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Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation

Proc Natl Acad Sci USA. 2015; pii: 201421204.

Chen SW, Drakulic S, Deas E, Ouberai M, Aprile FA, Arranz R, Ness S, Roodveldt C, Guilliams T, De-Genst EJ, Klenerman D, Wood NW, Knowles TP, Alfonso C, Rivas G, Abramov AY, Valpuesta JM, Dobson CM, Cremades N.

Proc Natl Acad Sci USA. 2015; pii: 201421204We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques.

Although the oligomers exist in a range of sizes, with different extents and nature of β-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their β-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the β-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.

RNA polymerase slippage as a mechanism for the production of frameshift gene products in plant viruses of the Potyviridae family

J Virol. 2015; pii: JVI.00337-15.

Rodamilans B, Valli A, Mingot A, San León D, Baulcombe D, López-Moya JJ, García JA.

J Virol. 2015; pii: JVI.00337-15Modifications of RNA sequences by nucleotide insertions, deletions or substitutions can result in expression of multiple proteins in overlapping open reading frames (ORF).

In the case of viruses, polymerase slippage results in the alteration of newly synthesized RNA. The mechanism has been well characterized in animal RN A viruses such as Ebolavirus (EBOV) or Hepatitis C virus (HCV). For plant viruses, polymerase slippage has been proposed as a general process for viral evolution in members of the Potyviridae family , although lack of experimental systems have precluded confirmatory data, and most pieces of evidence are indirect.

Distribution of DNA-condensing protein complexes in the adenovirus core

Nucleic Acids Res. 2015; pii: gkv187.

Pérez-Berná AJ, Marion S, Chichón FJ, Fernández JJ, Winkler DC, Carrascosa JL, Steven AC, Šiber A, San Martín C.

Nucleic Acids Res. 2015; pii: gkv187Genome packing in adenovirus has long evaded precise description, since the viral dsDNA molecule condensed by proteins (core) lacks icosahedral order characteristic of the virus protein coating (capsid).

We show that useful insights regarding the organization of the core can be inferred from the analysis of spatial distributions of the DNA and condensing protein units (adenosomes). These were obtained from the inspection of cryo-electron tomography reconstructions of individual human adenovirus particles. Our analysis shows that the core lacks symmetry and strict order, yet the adenosome distribution is not entirely random. The features of the distribution can be explained by modeling the condensing proteins and the part of the genome in each adenosome as very soft spheres, interacting repulsively with each other and with the capsid, producing a minimum outward pressure of ∼0.06 atm. Although the condensing proteins are connected by DNA in disrupted virion cores, in our models a backbone of DNA linking the adenosomes is not required to explain the experimental results in the confined state.

In conclusion, the interior of an adenovirus infectious particle is a strongly confined and dense phase of soft particles (adenosomes) without a strictly defined DNA backbone.

Characterization of interaction of magnetic nanoparticles with breast cancer cells

J Nanobiotechnology. 2015; 13(1): 16.

Calero M, Chiappi M, Lazaro-Carrillo A, Rodríguez MJ, Chichón FJ, Crosbie-Staunton K, Prina-Mello A, Volkov Y, Villanueva A, Carrascosa JL.

J Nanobiotechnology. 2015; 13(1): 16Different superparamagnetic iron oxide nanoparticles have been tested for their potential use in cancer treatment, as they enter into cells with high effectiveness, do not induce cytotoxicity, and are retained for relatively long periods of time inside the cells. We have analyzed the interaction, internalization and biocompatibility of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles with an average diameter of 15 nm and negative surface charge in MCF-7 breast cancer cells.

Cells were incubated with dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles for different time intervals, ranging from 0.5 to 72 h. These nanoparticles showed efficient internalization and relatively slow clearance. Time-dependent uptake studies demonstrated the maximum accumulation of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles after 24 h of incubation, and afterwards they were slowly removed from cells. Superparamagnetic iron oxide nanoparticles were internalized by energy dependent endocytosis and localized in endosomes. Transmission electron microscopy studies showed macropinocytosis uptake and clathrin-mediated internalization depending on the nanoparticles aggregate size. MCF-7 cells accumulated these nanoparticles without any significant effect on cell morphology, cytoskeleton organization, cell cycle distribution, reactive oxygen species generation and cell viability, showing a similar behavior to untreated control cells.

All these findings indicate that dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles have excellent properties in terms of efficiency and biocompatibility for application to target breast cancer cells.

Polyethylenimine-coated SPIONs trigger macrophage activation through TLR-4 signaling and ROS production and modulate podosome dynamics

Biomaterials. 2015; 52: 494-506.

Mulens-Arias V, Rojas JM, Pérez-Yagüe S, Morales MP, Barber DF.

Biomaterials. 2015; 52: 494-506Polyethylenimine (PEI) is widely used as transfection agent in preclinical studies, both in vitro and in vivo. Due to their unique chemical and physical properties, SPIONs (superparamagnetic iron oxide nanoparticles) have been thoroughly studied as nanocarriers. PEI appears to activate different immune cells to an inflammatory response (M1/TH1), whereas the SPION-induced response seems to be context-dependent; the immunogenicity of the combination of these components has not been studied.

Here we show that PEI-coated SPIONs (PMag) activate macrophages, as determined by measuring IL-12 secretion into culture medium and upregulation of several genes linked to the M1 phenotype. PMag-induced phosphorylation of p38 MAPK, p44/p42 MAPK and JNK, and upregulation of CD40, CD80, CD86 and I-A/I-E activation markers. PMag-induced macrophage activation depended partially on TLR4 (Toll-like receptor 4) and ROS (reactive oxygen species) signaling. Comparison of these responses with the LPS (lipopolysaccharide)-induced phenotype showed differences in gene expression profiling. PMag positively modulated podosome formation in murine macrophages, but hampered gelatin degradation by these cells.

In conclusion, PMag induced an M1-like phenotype that was partially dependent on both TLR4 and ROS. These results show the adjuvant potential of PMag and suggest their use in vaccination schedules.