Engineering of papaya mosaic virus (PapMV) nanoparticles through fusion of the HA11 peptide to several putative surface-exposed sites

Papaya mosaic virus has been shown to be an efficient adjuvant and vaccine platform in the design and improvement of innovative flu vaccines. So far, all fusions based on the PapMV platform have been located at the C-terminus of the PapMV coat protein. Considering that some epitopes might interfere with the self-assembly of PapMV CP when fused at the C-terminus, we evaluated other possible sites of fusion using the influenza HA11 peptide antigen. Two out of the six new fusion sites tested led to the production of recombinant proteins capable of self assembly into PapMV nanoparticles; the two functional sites are located after amino acids 12 and 187. Immunoprecipitation of each of the successful fusions demonstrated that the HA11 epitope was located at the surface of the nanoparticles. The stability and immunogenicity of the PapMV-HA11 nanoparticles were evaluated, and we could show that there is a direct correlation between the stability of the nanoparticles at 37°C (mammalian body temperature) and the ability of the nanoparticles to trigger an efficient immune response directed towards the HA11 epitope. This strong correlation between nanoparticle stability and immunogenicity in animals suggests that the stability of any nanoparticle harbouring the fusion of a new peptide should be an important criterion in the design of a new vaccine.     

A simple non-invasive protocol to establish primary cell lines from tail and toe explants for cytogenetic studies in Australian dragon lizards (Squamata: Agamidae)

Primary cell lines were established from cultures of tail and toe clips of five species of Australian dragon lizards: Tympanocryptis pinguicolla, Tympanocryptis sp., Ctenophorus fordi, Amphibolurus norrisi and Pogona vitticeps. The start of exponential cell growth ranged from 1 to 5 weeks. Cultures from all specimens had fibroblastic morphology. Cell lines were propagated continuously up to ten passages, cryopreserved and recovered successfully. We found no reduction in cell viability after short term (<6 months) storage at −80 °C. Mitotic metaphase chromosomes were harvested from these cell lines and used in differential staining, banding and fluorescent in situ hybridisation. Cell lines maintained normal diploidy in all species. This study reports a simple non-invasive method for establishing primary cell lines from Australian dragon lizards without sacrifice. The method is likely to be applicable to a range of species. Such cell lines provide a virtually unlimited source of material for cytogenetic, evolutionary and genomic studies.     

The Calcium-Dependent Interaction between S100B and the Mitochondrial AAA ATPase ATAD3A and the Role of This Complex in the Cytoplasmic Processing of ATAD3A

S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.The S100 proteins comprise a multigene family of low-molecular-weight EF-hand calcium binding and zinc binding proteins (5, 13, 16, 24, 33). To date, 19 different S100 proteins have been assigned to this protein family, and they show different degrees of similarity, ranging from 25 to 56% identity at the amino acid level. With S100B, S100P, and S100Z being the exceptions, the majority of the S100 genes are clustered on human chromosome 1q21 (33). Most S100 proteins serve as calcium sensor proteins that, upon activation, regulate the function and/or subcellular distribution of specific target proteins (13, 33, 47), and they are characterized by common structural motifs, including two low-affinity (K_D [equilibrium dissociation constant] of ∼10 μM to 100 μM) helix-loop-helix calcium binding domains (EF hands) that are separated by a hinge region and flanked by amino- and carboxy-terminal domains. The carboxy-terminal domain is variable among S100 proteins, and it typically is the site that is responsible for the selective interaction of each individual S100 protein with specific target proteins (30). S100 proteins are often upregulated in cancers, in inflammation, and in response to cellular stress (14, 16), suggesting that they function in cell responses to stress situations. Consistent with this hypothesis, stress situations were necessary to reveal phenotypes associated with the S100 knockout in mice (11, 14, 33, 56). Moreover, recent observations revealed a new function for the S100 protein family that included their ability to assist and regulate multichaperone complex-ligand interactions (41, 50, 51).One member of the S100 protein family, S100B, has attracted much interest in the past few years because, like other proteins implicated in neurodegeneration (e.g., amyloid, superoxide dismutase, and dual-specificity tyrosine phosphorylation-regulated kinase 1A), its gene is located within a segment of chromosome 21, which is trisomic in Down''s syndrome (DS). Its expression in the brain of mammals coincides with defined periods of central nervous system (CNS) maturation and cell differentiation (43). In oligodendrocyte progenitor cells (OPC), S100B expression is associated with differentiation, and S100B contributes to OPC differentiation in response to demyelinating insult (11). To understand the contribution of S100B to OPC differentiation, we searched for high-affinity S100B target proteins in this cell type by using far-Western analysis. A major and highly specific S100B target protein was identified, the mitochondrial ATAD3A protein.ATAD3A belongs to a new family of eukaryote-specific mitochondrial AAA⁺ ATPase proteins (17). In the human genome, two genes, Atad3A and Atad3B, are located in tandem on chromosome 1p36.33. The Atad3A gene is ubiquitous among multicellular organisms but absent in yeast. The Atad3B gene is specific to the human genome (27). ATAD3A is a mitochondrial protein anchored into the mitochondrial inner membrane (IM) at contact sites with the outer membrane (OM). Thanks to its simultaneous interaction with the two membranes, ATAD3A regulates mitochondrial dynamics at the interface between the inner and outer membranes and controls diverse cell responses ranging from mitochondrial metabolism, cell growth, and mitochondrial fission 20a, 25). The ATAD3A protein has also been identified as a mitochondrial DNA binding protein (23) and as a cell surface antigen in some human tumors (20, 21). The plasma membrane localization of ATAD3A in tumor cells is suggestive that ATAD3A mitochondrial routing can be compromised in pathological situations such as cancer. To understand the functional response resulting from the interaction between S100B and ATAD3A, we first characterized the minimal interaction domain on ATAD3A for S100B binding using thermodynamic studies of wild-type and ATAD3A variants as well as via nuclear magnetic resonance (NMR) spectroscopy techniques. These studies allowed us to further refine the consensus S100B binding motif, which is conserved in several other S100B target proteins, including the p53 protein and several newly discovered target proteins associated with the cell translational machinery. We next analyzed the cellular interaction of S100B with truncated ATAD3A mutants that harbor the S100B binding domain but that are deficient for mitochondrial import. These studies revealed that S100B could assist ATAD3A mutant proteins during cytoplasmic processing by preventing dysfunctional aggregation events. Our results are discussed in light of the possible function of S100B in assisting the cytoplasmic processing of proteins for proper folding and subcellular localization.     

Apoptotic Platelet Events Are Not Observed in Severe von Willebrand Disease-Type 2B Mutation p.V1316M

Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation.     

Evolution and Phylogenetic Diversity of Yam Species (Dioscorea spp.): Implication for Conservation and Agricultural Practices

Yams (Dioscorea spp.) consist of approximately 600 species. Presently, these species are threatened by genetic erosion due to many factors such as pest attacks and farming practices. In parallel, complex taxonomic boundaries in this genus makes it more challenging to properly address the genetic diversity of yam and manage its germplasm. As a first step toward evaluating and preserving the genetic diversity yam species, we use a phylogenetic diversity (PD) approach that has the advantage to investigate phylogenetic relationships and test hypotheses of species monophyly while alleviating to the problem of ploidy variation within and among species. The Bayesian phylogenetic analysis of 62 accessions from 7 species from three regions of Cameroon showed that most Dioscorea sections were monophyletic, but species within sections were generally non-monophyletic. The wild species D. praehensilis and cultivated D. cayenensis were the species with the highest PD. At the opposite, D. esculenta has a low PD and future studies should focus on this species to properly address its conservation status. We also show that wild species show a stronger genetic structure than cultivated species, which potentially reflects the management of the yam germplasm by farmers. These findings show that phylogenetic diversity is a promising approach for an initial investigation of genetic diversity in a crop consisting of closely related species.     

A Quantitative Technique to Compare and Classify Humpback Whale (Megaptera novaeangliae) Sounds

In an attempt to minimize observer bias, numerical taxonomy methods were used to describe and classify humpback whale sounds. The spectrograms (N = 1255) were digitized into a 16 × 21 binary matrix. The rows were 16 frequencies selected on a logarithmic scale (0.12–8 kHz). The columns were 21 time samples taken every 0.1 s. Each point of the matrix was coded 1 if it lay over part of the sound. Other binary variables were added to code for relative intensity within a sound, frequency modulation and amplitude modulation. The sounds were then compared using the Jaccard similarity coefficient for binary data, and classified with average linkage cluster analysis. This technique produced 115 clusters, which were compared with my aural and visual impressions of the sounds. I agreed with most major categories identified by cluster analysis, but many small clusters had to be fused to other categories. This was partially due to the technique used, and to the complexity of the repertoire under study. Improvements are proposed to further reduce observer bias in classification of sounds, and thus make studies of animal communication performed by different researchers or on different species more easily comparable.