that they paid for 7 nights but only stayed for 5, although other options would have had the same minimum rental period. I figured the extra charges would have amounted to $160. I was thinking that if they had seen a sheet like this they likely would have just left cash to cover it, though.
At this point I want things to end on a good note with this group. If I'm going to implement this new thing it will be for future rentals. I'm also going to write a separate sheet just on the recycling and leave it in our binder in case people aren't reading the guidelines.
I like your idea of the list. Not only does it really spell it out to them how important it is, hopefully it's also a great way of evidencing it to Airbnb should you need to charge them. Can you add it to your listing somehow so it's recorded 'officially?'.
As for these guest… they deliberately ignored at least 3 separate things you specifically told them, all of which directly cost you a lot of time and money. I don't think they deserve to get off so lightly
Good! I'm glad it comes across as friendly and not nit-picky. Thanks for the idea of the adding it to the listing. I just might do that.
Most of my bookings come in through VRBO (as this group did) so I'm dealing with them directly. Maybe I will re-think charging them. I also didn't factor in the cost of heating an empty house which would bring it up to over $200.
Not to mention the task of going through their icky garbage might have an additional "yuck" factor, not just "dropping it off".
We have the same. Very few clients are recycling even if we have all information on it.
When they are recycling it's almost everytime wrong done…
I don't think the list is a bad idea, especially since it sounds like your house is rather large. I would however, put some statement in there indicating that they need to let you know which services they would like to purchase in advance. [Hopeless probably.] This is the only way you can plan your own life to have the time to sort through and do extra cleaning.
I think having some sort of list would be good idea, and having some sort of method where they let you know ahead of time (by a certain deadline) that they have decided to take advantage of some of the "services" offered or they have declined them. Especially if you have to plan ahead to bring in extra help to play with garbage for several hours. That way they don't "pretend" to not have noticed the list and still leave things not taken care of.
Would you detail what is included in "kitchen cleanup"? I would be concerned a group may leave the fridge filled with meals in pots and pans, tupperware, etc, dirty dishes piled and caked on covering all the counters, etc. Would the $50 plus $10 still be worth it if they never picked a sponge and soap since the day the entered?
An excellent point. When we have a quick turn-around we would want to know what to expect in advance. It would have been a disaster to deal with this last group if another was coming right in.
Agree, maybe a charge per hour for cleaning. If it delays next guest's being able to check in, you might charge them for a day. These people deserve to be charged for their disrepect.
Oh! I'm sure this often happens! When I stay at other rental homes I would just do this anyway without be prompted, but many people seem to "ignore" the items left on the list.
Maybe I should do up a separate "kitchen" list, as well. These items are covered in out Check-out list. I just don't want to inundate people when they're on vacation.
That pretty well describes the fridge with this last group. It's an extra-large fridge and it was half-filled with leftovers, which was why I thought of that extra line to clean the fridge. Although there are a few unopened items that I think we might use up!
Oh I see - "We left you our food because we hate to throw away perfectly good food someone else can use." But they really mean "we didn't feel like scrubbing out all the containers we used to store the food." Sigh…
Personally your rules and lists are so complicated I would probably not stay with you.
I tend to agree. But to each his own. Only you can know if this will work for you. I know that it wouldn't for me in our rental.
I face similar dilemma. I just started hosting and have hosted two groups so far. The common problems I have are 1. uncleaned dishes/pans/pots; 2. stained towels.
I charge a very cheap cleaning fee of $60 for a house, and I tried to get quote for a professional clean and it would cost me at least $120. I fear high cleaning fee would deter guests from booking my place. What is everyone else's opinion?
Further, I put in the house manual that guests need to clean the dishes but they seemed to ignore that. I don't have a dish washer so I'm wondering could that be the problem? Maybe many people don't know how to clean dishes without a dish washer?
I also thought about the list of fees for additional cleaning, removing kid's drawings on wall, removing unreported stains on carpet, but I think putting them in my listing would deter guests from booking because many hosts don't have these rules in their listings. And just putting these in the house manual seems unfair because guests don't have a chance to know beforehand. Also, I feel I don't have a ground for claiming these fees because they are not in the house rule of my listing.
Anyway, I have spent way too much time to clean for both groups of guests so far. I plan to hire cleaner in the future because I won't have time to clean myself unless it is school holidays. I need advise on whether to increase my cleaning fee to my true cost or keep it low to attract more bookings and profit from the nightly rate?
I have similar experience, the guests I have hosted so far (only 2 groups) didn't clear the fridge and left lots of food/drinks for me. I feel it is such a waste, and I bought some of these for them, they just open the food and tasted a bit and decided it's not for them, but I confirmed what they would like before I bought for them (expansive ones not the low price ones). So I'm thinking maybe I should not offer breakfast because many are wasted. For example, my last group probably wasted $20 of food. All opened and consumed a little only. What do you guys think?
@Freya . I live in the house, so my solutions will be different than yours. I only put a finite amount of stuff into the fridge upon arrival, and they order what they want to eat in advance [mostly.] For milk, I keep a jug in my fridge and have cute little dairy bottles that I partially fill. I purchased tiny jam jars, Bonne Maman, orange marmalade and strawberry jam which are part of the croissant breakfast. To go with the rye rolls, I have mini cheeses. For one group, I made them vegan breakfast bread, a blueberry corn bread, and dispensed some blueberry jam for our family container into a smaller jar perfect for two people.
$20 is not in the budget for my place unless they stay for 5 nights. Here is the big difference. I replenish their rooms each day so I can see that they don't really use the milk, love the jams or not, and have or have not eaten the pieces of fruit. By dispensing things from larger containers, I know that the container hasn't been contaminated and don't have to throw much food away at all.
The last time I made a big trip, several places offered to stock the fridge with items of my choosing, and then I would reimburse them when I arrived. AirBNB is the only platform that I have seen provide any starter foods for self-catered properties.
I don't provide food or beverages. I do leave a dozen bottle of water, there is salt, pepper, and a few spices and herbs. It's too complex to guess what people want to eat. So far people have left my place clean, but I'm going to create a list of charges for cleaning up,when they don't. Not excessive, just a prod in case I let to a lazy bunch. If it's in my rules then I'm hoping air will agree to having the fees paid when necessary.
I wouldn't book a place if there was a charge sheet. I also HATE minibars in hotels.
Honestly, if you're absorbing these costs in the normal course of running your STR then you should build the cost into the rental price. Charges post-stay are really for extraordinary issues e.g. broken/damaged things.
Other hosts will disagree with me I think but to me, these are pretty normal and come under the heading of the cost of doing business.
This might be a dumb question but if guests are constantly leaving dirty dishes are you sure that you're providing them with the wherewithal to do so? (Plenty of dish soap, sponge, scrubbing thing, one of those brushes etc.? And dish towels, paper towel and so on). If find that guests rarely leave dirty dishes although even when they are washed and put away, they still need to be checked and sometimes washed again so even if guests are wonderful about it, it's still a necessary chore. Be sure too to leave only just enough of everything so that guests will need to do the dishes as they go.
Stained towels are common too but if they are white you can bleach them and 99% of the time, that works well. Bear in mind though that you will have to replace a few every year of so if you're fully booked.
Regarding your cleaning fee, it seems low for a full house rental. I'm assuming that you're getting a good rate for a full house so I doubt that upping the cleaning fee would deter guests. If you raise it to $100 you're only 'losing' $20 if you use a cleaning service and at whole house rates, you'd probably be happy to absorb that.
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text: Young Women Delaying Cervical Cancer Checks Experts Have Urged Women Concerned About Cervical Cancer To Seek Treatment As Soon As Possible 30.09.2014 Young women with symptoms of cervical cancer may be missing vital opportunities to treat the disease early by not going to their doctor soon enough, according to new figures. Researchers at King's College London and the University of Exeter Medical School carried out a study that is published today in the British Journal of General Practice. They interviewed 128 females under the age of 30, all of whom had recently been diagnosed with cervical cancer. Forty had received their diagnosis after seeking their GP's advice on symptoms; 86 had had their cancer picked up following routine cervical screening; and two had been diagnosed during treatment for an unrelated condition. It was discovered that 28 per cent of participants had waited more than three months to see a doctor even though they had concerns about their health, with women under the age of 25 most likely to delay. Ten of the women admitted they did not know what the symptoms of cervical cancer actually were. The most noticeable is usually vaginal bleeding outside the usual menstrual cycle, particularly after sexual intercourse, but signs can also include abdominal pain and unusual discharge. Senior author of the study Dr Lindsay Forbes said it is vital that people are made aware of the dangers of this form of cancer and educated on how to spot it early. "A better understanding of the importance of these symptoms could help promote earlier diagnosis and improve survival in this group," she pointed out. Around one in 34 women will get cervical cancer at some point in their lives and there were 919 deaths from it in 2012. However, prospects for survival are good if it can be caught before it progresses, which is why the NHS in England offers routine screening for it to women aged 25 to 64. There have recently been calls to lower this age limit to 16 following a string of cases in which much younger women were refused smear tests and went on to receive a cervical cancer diagnosis. Expert Opinion Early diagnosis and treatment plays a critical role in the chances of survival and recovery from cervical cancer and it is vital more is done to raise awareness of the disease and ensure young women know what symptoms to look out for and feel comfortable visiting their doctor to discuss their concerns. "Although cervical cancer is rare in young women, we have seen a number of examples of the disease not being spotted early enough by doctors, as medical professionals dismissed the warning symptoms as other problems due to the patients age. It is vital that GPs carefully check the symptoms of people suffering with the symptoms of cervical cancer regardless of age to ensure early diagnosis and an increased chance of survival." Julie Lewis, Partner Key contact Julie Lewis Partner +44 (0)370 1500 100 Email Julie Related articles 18.08.2017Settlement For Former Mill Worker Left With Noise-Induced Hearing Loss 18.08.2017Specialist Lawyers Instructed By Worried Mum After Council Seeks To Close Respite Care Centre 18.08.2017Family Calls For Lessons To Be Learned After Inquest Into Cyclist's Road Death 18.08.2017Lawyers Praise Decision To Fine Islington Council Following Personal Data Leak 17.08.2017Settlement For Family Of Elderly Man Who Died After Being Hit By Car As He Crossed The Road


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2 Oakdene Grove, Laverton, VIC 3028
Best match results for: CATEGORY = Roller Skating Clubs for Kids + LOCATION = Australia
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I recently had my daughters 9th birthday party at this venue and found the staff to be excellent. They have staff on the floor to help the children and also gave them tips on how to skate. There was a D.J. playing music and they played games with prizes.The party went for 2.5hrs and is very reasonably priced. Would highly recommend this venue!!
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Additional Details
- MLS Number: 216067145
- County: Collier
- Community Name: Riverstone
- Subdivsion: Riverstone
- MLS Area: NA21 - N/O Immokalee Rd E
- Year Built: 2013
- Listing Date: Saturday 29th of October 2016
Property DescriptionH.11223 Welcome to Riverstone, one of Naples hottest new developments! This great room Carlyle floor plan has it all! You will enjoy the light and bright colors along with the modern touches throughout. An extra bonus room offers room for all the kid's toys and games! Enjoy hosting all the hit parties in your large open kitchen with GE Profile Stainless Steel Appliances, large center island, and in-kitchen eating. If you would prefer more intimate seating, move on into the dining room with plenty of room for the entire family. At the end of the day, you will enjoy retreating into the large master bedroom or soaking in your oversized tub. Enjoy your large corner lot with an expansive grass area for your kids or grandchildren. Riverstone residents enjoy a plethora of amenities including; free-form resort-style pool, lap pool, large Jacuzzi, kids water play area, 5 tennis courts, indoor sports complex, fully-equipped fitness center, full-court basketball, walking/jogging path and so much more.
- General: Pets allowed
- Community: Pool table(s), Community clubhouse(s), Community exercise area(s), Community swimming pool(s), Community spa(s), Gated community
- Construction: Concrete block stucco, Stucco exterior
- Roof: Tile roof
- Exterior: Enclosed porch
- Fencing: Fenced
- Flooring: Carpet, Hardwood floors
- Laundry: Laundry sink, Laundry room/area
- Heat/Cool: Central air conditioning, Central heat
- Inclusions: Double oven, Microwave oven, Cooktop range, Disposal, Dishwasher, Refrigerator, Freezer, Clothes washer, Clothes dryer, Range and oven
- Interior: Pantry, Tub and separate shower, Dry bar, Walk-in closet(s), Security features, Fire sprinkler system, Built-in bookcase(s)
- Utilities: Public sewer service, Public water supply, TV cable available
- Location: Northern exposure
- Lot: Corner lot
- Parking: 3 car garage, Attached parking, Automatic garage door
- Rooms: Formal dining room, Breakfast area, Guest room, Great room, Foyer, Eat in kitchen
- Win/Doors: French door(s), Shutters
Property Map Go to street view!
Request Information on Single Family Riverstone Naples
Listing provided courtesy of John R. Wood Properties


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We've been together nearly a year now, he's 27 and I'm 25, and we haven't slept together yet and it's killing me!!
Anytime we get anywhere close he backs away and says he's very awkward about this stuff and then just cuddles me. I've been so patient and I am understanding but I'm beginning to find it really hard! As someone who enjoys sex as part of a healthy adult relationship, it's really starting to get to me.
Recently I tried to bring it up and said he has no idea how much I want him sometimes, he just responded saying how he's quite oblivious to things like that then changed the subject.
It's so unusual for a guy to wait this long. What can I do?
Most Helpful Guy
I think it's very unusual. Do you know if he's had bad experiences in the past and is afraid of getting hurt or something? Or maybe he's afraid that you won't like it. Try to make him feel very comfortable (you probably already have, but just saying). Tell him you want to understand his reasons and that this isn't clear for you and you deserve to know why.00|00|0
Yeah, that's really helpful actually about me deserving to know why. It is all just a big mystery to me at the moment! An explanation is the least i deserve :)
And this can be also a solution if he cooperates and give you an explanation. You'll be able to identify the causes of his behavior and try to work on them to finally have sex with him.


text:
- Description
- View Full Specifications
- Year:
- 2012
- Length:
- 24'
- Engine/Fuel Type:
- Single / Gas/Petrol
- Located In:
- Myrtle Beach, SC
- Hull Material:
- Fiberglass
- 54330-2941429
- Last Listed Price:
- Sold
This is the 2nd owner. Boat was purchased from sea ray dealer with less than 50hrs. Kept in boat shed and in freshwater. Shows extremely nice. Owners lack of time to use brings the boat back to market. 5.0L Mercruiser engine with duoprop. 110hrs. Reduced by $5,000 on may
Contact David Rogers @843-455-6807 for showing instructions or further questions.
Please contact David Rogers at 843-455-6807
Additional Specs, Equipment and Information:
Warranty: Lifetime
Hull Shape: Modified Vee
Beam: 8 ft 6 in
Minimum Draft: 1 ft 10 in
Maximum Draft: 3 ft 1 in
Deadrise: 21 ° at Transom
Dry Weight: 4850 lbs
Max Load Capacity: 1600 lbs
Total Power: 260 HP
Engine 1:
Engine Brand: MerCruiser
Year Built: 2012
Engine Model: 5.0L MPI Bravo III
Engine Type: Inboard/Outboard
Engine/Fuel Type: Gas/Petrol
Engine Hours: 110
Propeller: 3 blade propeller
Drive Type: Stern drive
Engine Power: 260 HP
Fresh Water Tanks: (9 Gallons)
Fuel Tanks: 1 (55 Gallons)
Number of heads: 1
Seating Capacity: 10
Cockpit speakers
CD player
Inside Equipment
Electric bilge pump
Manual bilge pump
Electrical Equipment
Electrical Circuit: 12V
Outside Equipment/Extras
Cockpit shower
Swimming ladder
Cockpit table
Bimini Top
Manufacturer Provided Description
The mission of our 240 Sundeck is to host a lot of guests easily and comfortably without losing the performance, good looks, and trailerability of a sport boat. No Problem! There's room for 10 people and up to 300 horsepower.
Key Features
- MerCruiser 5.0L MPI ECT w/Bravo III sterndrive is standard engine
- SmartCraft Diagnostics System standard.
- Transom seat that converts to a sun pad.
- Bow block-off door under windshield.
- Limited Lifetime Structural Warranty on hull and deck.
- Extended swim platform with rubber inserts.
- Standard bimini top with stainless-steel hardware.
- Sony Stereo and CD with iPod Connector.
- Easy-to-access transom with wakeboard/ski storage.
- Available in black, red, blue, burgundy, and pewter optional two-tone gel coat finish with Sundeck graphics.
- Sea Ray Owners Club membership (available in USA and Canada).
Hull and deck features
Beach Boarding Ladder, Forward Deck w/ Integral Hatch & Concealed StainlessSteel Four-Step Ladder Bow & Stern Eyes, Stainless Steel Cleats, Custom Sea Ray® Thru-Bolted - Stainless Steel (6) Pull Up Drain Plug, Brass-Threaded - Garboard Gel Coat, Arctic White Graphics Package Hardware, Stainless Steel Light, Stern - Removable Lights, Navigation - Stainless Steel Resin, High-Performance Vinylester Rub Rail, White PVC w/Stainless-Steel Insert Ski Tow Eye, Integral, Stainless-Steel - Transom Storage, Anchor w/Fiberglass Hatch & Stainless-Steel Latch Swim Platform, Concealed Stainless-Steel Three-Step Swim Ladder w/Rubber Inserts Vent System, Engine Compartment, Stainless Steel - Integral Windshield, Tempered/Tinted Curved Glass w/Walk-Thru Canvas (Sunbrella®): Bimini Top w/Boot & Stainless-Steel Ball & Socket Hardware Certifications: Meets Applicable USCG & ABYC Standards NMMA Certification Mercury® Installation Quality Certification
Technical features
Helm: Alarms, Engine - Oil & Water (Audible)
Keyless Dash Mechanical Horn Shift & Throttle Lever w/Trim (Chrome Accented)
Switch & Lanyard Stop Switch SmartCraft® Diagnostics: - Instrumentation, Custom Sea Ray® SmartCraft w/Glass Lenses,
Low-Glare Blue Night Lighting & Chrome Bezels -
4-in-1 Gauge (Includes: Fuel Level, Oil Pressure, Water Temp & Trim) - Speedometer - System Tachometer (Includes: Depth, Engine Alarms/Hour Meter, Fuel Consumption/Usage,
Trim Level, Troll Control & Voltage)
Steering, Power-Assisted Tilt Wheel
Switch Panel w/Weatherproof Lighted Chrome Toggle Switches,
Legend Backlighting & ISO Symbols Tilt & Trim,
Power Electrical Systems: Battery Tray w/Mount Fuse Block Wiring, Color-Coded w/Chafe Protection Engine Compartment: Bilge Pump, 12V Automatic Blower,
12V Bilge
Fire Extinguisher Discharge
Port Gel-Coated Engine Compartment Sound Insulated Engine Compartment
Fuel System: Fuel Lines, USCG - Type A-1 Fuel Tank w/Anti-Siphon Valve & Mechanical Sending Unit Water System: Pressure Water System w/Transom Shower & Sink Faucet Water Tank (9 Gallon)
The Company offers the details of this vessel in good faith but cannot guarantee or warrant the accuracy of this information nor warrant the condition of the vessel. A buyer should instruct his agents, or his surveyors, to investigate such details as the buyer desires validated. This vessel is offered subject to prior sale, price change, or withdrawal without notice.
Manufacturer Website
Other Boats from Myrtle Beach Yacht Sales
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44 Sedan Bridge
Miami, FL, United States
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410 Sundancer
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Jacksonville, FL, United States


text:
Alpinetrek expert Hannes
"This product is no longer available."
This is either an older model or we can or will no longer reorder this product from the manufacturer.
Don't give up – we have some other alternatives for you to choose from:
Snowlife - Foot Heat Packs - Toe warmer
Material information & features
- Design:
- Toe warmer
- Recommended use:
- All around
- Material:
- Activated charcoal fibers
- Extras:
- Warms the toes up to 5 hours
- Item No.:
- 209-0086


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The Federal Ministry of Youth Development was established in February 2007 with the vision of empowering Nigerian youth to become "self reliant and socially responsible". The Ministry ensures this by promoting the physical, mental and socio-economic development of the youth.
It provides sustainable framework for integrated planning and collaboration for the development of policies and programmes, and other initiatives that promote and enhance the development of young Nigerians.
The Ministry has six departments. These are: Network and Social Mobilization (NSM); Enterprise Development and Promotion (EDP); Education and Youth Development (EYD); Human Resources and Management (HRM); Finance and Accounts; and Planning, Monitoring and Information Management (PMI).
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- to formulate, monitor and review the National Youth Policy.
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The Federal Ministry of Youth Development has played its role as the advocate of the Nigerian youth effectively over the years and is poised to do more in future.


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Mission Statement
The mission of the North Carolina Psychoanalytic Foundation is to help individuals and our community understand and alleviate problems that lead to emotional suffering and disability and to enhance their capacity for personal development and improved community functioning.
NC Psychoanalytic Foundation
Phone: 919-847-2323
The mission of the North Carolina Psychoanalytic Foundation (NCPF) is to help individuals and our community understand and alleviate problems that lead to emotional suffering and disability and to enhance their capacity for personal development and improved community functioning.
About NCPF
Founded in 1995, NCPF is a 501(c)(3) public charity that strives to create a more resilient community by focusing the strengths of the psychoanalytic community in addressing critical needs of North Carolinians. We provide community outreach, programs, support for therapists in training, continuing education for therapists, and resource referral services.
Psychoanalysis is the most comprehensive treatment available today for healing mental distress and promoting personal growth and development. Psychoanalysis is a deep, insight-oriented form of psychotherapy in which patient and analyst work together to explore conscious and unconscious factors that create unhappiness in the form of painful symptoms, difficulties in work and relationships, or disturbances in self-esteem.
Psychoanalysis is often helpful when attempts at self-help, talking to family or friends, or briefer, less intensive treatments have not been adequate. The sustained and personal collaboration with an analyst can ease depression and anxiety, restore self-esteem, relieve inner conflict and lead to a greater capacity for love, work and play.
For more information about psychoanalysis and how it can be used to address issues in our community, visit our website here: www.ncpsychoanalysis.org/about.htm or watch our short video: An Introduction to Psychoanalysis.
Events and Programs
NCPF provides a range of services and programs including: Peaceful Schools-NC (bullying prevention initiative), Annual Courage Rocks! Be Bold Festival (bullying awareness festival event), Speakers' Bureau, Public Lectures/Workshops for Mental Health Professionals, Psychoanalytic Fellowships, Journalism Award, Open Sky Aikido Scholarships, and Psychoanalytic Night at the Movies (movie with discussion). For more information about our programs, visit our website: www.ncpsychoanalysis.org
Grants & Prizes
The David Raft Fellowship provides a student or trainee enrolled in or planning to enroll in one of the North Carolina Psychoanalytic Society's training divisions, a fellowship of up to $1,250 to help defray the costs of tuition.
The Priscilla Freeman Endowment provides grants and awards in support of children's and youth mental health programming and services.
To learn more about our grants, prizes, and awards, visit our website: www.ncpsychoanalysis.org/prizes.htm
NCPF relies on membership dues and tax-deductible donations for 75% of its program and operating expenses. As a 501(c)3 non-profit organization, all donations are tax-deductible. We receive support through fundraisers, such as the Great Human Race, direct donations (online or by mail), transfer of stocks, planned giving (listing NCPF as the beneficiary in wills, IRAs, life insurance policies, etc.), grants, and iGive (online shopping).
For more information about supporting NCPF, visit our website, send us an email, or call us at 919-847-2323.
- Kerry Waite
- 919-847-2323
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