R the dual fluorescence reporter assay, the fusion constructs containing the DsRed gene and miR1786, were designed to be co-expressed under control of the CMV promoter. Both constructs were co-transfected into 293FT cells using the calcium phosphate method. When the DsRed-miRNA is expressed and binds to the target site of the 39-UTR downstream of the GFP transcript, green fluorescence intensity decreases due to Title Loaded From File degradation of the GFP transcript. At 48 h post-transfection, dual fluorescence was detected by fluorescence microscopy and calculated by FACSCalibur flow cytometry (BD Biosciences). For flow cytometry, the cells were fixed in 4 paraformaldehyde and analyzed using FlowJo software (Tree Star Inc., Ashland, OR).Statistical AnalysesAll quantitative data were subjected to 24195657 analysis of variance (ANOVA) according to the general linear model (PROC-GLM) of the SAS program (SAS Institute, Cary, NC). All tests of significance were performed using the appropriate error terms according to the expectation of the mean square for error. Data are presented as mean 6 SEM unless otherwise stated. Differences in the variance between untreated and DES-treated oviducts were analyzed using the F test, and differences in the means were subjected to Student’s t test. Differences were considered significant at P,0.05.Author ContributionsConceived and designed the experiments: GS. Performed the experiments: CHL WL WJ JYL SMB JK. Analyzed the data: CHL WL JK FWB GS. Contributed reagents/materials/analysis tools: JYH. Wrote the paper: CHL WL FWB GS.
Alzheimer’s disease (AD) is associated with an imbalance in the production and clearance of the amyloid-b peptide (Ab) followed by Ab aggregation in the brain [1]. The aggregation ultimately ends in the formation of insoluble protein fibrils as components of amyloid plaques. Considerable evidence suggests that neurotoxic species are soluble oligomers or protofibrils of Ab that are present on or off aggregation pathways leading to fibril formation [2,3,4,5,6,7,8]. The 42-residue Ab42 fragment is in this R, this data suggests that Mtap may be acting in a regard more aggregation prone than the more prevalent but less active Ab40 fragment and an increase in the Ab42: Ab40 ratio is also associated with increased neurotoxicity [9]. Other evidence suggests that the rate of aggregation, and not only the aggregates that are present, acts to further enhance toxicity [10,11]. Ab can form a multitude of interconverting toxic aggregates both in vitro and in vivo [12,13,14]. However, in all cases, aggregate inhomogeneity and instability complicate research on correlations between aggregation, structure and toxicity. Different ways to stabilize intermediate aggregates by chemical cross linking [for instance [6]] or protein engineering [[15] and work cited therein] have therefore been devised.We recently engineered a double cysteine mutant of Ab (AbCC) for which aggregation is halted at the protofibrillar state [16], which is suggested to be the penultimate intermediate prior to amyloid fibril formation [13,14,17]. Briefly, AbCC was designed to test a structural model of aggregation [18] in which Ab adopts a hairpin conformation in aggregates on the path to fibril formation [18]. This model hypothesized that a conformational change in such aggregates results in the formation of seeds for runaway fibril polymerization. AbCC contains a double Ala21Cys/Ala30Cys mutation and a disulfide bond formed between the two cysteines locks the peptide into the hairpin conformation [16]. AbCC.R the dual fluorescence reporter assay, the fusion constructs containing the DsRed gene and miR1786, were designed to be co-expressed under control of the CMV promoter. Both constructs were co-transfected into 293FT cells using the calcium phosphate method. When the DsRed-miRNA is expressed and binds to the target site of the 39-UTR downstream of the GFP transcript, green fluorescence intensity decreases due to degradation of the GFP transcript. At 48 h post-transfection, dual fluorescence was detected by fluorescence microscopy and calculated by FACSCalibur flow cytometry (BD Biosciences). For flow cytometry, the cells were fixed in 4 paraformaldehyde and analyzed using FlowJo software (Tree Star Inc., Ashland, OR).Statistical AnalysesAll quantitative data were subjected to 24195657 analysis of variance (ANOVA) according to the general linear model (PROC-GLM) of the SAS program (SAS Institute, Cary, NC). All tests of significance were performed using the appropriate error terms according to the expectation of the mean square for error. Data are presented as mean 6 SEM unless otherwise stated. Differences in the variance between untreated and DES-treated oviducts were analyzed using the F test, and differences in the means were subjected to Student’s t test. Differences were considered significant at P,0.05.Author ContributionsConceived and designed the experiments: GS. Performed the experiments: CHL WL WJ JYL SMB JK. Analyzed the data: CHL WL JK FWB GS. Contributed reagents/materials/analysis tools: JYH. Wrote the paper: CHL WL FWB GS.
Alzheimer’s disease (AD) is associated with an imbalance in the production and clearance of the amyloid-b peptide (Ab) followed by Ab aggregation in the brain [1]. The aggregation ultimately ends in the formation of insoluble protein fibrils as components of amyloid plaques. Considerable evidence suggests that neurotoxic species are soluble oligomers or protofibrils of Ab that are present on or off aggregation pathways leading to fibril formation [2,3,4,5,6,7,8]. The 42-residue Ab42 fragment is in this regard more aggregation prone than the more prevalent but less active Ab40 fragment and an increase in the Ab42: Ab40 ratio is also associated with increased neurotoxicity [9]. Other evidence suggests that the rate of aggregation, and not only the aggregates that are present, acts to further enhance toxicity [10,11]. Ab can form a multitude of interconverting toxic aggregates both in vitro and in vivo [12,13,14]. However, in all cases, aggregate inhomogeneity and instability complicate research on correlations between aggregation, structure and toxicity. Different ways to stabilize intermediate aggregates by chemical cross linking [for instance [6]] or protein engineering [[15] and work cited therein] have therefore been devised.We recently engineered a double cysteine mutant of Ab (AbCC) for which aggregation is halted at the protofibrillar state [16], which is suggested to be the penultimate intermediate prior to amyloid fibril formation [13,14,17]. Briefly, AbCC was designed to test a structural model of aggregation [18] in which Ab adopts a hairpin conformation in aggregates on the path to fibril formation [18]. This model hypothesized that a conformational change in such aggregates results in the formation of seeds for runaway fibril polymerization. AbCC contains a double Ala21Cys/Ala30Cys mutation and a disulfide bond formed between the two cysteines locks the peptide into the hairpin conformation [16]. AbCC.
