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Conceived and designed the experiments: MF SD AVP. Performed the experiments: MF SD. Analyzed the data: MF SD AVP. Wrote the paper: MF SD AVP.
Mucopolysaccharidosis type IIIC or Sanfilippo syndrome type C (MPS IIIC, MIM #252930) is an autosomal recessive disorder caused by deficiency of the lysosomal membrane enzyme, heparan sulfate acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT, EC 188.8.131.52), which catalyses transmembrane acetylation of the terminal glucosamine residues of heparan sulfate prior to their hydrolysis by α-N-acetylglucosaminidase. Lysosomal storage of undegraded heparan sulfate in the cells of affected patients leads to neuronal death causing neurodegeneration and is accompanied by mild visceral and skeletal abnormalities, including coarse facies and joint stiffness. Surprisingly, the majority of MPS IIIC patients carrying missense mutations are as severely affected as those with splicing errors, frame shifts or nonsense mutations resulting in the complete absence of HGSNAT protein.
In order to understand the effects of the missense mutations in HGSNAT on its enzymatic activity and biogenesis, we have expressed 21 mutant proteins in cultured human fibroblasts and COS-7 cells and studied their folding, targeting and activity. We found that 17 of the 21 missense mutations in HGSNAT caused misfolding of the enzyme, which is abnormally glycosylated and not targeted to the lysosome, but retained in the endoplasmic reticulum. The other 4 mutants represented rare polymorphisms which had no effect on the activity, processing and targeting of the enzyme. Treatment of patient cells with a competitive HGSNAT inhibitor, glucosamine, partially rescued several of the expressed mutants.
Altogether our data provide an explanation for the severity of MPS IIIC and suggest that search for pharmaceutical chaperones can in the future result in therapeutic options for this disease.
Mucopolysaccharidosis III (also called Sanfilippo syndrome) is an autosomal recessive disease caused by lysosomal accumulation of heparan sulfate [reviewed in 1] and includes four allelic subtypes caused by the genetic deficiencies of heparan N-sulfatase (MPS III type A; MIM #252900), α-N-acetylglucosaminidase (MPS III type B; MIM #252920), heparan sulfate acetyl-CoA: α-glucosaminide N-acetyltransferase or HGSNAT (MPS III type C; MIM #252930), and N-acetylglucosamine 6-sulfatase (MPS III type D; MIM #252940). The majority of MPS IIIC patients have severe clinical manifestations with onset in infancy or early childhood. They rapidly develop progressive and severe neurological deterioration causing hyperactivity, sleep disorders, loss of speech accompanied by behavioral abnormalities, neuropsychiatric problems, mental retardation, hearing loss, and visceral manifestations, such as mild hepatomegaly, mild dysostosis multiplex, mild coarse facies, and hypertrichosis [2], [3]. A majority of patients experience severe mental retardation and die before adulthood but some survive to the fourth decade with progressive dementia and retinitis pigmentosa [1], [3]. In the very rare MPS IIIC patients with the onset of symptoms in adulthood the disease progression was similar in severity and time course to the forms with onset in childhood [4]. The birth prevalence of MPS IIIC in Australia, Portugal and the Netherlands was estimated at 0.07, 0.12 and 0.21 per 100,000, respectively [5], [6], [7].
Although from the moment of discovery MPS IIIC was recognized as a deficiency of an enzyme that transfers an acetyl group from cytoplasmically derived acetyl-CoA to terminal N-glucosamine residues of heparan sulfate within the lysosomes [8]–[10], the molecular defects causing the disease have not been characterized for almost three decades because the identification and cloning of HGSNAT has been hampered by low tissue content and instability of the enzyme. Recently our group and others cloned the gene coding for HGSNAT [11], [12] which paved the way for characterization of the molecular defects in MPS IIIC patients. To date, 54 HGSNAT sequence variants have been identified including 13 splice-site mutations, 11 insertions and deletions causing frame shifts and premature termination of translation, 8 nonsense, 18 missense mutations and 4 polymorphisms [reviewed in 13, http://chromium.liacs.nl/LOVD2/home.php?select_db=HGSNAT], While the HGSNAT transcripts with abnormal splicing, frame shifts and premature stop codons are rapidly degraded via the nonsense-mediated mRNA decay pathway [11], the phenotypic pathogenicity of missense mutations was impossible to predict. Here, in order to understand the biochemical effects of the missense mutations we have expressed mutant proteins in cultured human fibroblasts and COS-7 cells and found that all mutations result in misfolding of the enzyme. As a consequence, it is abnormally glycosylated, and is not targeted to the lysosome, but retained in the endoplasmic reticulum (ER).
The effect of HGSNAT mutations on the enzyme biogenesis and catalytic activity was studied by the transient expression of the mutant cDNA. Mutations were generated by site directed mutagenesis in the pCTAP-HGSNAT construct that expresses human HGSNAT with a C-terminal tandem affinity purification (TAP) tag consisting of a high affinity streptavidin-binding peptide (SBP) and a calmodulin-binding peptide (CBP) to allow purification of the recombinant protein using successively-applied streptavidin-resin and calmodulin-resin affinity purification steps or its detection with anti-CBP antibodies [14], [15]. In preliminary experiments we have ensured that a human wild-type HGSNAT carrying a TAP tag on its C-terminus had a lysosomal localization and showed catalytic activity similar to that of the untagged enzyme (not shown). The sequences of the constructs were verified to ensure the correct introduction of mutations. In total, 21 constructs bearing nucleotide changes identified in patients were transfected in COS-7 cells and assayed for N-acetyltransferase activity (Figure 1). All tested cells showed similar endogenous lysosomal β-hexosaminidase activity (not shown) but drastically different levels of the N-acetyltransferase activity. The four polymorphisms (P237Q, V481L, K523Q and A615T) displayed activity similar to that of the wild-type enzyme, whereas the activity of the rest of the mutants was below the detection level. Kinetics studies were further conducted using partially purified enzyme to determine if the P237Q and V481L mutants showing full enzymatic activity would have a different affinity for the substrate, however their Km and Vmax values were similar to those of the wild-type (Figure S1).
The expression of HGSNAT mutants was studied by Western blot analysis of cellular homogenates using anti-CBP antibodies (Figure 1B). All mutants were expressed at a level approximately similar to that of the wild-type HGSNAT, but showed a difference in their molecular mass. The four polymorphisms (P237Q, V481L, K523Q and A615T) showed a molecular mass of ~77 kDa, similar to that of the wild-type enzyme, whereas all missense mutants (C76F, L137P, G262R, N273K, P283L, R344C, R344H, W403C, G424S, E471K, M482K, A489E, S518F, S539C, S541L, D562V, P571L) had a smaller molecular mass of ~67 kDa. Since HGSNAT is predicted to have 5 potential N-linked glycosylation sites each potentially contributing ~2 kDa to the size of the mature enzyme [11], this molecular mass difference was consistent with the hypothesis that inactive mutants lacked proper glycosylation. To confirm this, the wild-type enzyme, 2 polymorphic variants and 2 inactive mutants were treated with endoglycosidase H which cleaves immature mannose-rich oligosaccharide chains added to glycoproteins in the ER [16] Analysis by Western blot (Figure 2) showed that upon deglycosylation the molecular mass of the wild-type enzyme and polymorphic mutants was reduced to 67 kDa, whereas the inactive mutants displayed only a subtle shift in their position on the gel, indicating that they already lacked most of the glycans. The homogenates were also treated with the peptide: N-Glycosidase F (PNGase F) which fully removes all sugars from mature glycoproteins (Figure 2B). In addition to performing enzymatic deglycosylation of cell homogenates we treated the COS-7 cells transiently expressing the wild-type enzyme, a polymorphic and an inactive mutant with tunicamycin, an inhibitor of N-acetylglucosamine transferase that blocks glycosylation of newly-synthesized proteins (Figure 2C). Results of both experiments were consistent with those obtained with Endoglycosidase H. Upon treatment with tunicamycin or PNGase F the size of the wild type HGSNAT and the enzyme containing the polymorphism was reduced by approximately ~10 kDa whereas the inactive mutant displayed only a minor change in electrophoretic mobility. Western blot analysis also revealed that homogenates of cells transfected with both wild-type HGSNAT and polymorphic mutants, but not of untransfected cells or cells transfected with inactive mutants contained an immunoreactive protein with a molecular mass of ~48 kDa. Since this form of HGSNAT contains the C-terminal CBP cross-reacting with the antibodies we estimated that it corresponds to the HGSNAT fragment missing ~300 amino acids on the N-terminus. It seems that the appearance of this form is associated with the proper glycosylation and lysosomal targeting of HGSNAT, but further studies are necessary to conclude whether it represents a product of intralysosomal maturation (with the N-terminal chain not being detected by our antibodies) or a degradation product. On the other hand, the 77-kDa form of HGSNAT separated by FPLC anion-exchange chromatography showed full enzymatic activity (Figure 3).
The effects of missense mutations on the subcellular localization of the enzyme were studied by confocal immunofluorescence microscopy. Immortalized human skin fibroblasts from a normal control were transfected with constructs expressing each of the missense mutations. The cells were allowed to express the mutant and polymorphic enzymes for 42 hours and were then fixed by paraformaldehyde. To identify the lysosomal-late endosomal compartment, prior to fixation the cells were treated with LysoTracker Red DND-99 dye. After fixation the cells were permeabilized with Triton X-100 and probed with anti-CBP antibodies to localize the HGSNAT and with antibodies against the ER marker calnexin and lysosomal marker LAMP-2.
Distinct punctate staining characteristic of lysosomal targeting of the protein was evident for the recombinant wild-type enzyme and all active mutants. Accordingly, both wild-type recombinant HGSNAT and polymorphic mutants almost completely co-localized with the lysosomal markers LysoTracker Red or LAMP-2 (representative data are shown in Figure 4). In contrast, the inactive mutants exhibited a diffuse pattern throughout the cell and were co-localized not with the lysosomal markers but with the ER marker calnexin (representative data are shown in Figure 4, see Figure S2 for the data on all mutants). Partial ER localization (typically 2–5% of the total, not shown) was also observed for the wild-type recombinant and active mutants and most likely represented a pool of the enzyme processed in the ER on its way to the lysosomes.
Since all the data were consistent with general folding defects and retention in the ER compartment of the HGSNAT mutants (C76F, L137P, G262R, N273K, P283L, R344C, R344H, W403C, G424S, E471K, M482K, A489E, S518F, S539C, S541L, D562V, P571L) we further tested whether competitive inhibitors of the enzyme that mimic the substrate binding in the active site would help to fold the enzyme in the ER so it can be properly modified and exported to the lysosome. We first tested several potential inhibitors of HGSNAT that were not expected to be highly toxic for the cultured cells and found that one of them, D-(+)-glucosamine hydrochloride, was a competitive inhibitor of the enzyme with a KI of 0.28 mM close to the KM value for the 4MU-βGlcN substrate (Figure S3). To establish the optimal concentration of the inhibitor in the culture medium and the length of the treatment, we have used the immortalized skin fibroblasts of previously reported MPS IIIC patient homozygous for the N273K mutation that causes misfolding of the enzyme (Figure 1 and Figure 4), since they were readily available in our lab [17]. Immortalized fibroblasts from a patient homozygous for a splice site mutation (c.1726+1G>A) [11] and normal immortalized fibroblasts were used as controls. We found that the treatment of fibroblasts from a patient homozygous for the N273K mutation with 14 mM glucosamine results in the progressive increase of N-acetyltransferase activity in cell homogenates which reaches 3 fold induction after 5 days of treatment (Figure 5A). The N-acetyltransferase activity in the normal cells and in the cells of the MPS IIIC patient homozygous for the splice site mutation c.1726+1G>A as well as the control β-hexosaminidase activity in all cell lines were not increased upon the treatment (not shown) suggesting that the observed induction of N-acetyltransferase activity was caused by rescuing the N273K mutant but not by the general induction of lysosomal enzymes. When the cells of the patient affected with the N273K mutation were treated for 5 days with increasing concentrations of glucosamine (Figure 5B) we found that the effect was proportional to the concentration of the inhibitor. At the glucosamine concentration of 14 mM (~50x KI) the N-acetyltransferase activity in the treated cells was 0.3 nmol/h mg which corresponds to ~7% of the average activity in normal control fibroblasts, but further increase of glucosamine concentration resulted in inhibition of cell growth. To understand whether similar effects could be also observed for other missense mutants the available primary cultures of skin fibroblasts from 9 MPS IIIC patients carrying missense HGSNAT mutations or a missense mutation in combination with a splice site or nonsense mutation were treated with glucosamine and assayed for N-acetyltransferase activity. Nine missense mutations were studied altogether (Figure 5C; patient genotypes are listed in the figure legend). Although the maximal effect for different cell lines was achieved at different concentrations of glucosamine and at different treatment time (not shown), for 8 of 9 cell lines a statistically significant increase in the activity was observed (Figure 5C), suggesting that some of the mutants were partially stabilized by the glucosamine, correctly processed and targeted to the lysosomal compartment.
The current work provides an explanation for the severe phenotype of MPS IIIC patients carrying missense mutations in the HGSNAT gene. Altogether, we studied the activity and biogenesis of HGSNAT mutants having 21 amino acid substitutions previously identified in MPS IIIC families and affecting 8 of the 11 transmembrane segments of the enzyme as well as its luminal and cytosolic domains (Figure 6). The missense mutations studied in this paper represent all of the currently identified missense HGSNAT mutations. The polymorphisms (P237Q, V481L, K523Q and A615T, shown in green in Figure 6) identified in MPS IIIC only in cis either with a splice site mutation or with a missense mutation [11], [13] were previously reported by us to result in catalytically active protein [13]. They were included in the current study because it was unclear whether they still could affect kinetic parameters or targeting of HGSNAT and therefore represent a clinically valuable phenotype. All variants were expressed as TAP-tagged proteins since no Western blotting or cellular immunostaining could be conducted using the patient fibroblasts due to a lack of specific antibodies against human HGSNAT. Our results show that the HGSNAT variants P237Q, V481L, K523Q and A615T are all correctly processed targeted to the lysosome and display full enzymatic activity. We conclude therefore that these four mutations represent rare polymorphisms in the HGSNAT gene and do not have clinical significance thus confirming our previous hypothesis [13].
Seventeen mutations (C76F, L137P, G262R, N273K, P283L, R344C, R344H, W403C, G424S, E471K, M482K, A489E, S518F, S539C, S541L, D562V and P571L, shown in red in Figure 6) result in production of misfolded HGSNAT protein that is abnormally glycosylated and not targeted to the lysosome. Seven of these mutations (G262R, P283L, W403C, M482K, A489E, S518F and P571L) are predicted to reside within the highly hydrophobic transmembrane domains of the protein. Four of them (G262R, W403C, M482K, A489E) introduce hydrophilic or charged residues inside the transmembrane domains which usually have a dramatic effect on the folding of the protein [reviewed in 18]. Three other changes (P283L, S518F and P571L) result in replacement of hydrophilic residues for hydrophobic ones that also could destabilize the transmembrane helix.
Six mutations (R344C, R344H, E471K, S539C, S541L and D562V) are found adjacent to the predicted transmembrane domains either on the cytoplasmic (D562V) or on the lumenal (R344C, R344H, E471K, S539C, and S541L) side and 4 mutations (C76F, L137P, N273K, and G424S) reside inside the hydrophilic lumenal domains of the enzyme. In most cases these mutations are predicted to have a drastic effect on protein folding since they involve replacements with amino acids significantly different in hydrophobicity (C76F, D562V, S541L), charge (R344C/H, N273K, E471K) or size (C76F, L137P, G424S). Thus, enzyme folding defects due to missense mutations, together with nonsense-mediated mRNA decay seem to be the major molecular mechanisms underlying MPS IIIC.
For at least 5 of the above changes (N273K, R344C, R344H, S518F and S541L) the active conformation can be stabilized by the competitive inhibitor of HGSNAT glucosamine resulting in part of the enzyme pool being properly processed and targeted to the lysosomes. L137P and P283L mutants may also be stabilized by the glucosamine treatment, however this could not be verified experimentally because in the available patient cell lines they were present together with the responsive mutations S518F and R344C, respectively. Only one cell line carrying E471K and D562V mutations did not show a significant increase in N-acetyltransferase activity in response to glucosamine. Further structural studies are needed to fully understand the difference in the effect of glucosamine on these mutants.
Although the spectrum of mutations in MPS IIIC patients shows substantial heterogeneity, some of the missense mutations have a high frequency within the patient population. Importantly, the two mutations, R344C and S518F, responsive to glucosamine-mediated refolding account for 22.0% and 29.3%, respectively, of the alleles among the probands of Dutch origin [19]. The S518F mutation has also been identified in a patient from Germany, while the R344C change was found in families from France, UK, Germany and Singapore. The responsive mutation R344H was found in 4 families from Eastern and Northern Europe (2 from Poland, one from Czech Republic and one from Finland) and the responsive mutation S541L was reported in 4 families from France, Ireland, Poland and Portugal. In general, the vast majority of patients is affected with at least one missense mutation interfering with the proper folding of the enzyme that could be partially rescued by the treatment of the cells with the competitive inhibitor of HGSNAT, glucosamine. We believe this makes MPS IIIC a good candidate for enzyme enhancement therapy [reviewed in 20], [21], where active site-specific inhibitors are used as pharmacological chaperones to modify the conformation of the mutant lysosomal enzymes usually retained and degraded in the ER in order to increase the level of the residual activity to a point sufficient to reverse the clinical phenotype. Together with inhibitors of heparan sulfate synthesis, pharmacological chaperones could potentially reduce storage of this polymer in the central nervous system to levels sufficient to stop neuronal death and reverse inflammation.
The current study was conducted with ethics approval from the review board of CHU Ste-Justine, University of Montreal.
The wild-type HGSNAT-TAP plasmid was obtained by subcloning the HGSNAT 1992 bp coding sequence into pCTAP vector (Stratagene). Briefly, a 3′ part of human pCMV-Script construct [11] missing the stop codon was amplified by PCR using primers 76-Cla-F 5′-TTG CTC TTA TAC TCA TGG TCT TTG TCA-3′ and TM76-R4 5′-ATA TGT CGA CGA GCC ATC CGA TTT TCC-3′ and then used to replace Cla I - Sal I segment of the same construct prior to subcloning into pCTAP using Hind III – Sal I sites. Missense mutants were constructed using QuikChange Lightning kit (Stratagene) with HGSNAT-TAP as a matrix (see Table S1 for primers). All primers were designed using QuikChange Primer Design Program (http://www.stratagene.com/qcprimerdesign). For all constructs the coding sequence included an extra 84 base pairs on their 5′ end (encoding 28 amino acids) as the sequence first described for HGSNAT gene [11], but for clarity the nomenclature used reflects a unified numbering system based on the sequence of GenBank entries NM_152419.2 and NG_009552.1 as in Fan et al. [12], Ruijter et al. [19], Fedele et al. [22] and Feldhammer et al. [13].
Skin fibroblast lines of MPS IIIC patients, obtained with a written informed consent [11], and COS-7 cells (ATCC) were cultured in Eagles's minimal essential medium supplemented with 10% (v/v) fetal calf serum (Wisent). Transfections were carried out using polyethyleneimine 25,000 (PEI, Polysciences inc.) or Lipofectamine LTX (Invitrogen). PEI was dissolved in water at 1 mg/ml and pH was adjusted to 6.8 with HCl. For transfection, a mixture of 2 µg DNA and 8 µg PEI in 0.4 ml of serum free medium was incubated for 15 min at room temperature and added to ~70% confluent cells growing in a 10 cm dish. The transfection media was replaced with growth media 18 h later. Lipofectamine LTX was used as described in the manufacturer's protocol. Patients (N273K and c.1726+1G>A) and control fibroblasts immortalized by transfection with retroviral vectors expressing the type 16 human papilloma virus E7 gene and the catalytic component of human telomerase were described before [17].
Patient and control skin fibroblasts were cultured as described above in growth media supplemented with various concentrations of D-(+)-glucosamine hydrochloride (Sigma G4875). Medium was replaced every day and at specified time cells were harvested and assayed for N-acetyltransferase activity and β-hexosaminidase activity as described below.
N-acetyltransferase enzymatic activity was measured using fluorogenic substrate, 4-methylumbelliferyl β-D-glucosaminide (4MU-βGlcN, Moscerdam, Rotterdam, The Netherlands) as previously described by He et al. [23]. The reaction mixture containing 5 µl of cell homogenate, 5 µl of 6 mM acetyl-CoA and 5 µl of 3 mM 4MU-βGlcN in McIlvain buffer (100 mM sodium citrate, 200 mM sodium phosphate, pH 5.7) was incubated at 37°C for 3–18 h. The reaction was terminated by adding 1.98 ml of 0.5 M Na2CO3/NaHCO3, pH 10.7, and fluorescence was measured and used to calculate the specific activity. Lysosomal β-hexosaminidase activity was measured as previously described [24]. Protein concentration was measured according to the method of Bradford [25] using a commercially available reagent (BioRad).
To remove N-linked glycans from HGSNAT, cell homogenates were treated with recombinant endoglycosidase H, or peptide: N-Glycosidase F (Endo H; PNGase F New England Biolabs). Briefly, the mix consisted of 10 µg of cell homogenate in 25 mM sodium phosphate buffer, pH 7.5, to which 1 µl (500 U) of concentrated Endo H or PNGase F was added before incubating at 37°C overnight and analysis by Western blot.
To inhibit glycosylation of newly-synthesized HGSNAT COS-7 cells transfected with plasmids coding for the wild-type enzyme and the C76F or P237Q variants were treated with 1 µg/ml of tunicamycin (Sigma) added 5 h after transfection and allowed to express the recombinant protein for 48 h in the presence of drug. Media and drug were changed after 24 h and the homogenates were analyzed by Western blot as described below.
Cell homogenates were sonicated and boiled in LDS sample buffer (Invitrogen) in the presence of 25 mM DTT. Proteins were resolved by SDS-polyacrylamide gel electrophoresis using NuPAGE 4–12% Bis-Tris gels (Invitrogen) and electrotransferred to PVDF membrane. Detection of TAP-tagged N-acetyltransferase protein was performed using anti-calmodulin binding peptide epitope tag (CBP) rabbit antibodies (Immunology Consultants Laboratory, dilution 130,000) and the Amersham ECL Western Blotting Detection Reagents (GE Healthcare) in accordance with the manufacturer's protocol.
COS-7 cells were harvested 42 h after transfection with the wild-type HGSNAT plasmid and suspended in lysis buffer (10 mM Tris-HCl, pH 7.5, 0.1% NP-40, 1 mM PMSF and Sigma P8340 protease inhibitor cocktail at 10 µl per 1 ml of cell suspension). The homogenate was sonicated, gently shaked at 4°C for 2 h and centrifuged at 13,000 rpm for 30 min. One ml of the supernatant containing 8 mg of total protein was applied to an ion-exhange Mono Q HR 5/5 column equilibrated with 10 mM Tris buffer, pH 8.2. The column was washed with 4 ml of the same buffer and then eluted using a 20 mL gradient of NaCl (0–0.5 M) at a flow rate of 0.5 ml/min. One ml fractions were collected and assayed for N-acetyltransferase activity. Thirty µl aliquots from each fraction were analyzed by SDS-PAGE and Western blot using anti-CBP antibodies as described above.
Immortalized control human skin fibroblasts were transfected with HGSNAT-TAP or plasmids coding for the HGSNAT mutants using Lipofectamine LTX (Invitrogen) as described in the manufacturer's protocol. Forty-two hours post-transfection cells were incubated for 1 hour with 1 µM Lysotracker Red DND-99 (Invitrogen) and then washed with ice-cold PBS. Cells were fixed with 4% paraformaldehyde, 4% sucrose in PBS for 5 min, and then rinsed 3 times with PBS. Cells were permeabilized by 0.25% Triton X-100 for 10 min and blocked for 1 h in 3% horse serum and 0.1% Triton X-100. Cells were either co-stained with rabbit anti-CBP (Immunology Consultants Laboratory; 1400) and mouse monoclonal anti-calnexin (Millipore; 1250) antibodies in 3% horse serum, with anti-CBP antibodies and mouse monoclonal antibodies against human LAMP-2 (Developmental Studies Hybridoma Bank; 1150), or with anti-CBP antibodies and Lysotracker Red DND-99. Cells were then counterstained with Oregon Green 488-conjugated anti-rabbit IgG antibodies or Texas-Red-conjugated goat anti-mouse antibodies (Molecular Probes; 11000). Slides were studied on a Zeiss LSM510 inverted confocal microscope (Zeiss). Images were processed using the LSM image browser software (Zeiss) and Photoshop (Adobe).
Lineweaver-Burk plot of substrate dependance for partially purified HGSNAT wild-type and P237Q and V481L mutants. COS-7 cells were harvested 42 hrs after transfection with the wild-type or mutant HGSNAT plasmids and suspended in lysis buffer (40 mM Tris-HCl, 300 mM KCl, pH 7.5, 0.1% NP-40, 1 mM PMSF and Sigma P8340 protease inhibitor cocktail at 10 µl per 1 ml of cell suspension). The homogenate was sonicated, gently shaked at 4°C for 2 h and centrifuged at 13,000 rpm for 30 min. The supernatant was first passed through an avidin-agarose column (Sigma A9207) then affinity purification of TAP-tagged HGSNAT was performed using streptavidin resin (Stratagene) according to the manufacturer's protocol. N-acetyltransferase activity was assayed as described in Material and Methods using 0.05 to 2.0 mM 4MU-βGlcN and 18 h incubation time. KM and VMAX values for all 3 enzymes were similar within the statistical error.
Localization of HGSNAT mutants expressed in cultured human skin fibroblasts by immunofluorescence microscopy. The cells transfected with wild-type or mutant HGSNAT-TAP constructs as indicated were fixed and stained with either mouse monoclonal anti-LAMP-2 antibodies, Lysotracker Red DND-99 or mouse monoclonal anti-calnexin antibodies (red) and rabbit polyclonal anti-CBP antibodies (green) as indicated. Slides were studied on a Zeiss LSM510 inverted confocal microscope. Magnification 630x. Panels show representative images showing co-localization of anti-CBP antibodies (green) and lysosomal and ER markers (red) for the active enzyme containing polymorphisms and all inactive mutants.
Dixon plot showing the inhibition of HGSNAT by glucosamine. COS-7 cells were harvested 42 hrs after transfection with the wild-type HGSNAT plasmid and N-acetyltransferase activity was measured in the homogenates for 3 h at 37°C in the presence of 2 mM AcCoA, 0.0375 to 1.5 mM 4MU-βGlcN and 0 to 2 mM D-(+)-glucosamine hydrochloride.
Primers for site-directed mutagenesis of HGSNAT-TAP plasmid.
The authors thank the patients and their families for participating in our study. We also thank Maryssa Canuel for helpful advice, Mila Ashmarina for critical reading and Eva Lacroix for help in preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This work was supported by research grants from March of Dimes Foundation, Sanfilippo Childrens Research Foundation and Canadian Institutes for Health Research (MOP84430) to A.V.P. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Pecan Apple Crisp
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Recipe by Manami
You can use any kind of nuts in the topping of this crisp, although pecans go so nicely with apples. Walnuts or sliced almonds would also be good choices. Courtesy of Diana's Desserts June 13, 2003.
See Discussion:
Ingredients Nutrition
- 6 large firm baking apples, peeled cored. quartered and sliced 1/2-inch thick. (McIntosh, Granny Smith or Fujis)
- 1⁄2 lemon, juice of
- 1⁄2 cup all-purpose flour
- 1⁄4 cup brown sugar
- 1 tablespoon granulated sugar
- 1⁄4 teaspoon ground cinnamon
- 1 dash ground nutmeg
- 1 cup pecans, toasted & coarsely chopped
- 1⁄2 cup unsalted butter, cut into pieces
- whipped cream
- vanilla ice cream (optional)
Directions See How It's Made
- Preheatoven to 350°F.
- Butter a deep 2-quart or 9x13" baking dish.
- Place the sliced apples in large mixing bowl.
- Stir in lemon juice.
- Toss well.
- Blend flour, both sugars, cinnamon and a dash of nutmeg in a food processor until well combined.
- Add butter and blend until mixture forms large clumps.
- Transfer to a bowl and work in pecans with your fingertips.
- Pour sliced apples into prepared baking dish.
- Spread the topping mixture over apples.
- Bake for 30 minutes or until apples are tender and the topping is browned.
- Serve warm with whipped cream or ice cream.

subset: commoncrawl


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Need Pettinice Or Satin Ice Fondant Asap....help!!!
Decorating By chrissysconfections Updated 17 Mar 2008 , 7:39pm by foxymomma521
Normally I buy my fondant online well in advance from any cake order but my DH just called me from work. He said a co-worker just asked him if I could do a wedding cake for him....in fondant with roses in 11-12 days!!!!
I can't get fondant from my normal supplier that fast and I'm trying not to go with the wilton brand. I know I can make MMF but honestly I don't like to and I'm thinking since he's so adament on fondant that he'd know the difference. Good thing is though that if need be he'd probably settle for Wilton brand simply because that's all that is available around here.
Does anyone know of any suppliers that would ship to me by say the 26th??
Thanks for any help!
IntoTheOven.com has expedited shipping, if you order it today, you can get it before the end of this week, and it is not outrageous...
I;m not sure where you are, but I order fondant all the time from Pfeil & Holing (cakedeco.com). They're about 3 hrs away from me in NY, but it arrives the next day!!
edited to add: and that's just regular shipping by UPS.
ditto to speed of IntoTheOven.
They're in Illinois and I'm in NC....usually get it in 3-4 days regular -- not expedited -- shipping.
Their prices are good too.
and if you are closer to CA -- the try Global Sugar Arts.
Doug, i think Global Sugar Art is in NY too? I know Caljava is in CA.
I just ordered from Into The Oven on Thursday and got my 10# of Chocopan this morning! I'm two states away and just used regular shiping. It is the second time I've ordered from them and have been very happy both times! They have good prices too!
I also get mine from Intotheoven.com and I get mine the same week I order
I order from CK Products who I consider to be slow on shipping, but it still gets here in 5 days, UPS ground.
I'm another IntoTheOven.com fan. I live in CA and mine is delivered in 5 working days. I believe they have a chart on their website that shows how long to estimate shipping, but I get mine 1 to 2 days before what it shows for my area.
Here Here on IntoTheOven! They are great when it comes to shipping! You can just do regular and it will get there in enough time. If you order today you'll probably have it by Thursday or Friday.
Doug, i think is in NY too? I know Caljava is in CA.
ok...so I'm left coast/right coast or is that west coast/east coast challenged....
oh heck...just point me to the chocolate!
I got my www.IntoTheOven.com order in 3 days with their regular shipping. There is a chart on their website showing you when UPS will deliver to ya.
Thank you all!! I just met with my husband's co-worker and he is going to have his future wife call me because he liked all the cakes I showed him...LOL Sounds to me like she already has him trained!
I'm hoping she calls tonight to finalize things because he said they may go with just BTC now too, he'd have to check with her. He said they already had the flowers and the cake topper for the cake so I'm assuming they are artifical and the previous decorator backed out for some reason. They mentioned a third party picking the cake up but I insisted on delivery since it's within our delivery free limits.
Hopefully if they call tonight and we can get the order rolling then I can order it tomorrow first thing if need be and have a backup plan if it's not here in time.
Thanks everyone!!
I like www.fondantsource.com I've placed about 10 orders with them and they were all delivered lightening fast. Use THANKYOU as a coupon code for $5 off a $20 order. Or DOLLAR for $1 off any order...
Heath was going to fix that so it wouldn't be blocked anymore... f o n d a n t s o u r c e . c o m


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Reviews For
Jack Black True Volume Thickening Shampoo
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Tell us your thoughts about Jack Black True Volume Thickening Shampoo so we can help you find the right products. With your review, you'll also receive 50 Rewards Points. Please note that it may take up to 72 hours for a review to appear on site. All reviews are subject to moderator approval.Write a Review
I have long blonde fine hair. And a lot of it. I decided to give this a try. I read other reviews that women can use this. I am sure that is true. But this shampoo left a film on my hair. Creating a very dull look to it. I used it twice then quickly returned. I gave it two stars because I feel you get a lot of shampoo for your money and the smell is nice.
My hair feels really nice after this shampoo. It works and makes hair a bit thicker. Good product, give it a go.
I am a girl and I love this shampoo, even if it is meant for men :)


text: unknown number: i can't believe you totally stood me up shae like wtf??? i brought you flowers and everything. what kind of girlfriend breaks up with their boyfriend through a bARISTA AT A CAFE. UR LITERALLY THE WORST. unknown number: OH AND BTW UR COOKIES ALWAYS KINDA SUCKED ASS LMAo nikki: um i'm sorry i think you've got the wrong number or something nikki: but whoever shae is, she sounds like a hoe lol


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This sweet vintage cross is made with two shades of 14K gold. Most of the cross is yellow gold and in the center there's an overlaid white gold cross. The yellow gold ends are textured. The white gold cross in the center is smooth. Makes for a very lovely contrast.
The cross measures 5/8 inch across by 1 1/8 inches high with bail. It weighs .8 gram. Back is marked 14K. Has also been acid-tested.
Excellent vintage condition.
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Fine to funky estate jewelry - Victorian to 1960s Mod - Antiques & Collectibles - FREE DOMESTIC SHIPPING - LAYAWAY Available


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It was Labor Day and we all was go to the lake so we headed up there the first day we set up camp. Then the next day we got the boat out and we went to find a boat ramp. Then we fond one but there was only one ramp and 20 people in front of us but we waited. Finally we got to lunch it but it wold not start. I was so mad because the weather was so nice and there was no waves or white caps. So we loaded it up and went back to camp. Then we went swimming and my dad worked on it the water was Reilly shallow. Then we got it working finely then we went to launch it. We got on the water the we got Reilly far out then the boat stopped working we got it working but it Reilly slow so we loaded it up again and went to camp my dad got it working again but we did not take it out because it was to late.
Then we went into a small town that was cold Wilson so we could get ice. The we got back to camp. We eight stake it was good. Then we went to sleep. We got up in the morning and it was so windy that it was shaking the camper so we cold not go boating that day but we went swimming and we found where Blake was camping so we went swimming together for 5 minuets because it started storming.
So we went to see if Blake could stay the night with me and he could so we got to the camper and we went down to the beach and played in the sand. We fond a tackle box that had nothing in it we where going to barry it but we didn't cuss it was time to eat we eight chicken. Then me and Blake went to the park to hang out.
Then it started to rain and there was a hill that got all muddy so we played on it tell 12:00 then we where all muddy so we went to take a shower. It was hard to get the mud out off our close. The we got in side and played on my iPod for a little bit then we wend to sleep it was late. Then we got up and went to the park for a little bit.
the we went to lunch the boat and take Blake home on the boat then we he got to his camp we went to go tubing it was fun wants we were don tubing we went to the docs to look then we swam and drove the boat for a wile then we went to load the boat up they where launching a big house boat it took a long time to launch it but it was fun to watch it. The we went home and the next day I went to school.


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Welcome to the Raise Bid coupon discount page. We have many deals and coupons for our customers that can be used throughout our website. Our deals change every month, so please check back often to reap the rewards.
October 2016 Coupon Codes
Enter any of the coupon codes in our shopping cart to receive your discount. Thank You, Happy Shopping!


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3780 Wynter Frost Walk Bethlehem, GA 30620
- Listing Status Active
- Neighborhood Wynterberry
- County Gwinnett
- Year Built 2009
- Sale Type Normal sale
This home is nestled in the perfect location! You will never want to leave the back yard as it features serine, and tranquil flowers and waterfall with a coy pond. You will want to buy this perfect home for the back yard alone. Well maintained landscape all year round. Spacious living area, and a bonus room tucked upstairs for those quite reading, workout or office days. MUST SEE IN PERSON!! Many upgrades in this home. Seeing is believing! More pictures to come of the inside soon.
- Listing Status Active
- Neighborhood Wynterberry
- County Gwinnett
- Year Built 2009
- Sale Type Normal sale
- Elementary Harbins / Gwinnett / Gwinnett County, GA
- Middle
- High Archer / Gwinnett / Gwinnett County, GA
Listing provided courtesy of CHAPMAN HALL REALTORS PROF.. Listing data displayed on this page comes from the Broker Reciprocity program of Georgia MLS. This information is considerd reliable but is not guaranteed.
Look Promising?
Want to schedule a showing? Have any questions? We're experts and ready to help!
Call us anytime at 404-965-4080
Listings identified with the FMLS IDX logo come from FMLS, are held by brokerage firms other than the owner of this website and the listing brokerage is identified in any listing details. Information is deemed reliable but is not guaranteed. ©2014 First Multiple Listing Service, Inc.
© 2014 North East Georgia MLS. the information provided is for the consumer's personal, noncommercial use and may not be used for any purpose other than to identify prospective properties that the consumer may be interested in purchasing. Some or all of the listings displayed may not belong to the firm whose web site is being visited.


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1 Will you charge:
A Top-up fees across the board maximum (£3,000)
B A flat rate for all courses below £3,000. (If so, what is it)
C Different rates for different courses. (If so,please specify range - ie minimum/maximum)
D No decision
2 If you are varying fees will they be:
A Lower for Sciences
B Lower for Arts/Humanities/social sciences
C Lower for foundation degrees
D Lower for vocationally-focused degrees
E A combination, please state (areas or specific courses)
3 Are you introducing bursaries?
A Yes
B No
4 How much for?
We already have bursaries of more than £300
5 If yes, will they be:
A Targeted at the most needy students
B Targeted at subjects which are short of students
C A mixture, please detail
6 If yes, are they funded
A From central university funds
B From a benefactor, private donations, etc?
C A mixture
7 Have you employed outside agencies to help you make the decision (market researchers/brand managers)?
A Yes
B No
8 Where will you spend the extra income?
A Better student facilities
B Better academic facilities
C Better academic pay
D Bursaries
E Bumping up research capacity
F Other, please state
9 Will top-up fees provide enough extra income for the university for the foreseeable future?
A Yes
B No
10 How long do you expect the limit on top-up fees to remain at £3,000?
A 1-3 years
B 4-6 years
C 7-10 years
D Over 10 years
Hard to predict
11 Do you think top-up fees will put less well-off students off going to university?
A Yes. "I think students will be more selective on the fee they are paying. They will look much closer at where they are going."
B No
12 How do you think Offa will affect your attempts to widen participation?
A Help
B Hinder
C Have no effect
D Undecided, please state


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Lille vs Marseille Prediction, Betting Tips and Preview
The great match we expect in the Pierre-Mauroy stadium, where meet Lille and Marseille. Lille plays most defensive football in the Ligue 1. They scored only 5 goals, but the same they conceded. Marseille is in awful form, they took only three points from five matches and lost two of three last matches. They have only four points far from Monpellier, the relegation zone team. Last year, both matches between these two teams, Marseille won. Match starts: 25.10.2015 16:00.
Lille is the most defensive team in the Ligue 1. Lille scored two goals only in one match of ten played. At home, Lille has two wins against Ajaccio 1:0 and Montpellier 2:0, two defeats from Nantes and PSG with 1:0 and one draw with Bordeaux 0:0. As a guest, Lille didn't win yet and scored only two goals in five matches. They drew two times by 0:0 with Monaco and Lyon and 1:1 with Rennes and Guingamp, and lost from Reims 1:0.
Marseille is now in the unbelievable 16th position and instead the fighting for the title or the positions that leads to Europe, they fear of the relegation. At home, they are three matches without a win. They beaten from Angers 2:1, drew with Lyon and Lorient 1:1 and won with huge 4:1 Bastia and even more convincing 6:0 over Troyes. As a guest, they defeated three times from Reims 1:0 Gungamp 2:0 and PSG 2:1 and drew with Toulouse 1:1.
Missing players
Lille have few missing players for this match against Marseille. Michael Frey, Benjamin Pavard and Mounir Obbadi are injured and they cannot help their team on this very hard match. Stoppila Sunzu is suspended and he also will not play for sure. Marko Basa is not fully fit and he is doubtful for the match.
On the other side Marseille dont have many missing players for this match. Abou Diaby and Lass Diarra are doubtful for the game and they probably will not play against Lille. All other players are fully fit and ready for the match. All of them are highly motivated to earn points here and for sure we expect very offensive playing from them.
Possible starting lineups
Lille: Enyeama; Amadou, Civelli, Basa; Sidibe, Balmont, Mavuba, Corchia; Boufal, Tallo, Bautheac
Marseille: Mandanda; Manquillo, N Kolou, Rolando, Mendy; Diarra, Silva, Alessandrini, Cabella, Barrada; Batshuayi
Lille vs Marseille Prediction
We expect very strong football match where both teams have chances to earn points. Lille play very strong football in defending and for sure it will be their main objective for this game. On the other side Marseille play very badly in this season and they have to play offensive on this match if want to earn points. It will be very hard job for them to score more then one goal Today. So our prediction and betting tips for match Lille vs Marseille Prediction: Under 2.5 goals. We expect exact score 1:1 so our second betting pick for this match is Both teams to score. Have a nice betting day and good luck in earning money with our football prediction and betting tips.


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Portugal's PM names new deputy in deal to avert coalition collapse
Portugal's prime minister said Saturday that foreign minister Paolo Portas, whose resignation this week sparked a political crisis, will remain in government and get a bigger role under a deal to hold the ruling coalition together.
"I have proposed that Paolo Portas take on the post of deputy prime minister with the responsibility of coordinating economic policies and relations with the 'troika' of Portugal's creditors," said Pedro Passos Coelho in a press conference alongside Portas.
Portas, who also leads the coalition's junior partner, the small conservative CDS-PP party, had resigned on Tuesday over disagreements with the coalition's economic policies.
His announcement had come just a day after finance minister Vitor Gaspar said he was quitting, throwing the country into a crisis.
The shock resignations raised the spectre of a fresh wave of instability in the eurozone's debt-laden periphery, and sent Portugal's 10-year borrowing rates to critical levels.
At the heart of the dispute are the painful economic reforms pushed by Passos Coelho's coalition in exchange for desperately needed loans from the "troika" of international creditors -- the European Union, the European Central Bank and the International Monetary Fund.
Portas, who wants a change in economic policy, objected to the premier's choice of new finance minister, Treasury Secretary Maria Luis Albuquerque, who was expected to pursue further austerity in defiance of mass protests.
Under details of the deal announced Saturday, Albuquerque was confirmed to the post.
Passos Coelho said the agreement with Portas was "solid" and would help "guarantee that the international engagements of Portugal will be respected".
"It is our obligation ... to ensure stability, which is indispensable to overcome the fragile political situation that we are experiencing," he said.
The political deal would still require the support of President Anibal Cavaco Silva, who has the power to dissolve parliament and call for elections.
Passos Coelho said the president would announce a cabinet reshuffle in the coming days.
Unions are piling on the pressure with hundreds of protesters answering their call, in a demonstration Saturday demanding the government's resignation.
"The situation has become unbearable. We want a real change, not musical chairs," said unemployed 38-year-old Edite Chainho, who was holding a banner reading "Government out".
The austerity measures including layoffs and higher social security contributions have thrown Portugal into a deeper recession with higher unemployment than had been expected, sparking mass protests and strikes.
The government expects the economy to contract by 2.3 percent by the end of the year, while the unemployment rate has soared to a record 18.2 percent.
Despite growing opposition to the reforms, the government is under pressure to present another 4.7 million euros in spending cuts to the troika when its auditors visit Portugal on July 15.
According to a survey published Saturday by the bi-weekly Expresso, 37.2 percent of those surveyed said they favour early elections, while 45.1 percent wanted the status quo or a new government headed by the president.


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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.
How to make my form mobile (phone) friendly?Asked by chemoreno on November 19, 2016 at 10:14 AM
I've created a form and used a widget so it can be used over the phone, but the users are having a hard time using it. The Date/Time won't fit and it's causing error as soon as you enter the Date because the Time is then cut off.
Please help.
JotForm Support
I can see that you already inserted the Mobile Responsive widget in your form. Please try enabling the "Make this form Responsive" option in the Form Designer as well.
Please insert the custom CSS code below in order for the Date Time field to be responsive:
@media only screen and (max-width: 40em) {
#label_10 {
width : 100% !important;
div#cid_10 {
display : block;
#label_21 {
width : 100% !important;
#cid_21 {
display : block !important;
span.allowTime-container {
display : block !important;
Here's a guide on How-to-Inject-Custom-CSS-Codes. Here's the result:
Hope that helps. Thank you.
Yes, it did helped!
The Dispatch Notification Form just worked beautifully after changes were made.
Although I struggled a little bit with one of the forms (AMG IC Run Sheet) when I modified the form and added the CSS code, because I had to adjust the width; or maybe I was just doing something wrong. But it appears to be working now as I intended. Can you check on this one for me please? If there's anything that I missed?
Thanks again..
JotForm Support
Hello Cherryl,
I did check your form "IC Run Sheet" and it does seems to display correctly in mobile device. I would suggest you to please move your question "Type of Vehicle Use" to a new line and that should give you enough width space to display the date/time filed correctly. Please check the screenshot below:
Thank you!


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Дата выпуска: 10.01.2010 г.
Продолжительность: 00:34:32
Описание: Priya is a petite brunette with huge tits. During a parent-teacher interview, she strips down. She fingers herself on his desk and even uses the ruler as a prop during her striptease. She keeps this going in front of him and he tries to avoid her, but she is willing to do anything to ensure that her child passes the class. She starts by giving him head and he licks her clit on his desk. She spreads her pussy lips wide for him and then mounts him in reverse. Soon he's got her doing doggy-style over his desk. They do many positions and there is constant oral sex and a tittie fuck till he nuts on her tits. Looks like Junior passes the class!
Формат: WMV
Качество: SiteRip
Видео: Windows Media Video 8 720x406 2428 kbps 29.97 fps
Аудио: Windows Media Audio 2 2 chanels 48.0 KHz 128 kbps
Размер: 660 мб


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"a huge thank you to all who work in this hospital"
About: Basingstoke and North Hampshire Hospital Basingstoke and North Hampshire Hospital Basingstoke RG24 9NA
Posted by Julia Annandale
At every turn we've been met by really outstanding care and kindness. Wonderful paramedics, doctors and nurses, fantastic medical treatment and so much time given to us by all when we needed it - thank you Basingstoke Hospital :)
Response from Basingstoke and North Hampshire Hospital 15 months ago
Thank you for taking the time to leave feedback regarding our cardiology services. I am always please to read heartfelt comments, like yours, on the care and treatment patient have received. I have shared these kind words with the team involved, who I am sure, will be delighted and greatly encouraged by them.
Mary Edwards, Chief Executive
- {{helpful}} of {{total()}} people think this response is helpful
- Was this response helpful? Yes | No
Thank you for your feedback.
See more responses from Basingstoke and North Hampshire Hospital


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Halifax have flatly denied receiving any approach from Super League strugglers Salford for highly-rated forward Sam Barlow.
Frenzied speculation over the weekend claimed the Reds had offered a transfer fee and the former Castleford hooker Wayne Godwin in a bid to land the former Siddal junior.
It is only a matter of weeks since Barlow signed a new two year deal at the Shay, apparently after rejecting approaches from both Salford and Fax's Championship rivals Featherstone.
"We have not had any discussions of any kind with Salford or any other Super League club regarding Sam Barlow," said Fax director Ian Croad, who said Barlow's contract does not include an automatic top flight get-out clause in the first 12 months.
"It's simple; As far as we are concerned Sam is a Halifax player for the next two seasons, end of story.
"I have no idea how these rumours start but we are building a team to win trophies, not dismantling one."
On the field, Featherstone lost their second Grand Final in three years when they were beaten by Sheffield in Sunday's Championship decider.
It was Rovers' second showpiece reverse of the year after losing to Fax in July's Northern Rail Cup final.


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This post has been updated.
There was a lot of love for Joe Brack's one-man show "My Princess Bride" after it debuted at the Fringe Festival this summer -- so much so that Brack and director Matty Griffiths decided to remount the production. But not everyone was a fan of the idea.
After receiving a cease-and-desist letter (the pair is not ready to say from whom, although people can probably guess), the play has been put on hold until further notice. Griffiths and Brack initially hoped to charge on, given that the play is really about Brack's own upbringing and his love for the book and movie. But, for the moment, the pair is taking the better-safe-than-sorry approach as they examine their options.
Before mounting the show, which the two men subsequently took on the road, Griffiths did some research to ensure they weren't infringing on copyright, and they were were "comfortable and confident it was fair use," he says.
"It's not a 'Princess Bride' redux," Brack says. "That's why the title is 'My Princess Bride.' It's the journey of my life."
While Brack does snippets of re-enactments, he intersperses such scenes with commentary. For example, during a solo parody of the iconic swordfight between Inigo Montoya and the man in black, Brack explains some of the history behind the obscure names of fencers that are thrown around in the dialogue.
But there is also plenty of personal material in the show: At one point Brack talks about the death of his grandmother in 2012.
The men are especially confused, because "My Princess Bride" neither shines a negative light on its source material nor constitutes a big-time production. With four performances a week for four weeks, Brack estimates that 1,000 people might have seen the show.
"In that scenario, we would have broken even -- at best," Brack says.
The law firm that emailed the cease-and-desist letter to Brack, Griffiths and Capital Fringe, which was to host the show, has not yet returned a request for comment.
While Brack and Griffiths stand by their belief that they have done nothing wrong, they had to cancel the show out of a sense of "caution and prudence," according to Griffiths, but more importantly because venues are understandably frightened by the risk. The men say that if another theater is willing to host them, they will mount the show.
In the meantime, the duo is awaiting word from their own attorney on next steps. Brack (who didn't realize he was wearing a black T-shirt with a faux nametag that reads "Hello my name is Inigo Montoya, you killed my father, prepare to die") has conflicted feelings. After exalting "The Princess Bride" for so many years, he says he feels lost.
"I'm gutted," Brack says. "The past two days have been so hard. And whenever I've been bummed out and sad, I watch 'The Princess Bride,' and I can't even do that now."
He owns three copies of the book, and he's reversed them in his bookcase to hide the titles.
"It feels like I've lost a friend," he says.


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Califon, NJ (PressExposure) October 03, 2007 -- Over the past few weeks, The National Metabolic & Longevity Research Center has been bombarded with questions â but not about how to lose weight like most people might think â but rather, how to look and feel younger. To help folks get off on the right foot, Christopher Guerriero shares with you the basic foundation to looking young and vibrantâ¦
The primary cause of all premature aging, is living an acid lifestyle. Once you switch from doing things that cause acid to build up in your body and start doing things to create an alkaline environment in your body, you'll immediately begin to look younger, you'll immediately begin to have more energy, and you'll immediately begin to feel far better about yourself. But what constitutes an alkaline lifestyle? Well, before we go into what an alkaline diet is, I'm going to go over what an acid diet is, which is a diet or a lifestyle or foods that you're putting into your body that are causing you to build up acid and build up stress inside of your system. Hereâs a brief list of foods you need to avoid - foods that actually cause your body to age faster:
>> Sugars and sugar-containing foods
>> yeasts, breads and pastries
>> raised baked goods, things like breads, rolls, and cakes
>> Alcoholic beverages, all alcoholic beverages cause acidity in the body
>> Condiments and sauces which are things like mustards, tomato sauce, soy sauce, horseradish, vinegar of all sorts, mayonnaise, salad dressing, things like that.
>> Processed or smoked meats, all processed and smoked meats cause acid in the body.
>> Sodas, depending on where you are in the world, you may call a soda a soft drink or a pop or something like that, but all sodas. There's absolutely nothing good in sodas. Every single ingredient in a soda causes an acid build up in your body.
...and that's just the short list.
But now it's time to learn about an alkaline lifestyle.
This is the lifestyle that's going to help you live a lot longer, it's going to help you be a lot healthier and happier during that long life, and it's going to help you look a lot stronger and sexier.
In order for your diet to alkalize your body, it must be rich in nutrient-dense foods, in clean water, in lean healthy protein, and it's also got to be free of most toxins. And I say âmost toxinsâ because in today's environment, it's very difficult to avoid all toxins because there are toxins in everything around us.
Foods that promote a strong, healthy looking, energy-rich, alkaline body are raw, preferably organic, vegetables, and this is the single most important step that you could take today to alkalize your system, eating fresh, raw, preferably organic, vegetables allows your body to actually use the live enzymes, the life inside of those vegetables, and the energy from those vegetables, to energize and build your health from within you. So you're building health and you're helping reduce signs of aging from the inside out.
Eat raw vegetables, preferably organic, all day long. Juice them at least one or two times a day if you can. If you can't juice them or you don't like eating vegetables on a regular basis, then get yourself a greens drink and drink that at least one or two times a day.
But that's it. That's the whole list. An alkaline diet consists of loads of fresh, raw, preferably organic, vegetables, lean sources of poultry in each meal like fish or chicken and turkey (and if you're getting chicken and turkey, try to make them organic or free-range because that means that you're avoiding the toxins in there), and egg whites.
And also fresh water, preferably distilled water because the distilled water is going to help pull out any of the impurities that are in your system.
If you don't want to get that strict yet, then at least begin taking out all the foods from the acid list, especially those loaded with preservatives and other chemicals, and then slowly work your way into adding the really nutrient-dense foods like the organic vegetables, the lean sources of clean protein and the fresh water.
For a full description of the top techniques to help you look years and years younger get the National Metabolic & Longevity Research Centerâs free audio titled: â17 Proven Ways To Look & Feel 10 Years Younger in 30 Days of Less!â at: http://www.MaximizeYourMetabolism.com/naturalyouth


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Unreachable Summit
Chapter Info
|Japanese Title:
|Romanized Title:
|Total Pages:
Chapter Chronology
|← Previous
|Next →
Unreachable Summit is the 329th chapter of Morikawa Jouji's manga series Hajime no Ippo.
- Characters Introduced: None
- Locations Introduced: None
- Techniques Introduced: None
- Mentioned Boxers: None
- Title Page Character(s): Makunouchi Ippo, Ricardo Martinez
With the jabs affecting his legs already, Ippo has no option but to walk directly into the attacks, while blocking in order to get within striking range. As he slowly walks forward, he receives the jabs, but his solid guard protects well against them. Once inside, Ippo begins weaving and starts performing his Dempsey roll. Martinez the effortlessly jabs Ippo's face over and over again, despite the fact that he is weaving his head at a high speed. With the Dempsey roll stopped, Martinez walks away and tells Stewart that he does not wish to shame the champion anymore and wants to end the spar. Kamogawa agrees, and tries to get Ippo out of the ring, but the boxer is in a state of unconsciousness and tries to hit Martinez before being stopped by his coach. As soon as he wakes up, Ippo thanks Martinez and leaves the gym.
Kamogawa states that it could not be helped since he was not yet up to the challenge, to which Ippo replies that he can not even put in word how far apart their levels are.
When he arrives home, Ippo is asked by his mother what happened to his face. When he takes a look in the mirror, he can see the effect "the world's strongest left" has on his face. After being so confident about Date's upcoming victory, Ippo begins to have doubts as he can not see Martinez losing.


text:
The home technology market is expanding exponentially, creating momentum and opportunities like never before – and nowhere is that potential better reflected than at your industry's premier trade event.
CEDIA 2017, dedicated 100% to home technology, gives you concentrated access to the new products, breakthrough innovations, and targeted training you need to drive your company competitively into the future.
Change is happening fast. Competing on the cutting edge requires that you understand current technology and get a glimpse of what's to come.
Whatever your role, CEDIA 2017 offers the training you need to move to the next level. From panels and workshops to lectures and labs, take advantage of a broad array of topics to create a customized training program for you and your team; you'll come away informed, inspired, and equipped for success.
CEDIA 2017 brings the home technology community together like no place else. Network with peers. Interact with thought leaders. Gain insight from influences. Meet collaborators. Don't go it alone – join more than 18,000 tech professionals for an unbeatable opportunity to connects.
Be a part of the biggest show in home technology. Learn about exhibiting at CEDIA. LEARN MORE
What is the CEDIA show? Discover networking, training, and innovation at the premier home technology tradeshow. ABOUT
Review the registration brochure and grow exponentially from all CEDIA 2017 has to offer!
Join us in San Diego, a lively, inviting city and an epicenter of technology that is home to many companies at the forefront of innovation. With its near-perfect weather, great hotels, convenient airport and excellent restaurants, San Diego is the ideal destination.
Members benefit big at the CEDIA show. Become a member and save money on training and more!
Learn more


text:
Christie Brinkley uses fillers to 'look my best'
Christie Brinkley used fillers to "look my best".
The 62-year-old American model - who was scouted at the age of 19 in a post office in Paris - has admitted she "occasionally" has cosmetic injections in an attempt to reverse any sun damage to her face and reduce the appearance of ageing because her career in the fashion industry puts pressure on her to look "good".
Speaking to PEOPLE about her beauty secret, the 'Timeless Beauty' author said: "I grew up in Malibu then my real job of modelling took me to beaches all over the world and under the sun. I really am aware of all the sun exposure I've had in my life and I'm interested in things that they can do to [reverse the sun's effects].
"Occasionally I'll do a little bit of filler.
"I'm a model. I want my skin to look its best ... As a model, if I don't look good, I'm not going to be able to be -- I don't want to say inspiration cause that sounds so full of myself."
And the blonde beauty - who has graced the cover of Sports Illustrated Swimsuit issue ion three consecutive issues - has revealed she has been slammed for her "fake" appearance, although Christie believes it is not solely down to the gruelling beauty procedures but additional skincare products,
She explained: "Some women on my Instagram will say, 'Oh, she's so fake. It's not her skincare, she gets filler.' And it's like okay if I only did filler and I didn't use my Recapture 360, I wouldn't have the moisturizing, I wouldn't have that bounce back ... it's the whole thing. You have to take care of the texture of your skin."
And the mother of three - who has daughters Alexa, 30, Sailor, 18, and son Jack, 21 - has revealed she has embarked on a healthy eating plan to take care of himself, although she will indulge in a glass of prosecco now and again.
She said: "You've got to take care of yourself and share what you do so others can take care of themselves as well. I try to be as vegan as I can be ... Occasionally I feel like I need a little piece of salmon. I think that there's nothing like a great oyster and a little glass of prosecco. But other than that it's a vegetable based diet with lots of greens and quinoa and some couscous."


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Spatial distribution and male mating success of
We found substantial variation in swarm size and in mating success between swarms. A strong correlation between swarm size and mating success was observed, and consistent with the hotspot model of lek formation, the
Characterizing the mating system of a species helps understand how this species has evolved and how selective pressures operate on male and female traits. The current study looked at male mating success of
Lekking behaviour is a frequent and conspicuous type of mating aggregation where males gather and display to prospective mates
In comparison with other territorial mating systems, the extreme clustering of lekking male territories raises the ultimate question of the benefits of this aggregative behaviour in contrast to its associated cost such as competition
Characterizing the mating system of a species helps understand how this species has evolved and how selective pressures operate on male and female traits
Swarm characteristics
During the survey, a total of 190 swarms were observed, sampled, and mapped across the focal village (Figure
Figure 1
The map of all swarms (N = 190) found during the survey overlaid on a satellite map (Google-Earth)
The map of all swarms (N = 190) found during the survey overlaid on a satellite map (Google-Earth). The full area bounded by the road (top left) and the swampy flood zone (bottom right) was included in the survey. The maximal North-South and East-West distances between the most extreme swarms are shown by the white arrows.
Figure 2
Swarm size dynamics (line graphs) over time in relation to mating dynamics (vertical bars)
Swarm size dynamics (line graphs) over time in relation to mating dynamics (vertical bars). The nine swarms were observed over one evening (18 August 2008) and divided arbitrarily into three panels to avoid clutter. Each color in each panel represents a distinct swarm measured from the first sighting of swarming in that evening until the last male disappeared. To accommodate variation between swarms in absolute size, the number of males in each swarm and time point was expressed as the fraction of the peak swarm size. Likewise the number of couples per interval of 3 minutes is presented as the fraction from the corresponding maximal value of that swarm. Note that the scale of the couples was compressed and values were jittered horizontally by up to ± 0.3 minute to avoid clutter.
Variation in swarm size
Of the 190 swarms that were mapped and characterized during the survey, 74 swarms were observed and photographed every three minutes from formation to the departure of the last male (see Methods). Peak swarm size and the cumulative male numbers (the sum of all males observed every 3 minutes throughout the duration of the swarm) were highly correlated (Figure
Figure 3
Distributions of peak swarm size (A) and the total number of couples per swarm (B)
Distributions of peak swarm size (A) and the total number of couples per swarm (B). Normal density curves are depicted based on the data means and standard deviations. The results of the Kolmogorov-Smirnov (KS) tests are reported under the mean and standard deviation. Panel A inset: Relationship between peak swarm size and cumulative swarm size (excluding swarms which were not observed throughout).
Accordingly, the effect of the marker type was evaluated in an ANOVA model accounting for the effects of date and the swarming site as random effects (Figure
Figure 4
Variation in peak swarm size (A) and the total number of couples per swarm (B) over time in each of the three zones
Variation in peak swarm size (A) and the total number of couples per swarm (B) over time in each of the three zones. The box of each box whisker plot extends from the first to the third quartiles (the interquartile range, IQR), the whiskers extend to extreme observations up to 1.5 times the IQR, beyond which observations are marked by squares. The median and the mean are marked by line and a dot. The color of the dot indicates the zone. Sample size per date ranged between 7 and 10 swarms. Insets: Variation in swarm size (A) and the total number of couples per swarm (B) among marker types.
Table 1
ANOVA results showing the effect of marker type (fixed), zone (random), and date (random) on peak swarm size and on total couples/swarm
Peak swarm size
Total couples/swarm
Marker Type
Res -2LL
The degrees of freedom for the F-test of "Marker Type" were 3 for the numerator and 37, and 33, for the denominator of swarm size and total couples, respectively (four swarms with size estimates had no complete measurement of total couples).
aF denotes that the standard F-test for fixed effects and Z denotes Wald-Z tests for the covariance parameter estimates
Table 2
ANOVA results testing the random effects of zone, swarm-site, and date on peak swarm size and on total couples/swarm
Peak swarm size
Total couples/swarm
Res -2LL
a Z denotes Wald-Z tests for the covariance parameter estimates
Variation in mating success between swarms
Overall, 1,456 mating couples were observed in 70 swarms (located at 30 swarming sites) over 11 days. The number of mating events per swarm varied from zero to 93, with a mode of six and a possible secondary mode around 50 (Figure
In order to evaluate the effect of swarming duration and swarm size on mating success, we used univariate and multivariate analyses. Mating success was significantly and positively related to swarm size, as indicated by the linear, univariate model (Figure
Figure 5
The effect of swarm size (A) and duration (B) on swarm mating success
The effect of swarm size (A) and duration (B) on swarm mating success. Simple linear regression (solid line) and locally-weighted scatterplot smoothing (LOESS, broken line) are used to evaluate the trends. Overall model statistics for the linear and polynomial regression models are shown (see text for details). Equations of the linear models are shown in blue.
Table 3
ANOVA results showing the effect of marker type, swarm size, swarm duration, and the square of swarm duration, as well as the random effects of date and swarming site on mating success (total couples/swarm)
DF (num/den)b
Swarm Size
Res -2LL
aF denotes the standard F-test for fixed effects and Z denotes Wald-Z tests for the covariance parameter estimates
bDegrees of freedom of the numerator (num) and denominator (den) of the F-tests
In the multivariate analysis, swarm size was the only significant factor, with swarm site no longer being significant (Table
The between-swarm variation in male body size (Nswarms = 10, Nmales = 491) was examined in relation to the random effects of date and swarm (within date) as well as the fixed effect of swarm "phase" (early, middle, or late; see Methods). None of these factors were significant (P > 0.26; data not shown). Virtually the same results were obtained when zone replaced date (data not shown). Finding no evidence for variation in body size between males of different swarms, no further testing was performed on the effect of male size on swarm mating success. However, no such relationship is apparent upon visual examination (Figure
Figure 6
Variation in male body size (wing length) among ten swarms
Variation in male body size (wing length) among ten swarms. Swarms are ordered according to the per-capita mating probability. The mean wing length of each swarm is marked by a dot, and its color indicates the zone. The box whisker plots are explained in Figure 4. Overall mean body size (2.81 mm) is indicated by the horizontal line. Sample size per swarm ranged between 21 and 95.
Spatial variation in swarm mating success
Despite extensive efforts to locate swarms throughout the predefined area, the swarms (N = 190) were concentrated in a central stretch along the village residential area with a higher density in the northern part of the village (Figure
Figure 7
Distribution of distance to the nearest swarm
Distribution of distance to the nearest swarm. To minimize the edge effect on the distribution, distances were computed for all swarms, but only those included in an inner diameter (N = 160) were included in the distribution shown above. Inset: the effect of overall swarm mating success on the average distance to the nearest 3 swarms to test that swarms cluster near highly successful swarms. Simple regression (solid line) and LOESS (broken line) are used to evaluate the trends. Quadratic and linear regression summary statistics are shown.
The role of male-male competition and female choice in shaping the mating systems in many lek-breeding animals is well-known but has been overlooked in mosquitoes. For example, little is known about the forces that constrain swarm spatial distributions and modulate the variation in mating success between and within swarms. Our main goals in the present study were to describe the variation in mating success between swarms and explore the factors that structure it. We expected to find large variation in swarm mating success between swarms and we presumed that this variation would reflect the difference in mating success of the males in different swarms. Surprisingly, our results reveal that despite substantial variation in mating success between swarms, the prospects of individual males to mate are independent from the swarm they are in. These results suggest that males may not be able to increase their individual mating success (fitness) by swarm selection. Consistent with this conclusion, we find no evidence that swarms cluster around a highly successful swarm, as there was no significant reduction in the average distance to the nearest three swarms with increasing mating success of a swarm. Further, significant variation in swarm size was found across space, and swarm site was the major factor that accounted for this variation. How then do these observations relate to the predictions of the different models for the evolution of leks?
Several different selective forces may have led to lekking behavior. The two major forces in play are male competition and female choice
The hotspot model of lek formation assumes that leks are male-initiated
The hotshot model of lek formation is male-initiated and argues that "poor quality" males gather around successful males to parasitize their greater attractiveness [29, and references therein]. Our data do not allow direct interpretations of the intrinsic mating success of individual males within swarms. However as suggested by a few authors
Leks may also form and evolve for reasons other that sexual ones. Yuval and Boukila
The mating systems of several insects are lek-based
The present study reports the swarming and mating behavior of
Study area
Our study on
Swarm survey
A swarm is defined as a collection of mosquitoes that fly in a cohesive stationary cloud, usually less than 1 m radius, after sunset on a given day. Swarms were surveyed at sunset by 30 trained observers across the entire village for 10 days. Every day, another sector of the village was covered. Swarms were sampled using an insect net, and individual mosquitoes were killed with chloroform, identified visually, and kept in 80% ethanol in 1.5-ml tubes. The location of the swarm, the time of collection, the landmarks under the swarm, and the height from the bottom of the swarm to the ground were recorded. Landmarks were classifieds by two criteria: the presence of contrasting light/dark surface (e.g., wells, grasses-footpath intersections) and projections (elevation) above ground (e.g., walls, woodpiles). This generated four classes of marker types, 'Flat-Contrast', 'Elevated-Contrast', 'Flat-No-contrast' and Elevated-No-contrast.' Observations were made on 190 swarming sites spread throughout the village. Swarm locations, households (compounds), and larval sites were mapped using a global positioning system (GPS) with measurements of latitude and longitude accurate to within 2 m. Collected specimens were identified by PCR to species and molecular form
Swarm size and mating success
After completion of the swarm survey (above) and the mapping of swarms throughout the entire village, three groups of ten swarms each in three different zones were selected for intensive observations. The zones were selected in high and low swarm-density areas. We started by randomly selecting one swarm in each zone, then the nearest nine swarms were subsequently selected. Each group of 10 swarms was surveyed for at least three days. The number of males in a swarm was determined at three-minute intervals from the beginning to the end of the swarm. Digital cameras were used to take pictures of the swarms and these pictures were subsequently used to estimate (by counting) the number of males in each swarm
Data Analysis
Statistical analyses were performed using SAS 9.2 (SAS Institute, Cary, NC). Goodness-of-fit tests with the normal distribution were performed using Kolmogorov-Smirnov (KS) tests. Certain analyses included fixed (e.g., marker type) and random variables (e.g., swarming site, date, and zone). If random effects were included, mixed models ANOVAs were implemented using PROC MIXED within SAS. Swarm sites and date were nested in zone, because only the swarms of a single zone could be observed each evening. Additionally, our classification of markers based on contrast and height above ground resulted in four categories (above), some of which were not found in all zones. Therefore, we evaluated the effect of zone and marker in separate models. The component of variance attributed to particular categorical (non-continuous) variables such as 'swarm site' was used as an estimate of its "effect" on the response variable. As for repeatability, it was computed as the relative contribution of the factor of interest to the total variance in the response variable using estimates of variance that were derived by PROC MIXED in SAS. This measure of effect size was calculated only for statistically significant categorical factors because the slope coefficient of continuous factors (e.g., swarm size) provides direct measure of the effect size of such factors on the response. To assess if effects were nonlinear, we used (i) locally weighted scatterplot smoothing (LOESS) side-by-side with regression analyses, (ii) polynomial regression according to the curvature suggested by the data and the LOESS, and (iii) lack-of-fit tests to determine if the non-linear component of the variance was significant. To minimize the edge effect in estimating the distance to the nearest swarm(s), distances were computed between all swarms (N = 190), but only swarms located in an inner rectangle (n = 160) were used (Figure
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
The work presented here was carried out in collaboration between all authors. ADi and TL designed the study. ADi carried out the field and laboratory work, participated in the analysis of data, interpreted the results, and drafted the paper. ASY, ADa and MD carried out the fieldwork and revised the manuscript. DLH participated in the data analysis and revised the manuscript. TL analysed the data, interpreted the results, and revised the manuscript. All authors have read and approved the final manuscript.
We are grateful to Boaz Yuval, Nick Manoukis and several anonymous reviewers for useful comments and suggestions to improve the manuscript. This investigation was funded by the Intramural Research Program of National Institutes of Allergy and Infectious Diseases, National Institute of Health and the Multilateral Initiative on Malaria.
Hotshots, hotspots, and female preference in the organization of lek mating systems
The evolution of leks
Manipulating lek size and composition using decoys: an experimental investigation of lek evolution models
Variation in male mating success at leks
Male competition and female choice interact to determine mating success in the bluefin killifish
Leks and mate choice
Lekking in birds and mammals: Behavioural and evolutionary issues
Natural selection and specialized chorusing behavior in acoustical insects
Evolution of competitive mate searching
Hotspots and the dispersion of leks
From individual behavior to population ecology
Male-male competition and mate choice in anuran amphibians
The swarming and mating behaviour of
The swarming and mating flight of Diptera
Mating systems of blood-feeding flies
Natural swarming behaviour in the molecular M form of
Reproductive isolation through swarm segregation in the molecular forms of
Mating behaviour in the mosquito,
The Evolution of Insect Mating Systems
The evolution of leks through female choice
Ecology, sexual selection, and the evolution of mating systems
Costs and consequences of variation in the size of ruff leks
The lekking game: can female choice explain aggregated male displays?
Male position and calling effort together influence male attractiveness in leks of the medfly, Ceratitis capitata (Diptera: Tephritidae)
Testing predictions for the evolution of lekking in the sandfly, Lutzomyia longipalpis
Tests of hypotheses for lek formation in a Hawaiian Drosophila
Observations on the swarming and mating behaviour of
Female mate choice across spatial scales: influence of lek and males attributes on mating success of blue-crowned manikins
Small males are more symmetrical: mating success in the midge
Male size does not affect mating success (of
Effects of body size on swarming behaviour and mating success of male
Sexual selection in mosquito swarms: may the best man lose?
Effect of larval crowding on mating competitiveness of
Age effects on the Insemination rate of
Age of
Genetic variation of male reproductive success in a laboratory population of
Intra-specific variation of sperm length in the malaria vector
An aggregation pheromone modulates lekking behavior in the vector mosquito
Flying in tune: sexual recognition in mosquitoes
Harmonic convergence in the love songs of the dengue vector mosquito
"Singing on the wing" as a mechanism for species recognition in the malarial mosquito
Temporal dynamics of mating and predation in mosquito swarms
Male swarms at landmarks and scramble competition polygyny in
Indirect mate choice, competition for mates, and coevolution of the sexes
Etude du comportement pour l'accouplement chez
Aestivation of the African malaria mosquito,
Simultaneous identification of species and molecular forms of the
Variation in metabolic rate of


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.
Google Spreadsheet Integration: It creates a new row in spreadsheet when upon editing a submissionAsked by ukff on September 08, 2013 at 12:53 PM
I've Integrated my form with Google Spreadsheet.
When I edit a form submission using jotform: edit: submit, the jotform data is correctly updated, however in Google Spreadsheet the last form that was submitted by a user is repeated. In other words when Google Spreadsheet is updated, the pointer in jotform must be pointing to the latest submission and NOT the record that was updated using the Edit function.
Thanks for contacting us.
I made some tests with one of my forms and I was able to see what you are talking about. When a submission is edited a new row is created in the Google spreadsheet:
However, please let me tell you that this is the way the Integration works. Everytime you edit a submission it will create a new row with the updated info in the spreadsheet.
However I would be more than glad to forward a feature request to our next level support team if you reallly need this to work in a different way.
Thanks for using Jotform.
Thanks, but I do not require a new feature. I believe there is a coding error.
Here's a better description:
A form has 5 submissions e.g. submission id: 1, 2, 3, 4 and 5.
In jotform edit: select submission id 3 (NB choose any submission id but NOT 5 i.e. do not choose the last/final submission id) and update it, and then submit the form.
In jotform you will see that submission id 3 has been correctly updated.
In Google Spreadsheet you will see that there is a new row and submission id 5 (and NOT 3) has been resubmitted.
Please test again and verify if same occurs for you.
Like Eliezer explained to you, this is how the Google spreadsheet integration work currently. Our internal spreadsheet that you see at the submission page will work in a different way. As you may know, we use an API for each 3rd party integration.
However, whether it's a bug or not, this can be something we can request to our developers so they can contact Google and get an updated API or see if they can fix it with the current, in order to allow our users to accomplish the same results as in our internal spreadsheet
At any rate, I've already opened a ticket to check this further. You should get an update about it on this thread
This issue is now fixed. Please let us know if the problem continues.


text:
Giveaway: Apple iPhone 5
0. phoneArena posted on 14 Dec 2012, 08:08
It's the holiday season and it's time for giveaways! This time, thanks to our openhearted partners from Radioshack, we'll be presenting one of you with a brand new AT&T iPhone 5 and an Otterbox Commuter Case to help you keep it looking new...
This is a discussion for a news. To read the whole news, click here
207. dwagner8 (Posts: 9; Member since: 05 Nov 2012)
My favorite strikes a good balance between protection and keeping the phone profile slim.
208. Adan_Ramos (Posts: 3; Member since: 25 Jun 2011)
My favorite case would have to be a hoc cases that verizon carries. Ive used them on my last 4 phones. I like this style of case because it allows me to carry my device on my belt clip when im at work and i can take it off the belt clip and put it in my pocket when im off work. its also great for when im on my lunch break and i want to watch something on my phone because they come with kickstands built in the case. the best thing about it is it keeps my phone size small unlike an otter box that makes phones much larger. it doesnt offer the most protection but it offers enough for me.
209. njdevil343 (Posts: 2; Member since: 28 Mar 2012)
Otterbox by far! So many times iv dropped my phone and it never has broke! More importantly, Otterbox's customer service has been great. The few times iv contacted them they were great!
210. uriel (Posts: 1; Member since: 13 Feb 2012)
I prefer genuine leather cases but I don't know of any so I have found otterbox to be a great replacement
211. Marino1310 (Posts: 1; Member since: 12 Dec 2012)
My favorite for protection is otterbocx and ballistic
212. ChocoJ (Posts: 10; Member since: 23 Jul 2012)
I use the Otterbox for my iPhone 4S, it's taken a few drops, and could use the extra love that case gives. For my Galaxy SIII, I use one of the Mophie cases, that way I don't have to waste time hunting for a charger.
213. kewlguy210 (Posts: 8; Member since: 28 Mar 2011)
Lifeproof is a good case and same are the otterbox ones.
214. mmini1 (Posts: 4; Member since: 05 Aug 2012)
I am a fan of cases that have both a hard shell and soft protective case together. Not only are they protective, but they allow multiple looks/textures to the phone.
215. JewBakaUCFG (Posts: 176; Member since: 25 Sep 2012)
Otter Box is awesome. I have a commuter on my GS3. Keeps it safe. Already protected it from a few falls. I really like that it comes with a one year warranty, too.
216. denmcdon (Posts: 90; Member since: 07 Jun 2009)
My favorite case is the Siedio active, good protection nice look and multiple color choices.
217. Manideepreddy (Posts: 14; Member since: 29 Nov 2011)
the bumper case is my fav. because the actual design of the phone visible and it even protects the phone
218. kfreeman_23 (Posts: 1; Member since: 12 Dec 2012)
I like the Otter Box commuter series because of the best possible protection with a pocket friendly size
219. random2491 (Posts: 7; Member since: 06 Aug 2012)
Otterbox because of its durability and reliability.
220. chadcy (Posts: 29; Member since: 19 Nov 2012)
a protective case that guards against screen smudges and drops. lifeproof.
221. housry23 (Posts: 132; Member since: 03 Jun 2012)
My favorite type of cases are bumper cases. They offer enough protection with minimal increase in girth. I especially love the aluminum bumpers with chrome buttons. To protect the rest of the device, I usually use a clear skin of some sort.
222. calmaroc (Posts: 4; Member since: 05 Dec 2012)
iReally want this phone :-)
223. steven252000 (Posts: 64; Member since: 10 Feb 2010)
I like my Otter Box case the best because it feels very sturdy although a little thick. However, I do not like the clip/holster. I really prefer the holster/clip that I had on my Seidio case.
224. Macnasty (Posts: 1; Member since: 12 Dec 2012)
Definitely tech21's D30 cases. Always protects my device no matter what.
225. StrikeZero (Posts: 4; Member since: 07 Dec 2010)
My favorite case is lifeproof.
226. cowboysfan032970 (Posts: 7; Member since: 09 Jun 2012)
My favorite is the otter commuter series beause it protects without the bulk!
227. phanboi (Posts: 4; Member since: 04 Nov 2012)
My favorite case is the Otterbox defender because of it's toughness and durability :D
228. Izzy_V (Posts: 216; Member since: 07 Jul 2012)
I don't use cases. Never had the need to.
229. elberthung (Posts: 6; Member since: 19 Nov 2012)
My favorite case is the one sell on eBay lol. Cuz its cheap!
230. baybrady (Posts: 7; Member since: 29 Oct 2012)
A thin case is a good case.
231. astronaut (Posts: 6; Member since: 10 May 2012)
Incipio is my favorite because, no reason
232. bgapfire (Posts: 9; Member since: 08 Jun 2010)
I enjoy the Otterbox only for its protection of the phone. When casual, I enjoy Spigen cases.
233. mas11 (Posts: 1034; Member since: 30 Mar 2012)
Otterbox Defender, there is no way my screen will crack with this thing on.
234. dashiz09 (Posts: 7; Member since: 24 May 2012)
Otterbox's Commuter Case because it protects my phone.
235. dchan149 (Posts: 4; Member since: 28 Nov 2012)
Incipio Feather. #showmesomelove!
236. bovcrx (Posts: 83; Member since: 06 Mar 2012)
I like otterbox because they make holsters for their heavy duty cases.


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.
Dropbox won't authenticate when using Safari on Mac.Asked by Carly0826 on January 15, 2015 at 05:51 PM
My dropbox says that jot form has been given access but it still won't authenticate on this end when I press the authentic button? Help!
JotForm Support
I just tried it and it is working, what browser are you using? It might be related to browser issues, you could clear browsers cache, or try in another browser, we recommend using Google Chrome and FireFox. Let us know what happens.
Hi I am using safari on my mac. should I try another?
JotForm Support
Please do, I just tried it in a browser emulator using mac and safari, it did not work, I will report this to our second level.
Google Chrome worked. thank you!
Hi Carly0826,
On behalf of my colleague, you are most welcome. Thank you, you for informing us that you were able to perform the task in another browser.
As stated the issue regarding it not working in Safari has been passed on the our second level support.
Any update on the issue will be posted on this thread.
Thank you.
JotForm Support
The dropbox integration on Safari bug has now been fixed. Thanks for reporting it