Combination of bioanalytical approaches and quantitative proteomics for the elucidation of the toxicity mechanisms associated to TiO2 nanoparticles exposure in human keratinocytes.

Titanium dioxide nanoparticles (TiO2-NPs) are being used in several consumer products. The high refractive index of nano-scaled titanium dioxide particles allows them to protect from UV radiation, and so, they can be found as one of the main components of cosmetics and suncreens. Many studies have reported the potential toxicological effects associated to TiO2-NPs such as ROS generation, DNA damage, apoptosis and cell cycle arrest, among others. The continuous and systematic use of TiO2-NPs in cosmetic products requires a full comprehension of the risks involving their sustained contact with the human skin. Thus, it is important to evaluate not only the hazardous effects but to elucidate the biomolecular mechanisms involved in such effects. Based on this premises, we have evaluated the potential toxicity of TiO2-NPs using a human epithelial cell culture (HaCaT cells) as in-vitro model, together with different bioanalytical approaches and mass spectrometrybased quantitative proteomics, to gain a deeper insight into the molecular mechanisms of toxicity associated to TiO2-NPs exposurepre-print451 K

Vitamin C activates young LINE-1 elements in mouse embryonic stem cells via H3K9me3 demethylation

This work was supported by grants from the Wellcome Trust/Royal Soci‑ ety (101225/Z/13/Z) and MRC (MR/X008487/1) to M.R.B.; and BBSRC (BB/ T000031/1) to M.R.B. and J.M.F.Background Vitamin C (vitC) enhances the activity of 2-oxoglutarate-dependent dioxygenases, including TET enzymes, which catalyse DNA demethylation, and Jumonji-domain histone demethylases. The epigenetic remodelling promoted by vitC improves the efficiency of induced pluripotent stem cell derivation, and is required to attain a ground-state of pluripotency in embryonic stem cells (ESCs) that closely mimics the inner cell mass of the early blastocyst. However, genome-wide DNA and histone demethylation can lead to upregulation of transposable elements (TEs), and it is not known how vitC addition in culture media affects TE expression in pluripotent stem cells. Results Here we show that vitC increases the expression of several TE families, including evolutionarily young LINE-1 (L1) elements, in mouse ESCs. We find that TET activity is dispensable for L1 upregulation, and that instead it occurs largely as a result of H3K9me3 loss mediated by KDM4A/C histone demethylases. Despite increased L1 levels, we did not detect increased somatic insertion rates in vitC-treated cells. Notably, treatment of human ESCs with vitC also increases L1 protein levels, albeit through a distinct, post-transcriptional mechanism. Conclusion VitC directly modulates the expression of mouse L1s and other TEs through epigenetic mechanisms, with potential for downstream effects related to the multiple emerging roles of L1s in cellular function.Wellcome Trust/Royal Society (101225/Z/13/Z)MRC (MR/X008487/1)BBSRC (BB/T000031/1

Integration of Transcriptomics and Metabolomics to Reveal the Molecular Mechanisms Underlying Rhodium Nanoparticles-Based Photodynamic Cancer Therapy

Rhodium nanoparticles have recently been described as promising photosensitizers due to their low toxicity in the absence of near-infrared irradiation, but their high cytotoxicity when irradiated. Irradiation is usually carried out with a laser source, which allows the treatment to be localized in a specific area, thus avoiding undesirable side effects on healthy tissues. In this study, a multi-omics approach based on the combination of microarray-based transcriptomics and mass spectrometry-based untargeted and targeted metabolomics has provided a global picture of the molecular mechanisms underlying the anti-tumoral effect of rhodium nanoparticle-based photodynamic therapy. The results have shown the ability of these nanoparticles to promote apoptosis by suppressing or promoting anti- and pro-apoptotic factors, respectively, and by affecting the energy machinery of tumor cells, mainly blocking the β-oxidation, which is reflected in the accumulation of free fatty acids and in the decrease in ATP, ADP and NAD+ levels.Depto. de Química AnalíticaFac. de Ciencias QuímicasTRUEMinisterio de Ciencia, Innovaciónpu

Cancer cell targeting and therapeutic delivery of silver nanoparticles by mesoporous silica nanocarriers: insights into the action mechanisms by quantitative proteomics

The aim of the present work is to provide an approach to safely deliver silver nanoparticles (AgNPs) as cytotoxic agents into cancer cells, and to provide a deeper insight into the cellular mechanisms affected by such targeted delivery. The use of mesoporous silica nanoparticles (MSNs) as nanovehicles decorated with transferrin (Tf, targeting agent) provides a nanoplatform for the nucleation and immobilization of AgNPs (MSNs-Tf-AgNPs). We have performed the physico-chemical characterization of the nanosystems and evaluated their therapeutic potential using bioanalytical strategies to estimate the efficiency of the targeting, the degree of cellular internalization in two cell lines with different TfR expression, and the cytotoxic effects of the delivered AgNPs. In addition, cellular localization of the nanosystems in cells has been evaluated by a transmission electron microscopy analysis of ultrathin sections of Human hepatocarcinoma (HepG2) cells exposed to MSNs-Tf-AgNPs. The in vitro assays demonstrate that only the nanosystem functionalized with Tf is able to transport the AgNPs inside the cells which overexpress transferrin receptors. Therefore, this novel nanosystem is able to deliver AgNPs specifically to cancer cells overexpressing Tf receptors and offers the possibility of a targeted therapy using reduced doses of silver nanoparticles as cytotoxic agents. Then, a quantitative proteomic experiment validated through the analysis of gene expression has been performed to identify the action molecular mechanisms associated with the chemotherapeutic potential of the MSNs-Tf-AgNPs nanocarriers

Mesoporous Silica Nanoparticles as a Potential Platform for Vaccine Development against Tuberculosis.

The increasing emergence of new strains of Mycobacterium tuberculosis (Mtb) highly resistant to antibiotics constitute a public health issue, since tuberculosis still constitutes the primary cause of death in the world due to bacterial infection. Mtb has been shown to produce membrane-derived extracellular vesicles (EVs) containing proteins responsible for modulating the pathological immune response after infection. These natural vesicles were considered a promising alternative to the development of novel vaccines. However, their use was compromised by the observed lack of reproducibility between preparations. In this work, with the aim of developing nanosystems mimicking the extracellular vesicles produced by Mtb, mesoporous silica nanoparticles (MSNs) have been used as nanocarriers of immunomodulatory and vesicle-associated proteins (Ag85B, LprG and LprA). These novel nanosystems have been designed and extensively characterized, demonstrating the e ectiveness of the covalent anchorage of the immunomodulatory proteins to the surface of the MSNs. The immunostimulatory capacity of the designed nanosystems has been demonstrated by measuring the levels of pro- (TNF) and anti-inflammatory (IL-10) cytokines in exposed macrophages. These results open a new possibility for the development of more complex nanosystems, including additional vesicle components or even antitubercular drugs, thus allowing for the combination of immunomodulatory and bactericidal e ects against Mtb.post-print2418 K

Structure of the receptor-binding carboxy-terminal domain of the bacteriophage T5 L-shaped tail fibre with and without its intra-molecular chaperone

Bacteriophage T5, a Siphovirus belonging to the order Caudovirales, has a flexible, three-fold symmetric tail, to which three L-shaped fibres are attached. These fibres recognize oligo-mannose units on the bacterial cell surface prior to infection and are composed of homotrimers of the pb1 protein. Pb1 has 1396 amino acids, of which the carboxy-terminal 133 residues form a trimeric intra-molecular chaperone that is auto-proteolyzed after correct folding. The structure of a trimer of residues 970–1263 was determined by single anomalous dispersion phasing using incorporated selenomethionine residues and refined at 2.3 Å resolution using crystals grown from native, methionine-containing, protein. The protein inhibits phage infection by competition. The phage-distal receptor-binding domain resembles a bullet, with the walls formed by partially intertwined beta-sheets, conferring stability to the structure. The fold of the domain is novel and the topology unique to the pb1 structure. A site-directed mutant (Ser1264 to Ala), in which auto-proteolysis is impeded, was also produced, crystallized and its 2.5 Å structure solved by molecular replacement. The additional chaperone domain (residues 1263–1396) consists of a central trimeric alpha-helical coiled-coil flanked by a mixed alpha-beta domain. Three long beta-hairpin tentacles, one from each chaperone monomer, extend into long curved grooves of the bullet-shaped domain. The chaperone-containing mutant did not inhibit infection by competition.This research was sponsored by grants BFU2011-24843, BIO2011-14756-E, BFU2014-53425P (Mark J. van Raaij), and BFU2014-55475R (José R. Castón) and the BioFiViNet network (FIS2011-16090-E) from the Spanish Ministry of Economy and Competitiveness, grant S2013/MIT-2807 (José R. Castón) from the Comunidad Autónoma de Madrid and a joint networking grant from CSIC (2011FR0016; Mark J. van Raaij) and CNRS (2011EDC25326; Pascale Boulanger). Carmela Garcia-Doval was the recipient of a pre-doctoral FPU fellowship from the Spanish Ministry of Education, Culture and Sports and José M. Otero of a post-doctoral Plan I2C fellowship from the Xunta de Galicia. The research leading to these results has also received funding from the European Community’s Seventh Framework Programme (FP7/2007–2013) under BioStruct-X (grant agreement number 283570). We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer Reviewe

Antimycobacterial effect of selenium nanoparticles on Mycobacterium tuberculosis.

Tuberculosis remains the leading cause of death from a single infection agent worldwide. In recent years, the occurrence of tuberculosis cases caused by drug-resistant strains has spread, and is expected to continue to grow. Therefore, the development of new alternative treatments to the use of antibiotics is highly important. In that sense, nanotechnology can play a very relevant role, due to the unique characteristics of nanoparticles. In fact, different types of nanoparticles have already been evaluated both as potential bactericides and as efficient drug delivery vehicles. In this work, the use of selenium nanoparticles has been evaluated to inhibit the growth of two types of mycobacteria: Mycobacterium smegmatis and Mycobacterium tuberculosis. The results showed that selenium nanoparticles are able to inhibit the growth of both types of mycobacteria by damaging their cell envelope integrity. These results open a new opportunity for the use of this type of nanoparticles as antimycobacterial agents by themselves, or for the development of novel nanosystems that combine the action of these nanoparticles with other drugs

Nueva localidad de Bulinus truncatus (Audouin, 1827) (Gastropoda: Planorbidae), hospedador intermediario de Schistosoma haematobium, y su distribución en la península Ibérica

This paper reports a new population of Bulinus truncatus (Audouin, 1827) (Gastropoda, Planorbidae) found in the province of Almería (Southeast Spain). B. truncatus is an intermediate host of Schistosoma haematobium, the trematode which causes urinary schistosomiasis in humans. Individuals were identified to species level by double-nested PCR, resulting in 100% homology. This population is located under the driest climate conditions of the Iberian Peninsula. Data on the distribution of this species in the Iberian Peninsula was gathered and is provided in this paper. Improved knowledge of the distribution of Bulinus truncatus is key to assess the risk of new outbreaks of schistosomiasis in the Iberian Peninsula.En este trabajo se reporta una nueva población de Bulinus truncatus (Audouin, 1827) (Gastropoda, Planorbidae) en la provincia de Almería (Sureste de España). Se trata de una especie que presenta considerable interés tanto zoológico como epidemiológico por su papel como hospedador intermediario de Schistosoma haematobium, responsable de la esquistosomiasis urogenital humana. Los ejemplares fueron determinados a nivel específico mediante PCR doble anidada, con un 100% de homología. Se trata de la población localizada más al Sureste y en condiciones de mayor aridez en la península Ibérica. Se recopilaron los datos de presencia publicados para conocer su distribución en la península. El conocimiento de la distribución de Bulinus truncatus es una pieza clave para evaluar el riesgo de nuevos focos de esquistosomiasis en la península Ibérica

Landscape evolution and geodynamic controls in the Gulf of Cadiz (Huelva coast, SW Spain) during the Late Quaternary

The coastal evolution of the El Abalario area (Huelva, southern Spain) during the Late Pleistocene and Holocene is reinterpreted after a refinement of the available geochronology by means of optically stimulated luminescence (OSL) dating. New data come from the analysis of soft sediment deformation, palaeosols, geomorphological mapping, and published seismic surveys on the onshore and offshore Gulf of Cadiz. The present structure of El Abalario dome resulted from the complex interaction of littoral-catchment processes and sea-level changes upon an emergent coastal plain, conditioned by the upwarping of the underlying Pliocene - Pleistocene prograding deltaic sequence. Upwarping is probably related to escape of over-pressurized fluids, accompanied by dewatering, prior to (?) and during OIS (Oxygen Isotopic Stage) 5. Continued upwarping produced the large NW - SE gravitational fault of Torre del Loro (TLF) in the southwestern flank of the dome, roughly parallel to the present coastline during OIS 5 - OIS 4. The resulting escarpment favoured the accumulation of aeolian sand dunes (units U1, U2, and U3) from OIS 5 to early OIS 1. Unit U1 (OIS 5) ends upwards in a supersurface with a thick weathering profile that suggests moist and temperate climatic conditions. Unit U2 accumulated mainly during OIS 4 and OIS 3 with prevailing W/E winds. The supersurface between U2 and U3 records a part of OIS 2, with relative low sea level. Sedimentation of unit U3 took place during the Last Deglaciation (radiocarbon and OSL ages) with prevailing W/SW winds, under a temperate moist climate, that became more arid towards the top (Holocene). A major supersurface with an iron crust-like layer (SsFe) developed during the Holocene Climatic Optimum (OIS 1) under wetter and more temperate conditions than before, fossilizing the TLF. The supersurface is covered by younger aeolian dunes (U4, U5, U6, and U7) transported by W - SW winds since the Late Neolithic - Chalcolithic cultural period (̃5.0 ky cal BP).Financial support from Spanish Projects BTE2002-1065 and BTE2002-1691. This is a contribution to IGCP 495 and to the INQUA “Coastal and Marine Processes Commission”