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Abstract Background The Wnt signaling pathway is activated by mutations in the APC and β-catenin genes in many types of human cancer. β-catenin is stabilized by these mutations and activates transcription in part by acting as a bridge between Tcf/LEF proteins and the HD2 domain of the BCL9 coactivator. We have previously described oncolytic adenoviruses with binding sites for Tcf/LEF transcription factors inserted into the early viral promoters. These viruses replicate selectively in cells with activation of the Wnt pathway. To increase the activity of these viruses we have fused the viral transactivator E1A to the BCL9 HD2 domain. Methods Luciferase assays, co-immunoprecipitation and Western blotting, immunofluorescent cell staining and cytopathic effect assays were used to characterize the E1A-HD2 fusion protein and virus in vitro. Growth curves of subcutaneous SW620 colon cancer xenografts were used to characterize the virus in vivo. Results The E1A-HD2 fusion protein binds to β-catenin in vivo and activates a Tcf-regulated luciferase reporter better than wild-type E1A in cells with activated Wnt signaling. Expression of the E1A-HD2 protein promotes nuclear import of β-catenin, mediated by the strong nuclear localization signal in E1A. Tcf-regulated viruses expressing the fusion protein show increased expression of viral proteins and a five-fold increase in cytopathic effect (CPE) in colorectal cancer cell lines. There was no change in viral protein expression or CPE in HeLa cells, indicating that E1A-HD2 viruses retain selectivity for cells with activation of the Wnt signaling pathway. Despite increasing the cytopathic effect of the virus in vitro, fusion of the HD2 domain to E1A did not increase the burst size of the virus in vitro or the anti-tumor effect of the virus in an SW620 xenograft model in vivo. Conclusion Despite an increase in the nuclear pool of β-catenin, the effects on viral activity in colon cancer cells were small, suggesting that factors acting downstream of β-catenin are limiting for viral replication and toxicity in these cells. The approach of fusing E1A to a protein domain implicated in oncogenic signaling could be used to selectively increase the activity of oncolytic viruses targeting several other pathways defective in cancer.

A multilaboratory testing study was conducted on AOAC First Action Official MethodSM 2016.15: Quantification of Whey Protein Content in Infant Formula Powders by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE). Nineteen laboratories participated in the analysis of duplicate blind-coded samples of 15 formula powder products for infants and young children. Electrophoregrams were recorded at UV220 nm and integrated. The normalized peak areas of whey and casein proteins were summed separately to calculate total whey protein content. Apart from one sample [NIST Standard Reference Material (SRM) 1849a], relative standard deviation of repeatability (RSDr) and reproducibility (RSDR) ranged from 0.83 to 2.11% and from 2.18 to 4.22%, respectively, and Horwitz ratios ranged from 1.02 to 1.85, meeting the precision limits specified in the whey protein Standard Method Performance Requirements and in the guidelines recommended for the Horwitz ratio. In these samples, the measured whey protein content was between 98 and 108% of the declared value. NIST SRM 1849a showed atypical results, with elevated RSDr (3.51%), RSDR (5.94%), Horwitz ratio (2.62), and recovery (134%). There is no clear reason for this. The percent whey protein value for NIST is calculated from the formulation and is not a reference or certified value. Multiple instrument models and makes, as well as capillary sources, were used in this collaborative study, demonstrating the robustness of the method. The method is fit-for-purpose for the quantification of whey protein content in milk-based formula powder products for infants and young children. It is not applicable to the analysis of hydrolyzed or plant protein-based infant formulas. [ABSTRACT FROM AUTHOR]

The article focuses on a study reagrding the quantification of whey protein content in infant formulas by sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE). It mentions that proteins in infant formula samples are denatured by anionic surfactant SDS and states that infant formula electrophoregrams are optimized by batch analysis. It also mentions the use of acidic wash solution and SDS gel buffer for quantification of whey protein.

The Wnt signaling pathway plays key roles in development and adult homeostasis. Wnt proteins are secreted, lipid-modified glycoproteins. They can form morphogen gradients that are regulated at the level of protein secretion, diffusion, and internalization. These gradients can only exist if the hydrophobic Wnt proteins are prevented from aggregating in the extracellular environment. Heparan sulfate proteoglycans (HSPGs) are necessary for proper activity of Wnt proteins and influence their distribution along the morphogenetic gradient. In this study, we show that HSPGs are able to maintain the solubility of Wnt proteins, thus stabilizing their signaling activity. Our results suggest that the role of HSPGs is not only to concentrate Wnt molecules at the cell surface but also to prevent them from aggregating in the extracellular environment. Developmental Dynamics 239:184-190, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Embryonic stem cells (ESCs) form descendants of all three germ layers when differentiated as aggregates, termed embryoid bodies. In vivo, differentiation of cells depends on signals and morphogen gradients that provide instructive and positional cues, but do such gradients exist in embryoid bodies? We report here the establishment of anteroposterior polarity and the formation of a primitive streak-like region in the embryoid body, dependent on local activation of the Wnt pathway. In this region, cells undergo an epithelial-to-mesenchymal transition and differentiate into mesendodermal progenitors. Exogenous Wnt3a protein posteriorizes the embryoid body, resulting in predominantly mesendodermal differentiation. Conversely, inhibiting Wnt signaling promotes anterior character and results in neurectodermal differentiation. The activation of Wnt signaling and primitive streak formation requires external signals but is self-reinforcing after initiation. Our findings show that the Wnt pathway mediates the local execution of a gastrulation-like process in the embryoid body, which displays an unexpected degree of self-organization. [ABSTRACT FROM AUTHOR]

A multilaboratory testing study was conducted on AOAC First Action Official Method SM 2016.15: Quantification of Whey Protein Content in Infant Formula Powders by Sodium Dodecyl Sulfate-Capillary Gel Electrophoresis (SDS-CGE). Nineteen laboratories participated in the analysis of duplicate blind-coded samples of 15 formula powder products for infants and young children. Electrophoregrams were recorded at UV220 nm and integrated. The normalized peak areas of whey and casein proteins were summed separately to calculate total whey protein content. Apart from one sample [NIST Standard Reference Material (SRM) 1849a], relative standard deviation of repeatability (RSDr) and reproducibility (RSDR) ranged from 0.83 to 2.11% and from 2.18 to 4.22%, respectively, and Horwitz ratios ranged from 1.02 to 1.85, meeting the precision limits specified in the whey protein Standard Method Performance Requirements and in the guidelines recommended for the Horwitz ratio. In these samples, the measured whey protein content was between 98 and 108% of the declared value. NIST SRM 1849a showed atypical results, with elevated RSDr (3.51%), RSDR (5.94%), Horwitz ratio (2.62), and recovery (134%). There is no clear reason for this. The percent whey protein value for NIST is calculated from the formulation and is not a reference or certified value. Multiple instrument models and makes, as well as capillary sources, were used in this collaborative study, demonstrating the robustness of the method. The method is fit-for-purpose for the quantification of whey protein content in milk-based formula powder products for infants and young children. It is not applicable to the analysis of hydrolyzed or plant protein-based infant formulas.