The antigenic variation of influenza virus represents a major health problem. However, the extracellular domain of the minor, virus-coded M2 protein is nearly invariant in all influenza A strains. We genetically fused this M2 domain to the hepatitis B virus core (HBc) protein to create fusion gene coding for M2HBc; this gene was efficiently expressed in Escherichia coli. Intraperitoneal or intranasal administration of purified M2HBc particles to mice provided 90-100% protection against a lethal virus challenge. The protection was mediated by antibodies, as it was transferable by serum. The enhanced immunogenicity of the M2 extracellular domain exposed on HBc particles allows broad-spectrum, long-lasting protection against influenza A infections.

Beta-catenin-dependent or canonical Wnt signals are fundamental in animal development and tumor progression. Using Xenopus laevis, we report that the BTB/POZ zinc finger family member Kaiso directly represses canonical Wnt gene targets (Siamois, c-Fos, Cyclin-D1, and c-Myc) in conjunction with TCF/LEF (TCF). Analogous to beta-catenin relief of TCF repressive activity, we show that p120-catenin relieves Kaiso-mediated repression of Siamois. Furthermore, Kaiso and TCF coassociate, and combined Kaiso and TCF derepression results in pronounced Siamois expression and increased beta-catenin coprecipitation with the Siamois promoter. The functional interdependency is underlined by Kaiso suppression of beta-catenin-induced axis duplication and by TCF-3 rescue of Kaiso depletion phenotypes. These studies point to convergence of parallel p120-catenin/Kaiso and beta-catenin/TCF signaling pathways to regulate gene expression in vertebrate development and possibly carcinogenesis.

The Wnt/β-catenin signalling pathway shares a key component, β-catenin, with the cadherin-based adhesion system. The signalling function of β-catenin is conferred by a soluble cytoplasmic pool that is unstable in the absence of a Wnt signal, whilst the adhesion function is based on a cadherin-bound, stable pool at the membrane. The cadherin complex is dynamic, allowing for cell-cell rearrangements such as epithelial-mesenchymal transition (EMT), where the complex turns over through internalisation. Potential interplay between the two pools remains poorly understood, but cadherins are generally considered negative regulators of Wnt signalling because they sequester cytoplasmic β-catenin. Here we explore how cellular changes at EMT affect the signalling capacity of β-catenin using two models of EMT: hepatocyte growth factor (HGF) treatment of MDCK cells, and gastrulation in embryonic development. We show that EMT not only provides a pool of signalling-competent β-catenin following internalisation of cadherin, but also significantly facilitates activation of the Wnt pathway in response to both Wnt signals and exogenous β-catenin. We further demonstrate that availability of β-catenin in the cytoplasm does not necessarily correlate with Wnt/β-catenin pathway activity, since blocking endocytosis or depleting endogenous cadherin abolishes pathway activation despite the presence of β-catenin in the cytoplasm. Lastly we present data suggesting that cadherins are required for augmented activation of the Wnt/β-catenin pathway in vivo. This suggests that cadherins play a crucial role in β-catenin-dependent transcription.

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a na€ ıve pluripotent state. Efforts have been made to trigger na€ ıve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of na€ ıve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor 1 Ascorbic Acid 1 Forskolin) facilitating rapid induction of transgene-free na€ ıve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted na€ ıve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated na€ ıve pluripotency genes, and exhibited dependence on signaling pathways similar to na€ ıve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward na€ ıve pluripotency. Collectively, we demonstrate an alternate route to capture na€ ıve pluripotency in hESCs that is fast, reproducible, supports na€ ıve mESC derivation, and allows efficient differentiation. STEM CELLS 2015;33:2686-2698 SIGNIFICANCE STATEMENTNa€ ıve pluripotency, commonly displayed by mouse embryonic stem cells (ESCs), holds several advantages over stem cells exhibiting a primed pluripotent state such as human ESCs, which already show a bias towards certain lineages. We report the formulation of a novel culture condition with minimal components facilitating rapid, robust and efficient induction of na€ ıve pluripotency in primed human ESCs. These novel na€ ıve human ESCs were karyotypically normal, underwent efficient single cell passaging, exhibited a unique epigenetic and lncRNA profile and unbiased lineage-specific differentiation similar to mouse ESCs. This na€ ıve state of pluripotency is important for possible future regenerative cell applications including efficient genome engineering and targeted gene correction.

Matrix protein 2 (M2) of influenza A is a tetrameric type III membrane protein that functions as a proton-selective channel. The extracellular domain (M2e) has remained nearly invariable since the first human influenza strain was isolated in 1933. By linking a modified form of the leucine zipper of the yeast transcription factor GCN4 to M2e, we obtained a recombinant tetrameric protein, M2e-tGCN4. This protein mimics the quaternary structure of the ectodomain of the natural M2 protein. M2e-tGCN4 was purified, biochemically characterized, and used to immunize BALB/c mice. High M2e-specific serum IgG antibody titers were obtained following either intraperitoneal or intranasal administration. Immunized mice were protected fully against a potentially lethal influenza A virus challenge. Antibodies raised by M2e-tGCN4 immunization specifically bound to the surface of influenza-infected cells and to an M2-expressing cell line. Using a M2e peptide competition enzymelinked immunosorbent assay with M2-expressing cells as target, we obtained evidence that M2e-tGCN4 induces antibodies that are specific for the native tetrameric M2 ectodomain. Therefore, fusion of an oligomerization domain to the extracellular part of a transmembrane protein allows it to mimic the natural quaternary structure and can promote the induction of oligomer-specific antibodies.Influenza has one of the highest infection rates of all human viruses and can kill healthy persons of all ages (1). It is estimated that influenza infection during seasonal epidemics kills 1 in 1000 infected individuals, whereas an unpredictable pandemic is likely to kill millions. In addition, increased hospitalization and absenteeism from school and work are direct consequences of the flu. At present, the best way to protect against influenza is to vaccinate against the ever-mutating strains (2). However, antigenic drift and occasional shift of the two major membrane glycoproteins, hemagglutinin and neuraminidase, make vaccine production cumbersome and necessitate yearly revision of the vaccine seed strains by the World Health Organization.Influenza A also encodes a third integral membrane protein, M2, 2 a homotetramer, the subunit of which has a small external domain (M2e) of 23 amino acid residues (3). Natural M2 protein is present in a few copies in the virus particle but in abundance on virus-infected cells (4). In contrast to hemagglutinin and neuraminidase, M2e is almost nonimmunogenic (5), and its sequence is highly conserved. Capitalizing on these properties, we developed a universal influenza A vaccine by linking the M2e peptide to a virus-like particle based on the hepatitis B virus core (HBc) (6). In this context, M2e is highly immunogenic, and the M2e-HBc vaccine induces antibodies that protect mice against influenza-induced death and morbidity.Oligomeric proteins found in vaccines derived by inactivating or attenuating a pathogen often function as their major antigenic determinant. Conformational epitopes embedded in the quaternary structures may critically con...

No abstract

The pronephric kidney controls water and electrolyte balance during early fish and amphibian embryogenesis. Many Wnt signaling components have been implicated in kidney development. Specifically, in Xenopus pronephric development as well as the murine metanephroi, the secreted glycoprotein Wnt-4 has been shown to be essential for renal tubule formation. Despite the importance of Wnt signals in kidney organogenesis, little is known of the definitive downstream signaling pathway(s) that mediate their effects. Here we report that inhibition of Wnt/β-catenin signaling within the pronephric field of Xenopus results in significant losses to kidney epithelial tubulogenesis with little or no effect on adjoining axis or somite development. We find that the requirement for Wnt/β-catenin signaling extends throughout the pronephric primordium and is essential for the development of proximal and distal tubules of the pronephros as well as for the development of the duct and glomus. Although less pronounced than effects upon later pronephric tubule differentiation, inhibition of the Wnt/β-catenin pathway decreased expression of early pronephric mesenchymal markers indicating it is also needed in early pronephric patterning. We find that upstream inhibition of Wnt/β-catenin signals in zebrafish likewise reduces pronephric epithelial tubulogenesis. We also find that exogenous activation of Wnt/β-catenin signaling within the Xenopus pronephric field results in significant tubulogenic losses. Together, we propose Wnt/β-catenin signaling is required for pronephric tubule, duct and glomus formation in Xenopus laevis, and this requirement is conserved in zebrafish pronephric tubule formation.

No abstract