Mechanical Stability and Reversible Fracture of Vault Particles

Vaults are the largest ribonucleoprotein particles found in eukaryotic cells, with an unclear cellular function and promising applications as vehicles for drug delivery. In this article, we examine the local stiffness of individual vaults and probe their structural stability with atomic force microscopy under physiological conditions. Our data show that the barrel, the central part of the vault, governs both the stiffness and mechanical strength of these particles. In addition, we induce single-protein fractures in the barrel shell and monitor their temporal evolution. Our high-resolution atomic force microscopy topographies show that these fractures occur along the contacts between two major vault proteins and disappear over time. This unprecedented systematic self-healing mechanism, which enables these particles to reversibly adapt to certain geometric constraints, might help vaults safely pass through the nuclear pore complex and potentiate their role as self-reparable nanocontainers.     

Bacterial contamination of in vitro plant cultures: confounding effects on somaclonal variation and detection of contamination in plant tissues

Bacterial contamination represents a serious problem for plant tissue culture research and applications. Bacterial interference with normal plant physiology and morphology can generate misleading conclusions if the presence of bacteria is ignored. Bacterial contaminants in in vitro plant culture are typically detected by direct observation; thus, it is assumed that cultures without visible symptoms are bacteria free. Here, we demonstrate that contaminating Bacillus DNA in plant DNA solutions from asymptomatic plants can interfere with the analysis of somaclonal variation in chrysanthemum. We studied somaclonal variation in chrysanthemum using short semi-specific PCR primers based on conserved motifs in NBS–LRR disease resistance genes and in mobile elements. Instead of true somaclonal variation we found three polymorphic bands derived from contaminant bacterial DNA in plant extracts. Although the detection of asymptomatic bacteria in in vitro plant cultures is a major issue, we found that it has not been adequately addressed to date, particularly for studies on somaclonal variation. We reviewed the most commonly cited contaminant bacteria in in vitro plant culture and designed specific 16S rRNA gene-based PCR primers for the main genera causing contamination (Bacillus, Pseudomonas, Staphylococcus, Lactobacillus, Erwinia/Enterobacter and Xanthomonas). Using a panel of pure bacterial DNAs, artificial mixes of bacterial/plant DNAs, and in vitro plant cultures with and without visible contamination we demonstrated that our primers are in most instances both reliable and sensitive, and appropriate for the identification and tracking of the most frequent bacterial contaminants in plant in vitro cultures. Implications of bacterial identification to molecular analysis of somaclonal variation and plant culture decontamination are discussed.     

Bycatch species composition over time by tuna purse-seine fishery in the eastern tropical Atlantic Ocean

Within the Ecosystem-based fisheries management framework, we evaluated the changes over time in bycatch species of the European tuna purse-seine fishery operating in the eastern Atlantic Ocean. Bycatch data was collected during two scientific observer programs conducted in the late 1990s and in the late 2000s. Over these two time periods, we compared the temporal trends in bycatch species composition, the probability of occurrence of functional groups per fishing set, the spatio-temporal species richness and the potential impact on several species listed in the red list of the International Union for Conservation of Nature (IUCN). The analyses were performed separately on the two main fishing modes of the fleet, i.e. sets on free-swimming school sets and on fish aggregating devices (FADs). Owing data quality constraints, we did not estimate bycatch rates. Ours results showed that the species composition of sharks caught on FADs decreased over time. The total species richness was higher for FAD sets than for free-swimming school sets (87 vs. 61 species respectively), such difference is common between fishing modes worldwide. For the species catalogued as threatened by the IUCN, in free-swimming schools, 25.5 % of the species caught during first period increased to 30.4 % during second period, while for FAD-fishing the increase was from 28.8 % during first period to 34.9 % in second period. Ours findings suggest that tropical tuna purse-seine fisheries should include ecosystem-based governance of bycatch. Effective tuna management will require a combination of technological improvements for mitigating incidental catch of vulnerable species, best use of byproduct species, regulations in fishing practices and in spatial distribution of fishing effort, and international agreements that, together, can monitor and manage bycatch, reducing the negative fishing effects on the epipelagic ecosystem biodiversity.     

Structure and Immunogenicity of a Peptide Vaccine,Including the Complete HIV-1 gp41 2F5 Epitope: IMPLICATIONS FOR ANTIBODY RECOGNITION MECHANISM AND IMMUNOGEN DESIGN*

The membrane-proximal external region (MPER) of gp41 harbors the epitope recognized by the broadly neutralizing anti-HIV 2F5 antibody, a research focus in HIV-1 vaccine development. In this work, we analyze the structure and immunogenic properties of MPERp, a peptide vaccine that includes the following: (i) the complete sequence protected from proteolysis by the 2F5 paratope; (ii) downstream residues postulated to establish weak contacts with the CDR-H3 loop of the antibody, which are believed to be crucial for neutralization; and (iii) an aromatic rich anchor to the membrane interface. MPERp structures solved in dodecylphosphocholine micelles and 25% 1,1,1,3,3,3-hexafluoro-2-propanol (v/v) confirmed folding of the complete 2F5 epitope within continuous kinked helices. Infrared spectroscopy (IR) measurements demonstrated the retention of main helical conformations in immunogenic formulations based on alum, Freund''s adjuvant, or two different types of liposomes. Binding to membrane-inserted MPERp, IR, molecular dynamics simulations, and characterization of the immune responses further suggested that packed helical bundles partially inserted into the lipid bilayer, rather than monomeric helices adsorbed to the membrane interface, could encompass effective MPER peptide vaccines. Together, our data constitute a proof-of-concept to support MPER-based peptides in combination with liposomes as stand-alone immunogens and suggest new approaches for structure-aided MPER vaccine development.     

Acetato and formato copper(II) complexes with 4,4′-dimethyl-2,2′-bipyridine and 5,5′-dimethyl-2,2′-bipyridine: Synthesis, crystal structure, magnetic properties and EPR results. A new 1D polymeric water chain

By the reactions of Cu(AcO)₂·H₂O and Cu(HCOO)₂·4H₂O with 4,4′-dimethyl-2,2′-bipyridine and 5,5′-dimethyl-2,2′-bipyridine the compounds [Cu(AcO)₂(4,4′-Me₂-2,2′-bipy)]·1/2H₂O (1), [Cu(AcO)₂(5,5′-Me₂-2,2′-bipy)(H₂O)] (2), [Cu(HCOO)(μ-HCOO)(4,4′-Me₂-2,2′-bipy)]_n·nH₂O (3) and [Cu(HCOO)(μ-HCOO)(5,5′-Me₂-2,2′-bipy)]_n·2nH₂O (4) were obtained. In the acetate complexes, 1 and 2, the geometry around copper is distorted octahedral and square pyramidal, respectively. Dimeric units of different geometry are formed in both cases through hydrogen bonds in which non-coordinated (in 1) and coordinated (in 2) water molecules are involved. The structures of 3 and 4 consist of polymeric monodimensional chains of square pyramidal copper units linked by axial-equatorial syn-anti (3) or anti-anti (4) bridging formate groups. Water molecules form hydrogen bonds with formate groups of the same chain in compound 3. In compound 4 the water molecules link the polymeric contiguous chains of complex through hydrogen bonds with oxygen atoms of formate groups and they are also linked between them, forming monodimensional water chains which run parallel to the complex chains. Sheets parallel to the ac plane are formed by alternating chains of water and polymeric complex. Magnetic properties and EPR spectra for these compounds have been studied.     

Hexapeptides that interfere with HIV-1 fusion peptide activity in liposomes block GP41-mediated membrane fusion

Upon receptor-mediated activation, the gp41 hydrophobic, conserved fusion peptide inserts into the target membrane and promotes the kind of perturbations required for the progression of the HIV-cell fusion reaction. Using a synthetic combinatorial library we have identified all d-amino acid hexapeptide sequences that inhibited the fusion peptide capacity of perturbing model membranes. Two hexapeptides that effectively inhibited the fusion peptide in these systems were subsequently shown to inhibit cell-cell fusion promoted by gp41 expressed at cell surfaces. These observations might be of importance for understanding the mechanisms underlying fusion peptide activity and suggest new strategies for screening compounds that target these viral sequences.     

A Novel Liposome-Based Nanocarrier Loaded with an LPS-dsRNA Cocktail for Fish Innate Immune System Stimulation

Development of novel systems of vaccine delivery is a growing demand of the aquaculture industry. Nano- and micro- encapsulation systems are promising tools to achieve efficient vaccines against orphan vaccine fish diseases. In this context, the use of liposomal based-nanocarriers has been poorly explored in fish; although liposomal nanocarriers have successfully been used in other species. Here, we report a new ∼125 nm-in-diameter unilamellar liposome-encapsulated immunostimulant cocktail containing crude lipopolysaccharide (LPS) from E. coli and polyinosinic:polycytidylic acid [poly (I:C)], a synthetic analog of dsRNA virus, aiming to be used as a non-specific vaccine nanocarrier in different fish species. This liposomal carrier showed high encapsulation efficiencies and low toxicity not only in vitro using three different cellular models but also in vivo using zebrafish embryos and larvae. We showed that such liposomal LPS-dsRNA cocktail is able to enter into contact with zebrafish hepatocytes (ZFL cell line) and trout macrophage plasma membranes, being preferentially internalized through caveolae-dependent endocytosis, although clathrin-mediated endocytosis in ZFL cells and macropinocytocis in macrophages also contribute to liposome uptake. Importantly, we also demonstrated that this liposomal LPS-dsRNA cocktail elicits a specific pro-inflammatory and anti-viral response in both zebrafish hepatocytes and trout macrophages. The design of a unique delivery system with the ability to stimulate two potent innate immunity pathways virtually present in all fish species represents a completely new approach in fish health.