Thymol and Thymus vulgaris extract protects human keratinocyte cell line (HaCaT) from UVA and UVB damage

Objective: The aims of our study were to characterize ultraviolet (UV)A- and UVB-induced damages in a keratinocytes cell line (HaCaT), and to evaluate the protective capacities two plant-derivative compounds, namely Thymus vulgaris L leaf extract and thymol, its major component. A polyphenol rich diet has gained wide attention and it is now considered to be a protective agent for human skin, which can be over-exposed to environmental factors and in particular UV light. Methods: Cells were pretreated for 1 h, in serum-free medium, with thymol (1 \u3bcg/ml) or Thymus vulgaris L (1.82 \u3bcg/ml) then exposed to different UVA (8-24 J/cm2) or UVB doses (0.016-0.72 J/cm2). Immediately after the UV exposure the intracellular redox status was evaluated by reactive oxygen species quantification and apoptotic events. Genotoxic aspects were evaluated 24 h after the end of irradiations using the alkaline comet assay and the immunostaining of phosphorylated H2AX histone protein (detected 1 h after the end of UV exposure). Results: The pre-treatment of our experimental model with the two substances confirmed an antioxidant action and anti-apoptotic effect by reducing the cells percentage (sub-G1 phase). Furthermore, thymol and extract of Thymus vulgaris L were able to reduce genotoxic damage. The alkaline comet assay showed that the two substances were capable to decrease DNA damage. Also in this case, Thymus vulgaris L extract is more effective than thymol in decreasing genotoxicity markers. Conclusions: Our results confirmed the more oxidant UVA and more genotoxic UVB effects. Regarding the protective effect of thymol and Thymus vulgaris L extract, data obtained proved their antioxidant and free-radical scavenging ability as known for phenolic (which our compounds belong to) and polyphenolic compounds. Thymol and mainly Thymus vulgaris L extract were also able to reduce the direct genotoxic damage

Assessment of Toxicity of Myristicin and 1’-Hydroxymyristicin in HepG2 Cell Line

Background and Objective: Myristicin belongs to a class of potentially toxic chemicals (alkoxy substituted allylbenzenes) and despite the structural analogy with safrole, data on this compound are very controversial and unclear. In this study assessed the cytotoxic and genotoxic potential of myristicin and 1\u2019-hydroxy-myristicin after 24 h of exposure in HepG2 cells. Methodology: The compounds were tested up to 600 \u3bcM concentration, for 24 h. The genotoxicity was assessed with alkaline and neutral comet assay and micronucleus assay. The data were analysed by one-way ANOVA. Results: It is to be emphasized that only the synthetic Phase 1 metabolite (1\u2019-hydroxymyristicin) showed a genotoxic effect starting from the concentration of 150 \u3bcM both in comet and micronucleus tests. However, it is important to point out that the same concentration cause a statistically significant (p<0.001) apoptotic process. Conclusion: The consumption of a traditional diet determines very low levels of exposure to the parent myristicin. This fact implies as the primary metabolic pathway the O-demethylation (5-allyl-2,3-dihidroxyanisole) and not to Phase I metabolism, which leads to the conclusion that this substance could not present a significant risk to humans

Rhus coriaria l. Fruit extract prevents UV-A-induced genotoxicity and oxidative injury in human microvascular endothelial cells

Rhus coriaria L. (sumac) is a small plant widely diffused in the Mediterranean region. Its fruit are often consumed as a spice but are also present in traditional medicine of several countries. Recently, interest in this plant has increased and many scientific works reported its beneficial effects including antioxidant and anti-inflammatory properties. Plant extracts can be successfully used against ultraviolet rays, which are able to reach and damage the human skin; however, sumac extracts were never applied to this usage. Thus, in this study, we used a macerated ethanol extract of Rhus coriaria L. dried fruit (mERC) to demonstrate its preventive role against the damage induced by ultraviolet-A rays (UV-A) on microvascular endothelial cells (HMEC-1). In vitro effects of the extract pre-treatment and UV-A exposure were evaluated in detail. The antioxidant capacity was assessed by reactive oxygen species (ROS) formation and cellular antioxidant activity measurement. Genoprotective effects of mERC were investigated as well. Our findings indicate that the extract acts as a cell cycle inhibitor or apoptosis inducer, according to the level of damage. The present work provides new insights into the usage of Rhus coriaria extracts against skin injuries

Effects of UV rays and natural compound repairs using an ex-vivo human skin model: morphological and genotoxicological analysis

Among the key factors in skin disorders such as wrinkling, dryness and photo-aging, the exposure to solar ultraviolet (UV) radiation plays a central role (1). Recently, compounds rich in polyphenols such as Thymus Vulgaris Leaf (TVL) extract and its major component Thymol (T) have been proposed in the prevention of UV-induced skin damages (2). Experiments were carried out in a human ex-vivo skin model, in which biopsies were obtained from aesthetic surgery of healthy 20-40 year-old women (n=6) after written informed consent (3). After 24 h, samples were pre-treated for 1 h with comparable concentrations of two compounds (TVL: 1.82 \ub5g/mL and T: 1 \ub5g/mL) before being irradiated with different UVB doses (0.24 J/cm2 to 0.72 J/cm2) or UVA radiation (8 J/cm2 to 32 J/cm2). Samples were harvested 24 h after irradiation and were processed both for light and transmission electron microscopy.Cell proliferation, Lactate Dehydrogenase assay, alkaline comet test, and histone H2AX phosphorylationwere evaluated. Both UVB and UVA induced an early inhibition of cell proliferation and DNA damage compared with respective controls. In particular, UVB rays were always more cytotoxic and genotoxic than UVA. The T-pretreatment showed a reduction of UVB-induced structural/ultrastructural and genotoxic damages. These results suggest that polyphenol fraction of tested substances may be useful for skin photoprotection after UV radiation damage in an ex-vivo human skin model. The present study suggests that this experimental setting can be a reliable approach for safety evaluation of UV skin exposure

Effetti tossicologici del particolato ultrafine emesso da impianti residenziali a biomassa : note sul progetto TOBICUP

This work summarises the main findings of the TOBICUP (Toxicity of BIomass Combustion generated Ultrafine Particles) project. The project has investigated the physio-chemical features and toxicological response of ultrafine particles (UFPs) from biomass-fuelled domestic stoves. Experimental determinations consider both UFP samples collected at the stack of wood log and pellet stoves and environmental samples collected at a site where biomass burning for domestic heating is the main source of airborne UFPs. Results for the stack samples show that combustion in pellet stoves is more complete, producing UFPs that determine toxicological responses per unit input energy less relevant compared with wood log stoves. Results for the environmental samples show a larger contribution from domestic heating to airborne UFPs in wintertime, traced by the higher content of levoglucosan and potassium than in summertime. However,toxicological response are influenced by the different reactivity of the atmosphere at seasonal level: in wintertime, genotoxic effects prevail due to the larger concentration of PAH and levoglucosan; in summertime, pro-inflammatory effects are more relevant due to the higher degree of oxidation of UFPs, favoured by the stronger photochemical processes occurring in the warm season

Cryo-EM and the elucidation of new macromolecular structures: Random Conical Tilt revisited

Cryo-Electron Microscopy (cryo-EM) of macromolecular complexes is a fundamental structural biology technique which is expanding at a very fast pace. Key to its success in elucidating the three-dimensional structure of a macromolecular complex, especially of small and non-symmetric ones, is the ability to start from a low resolution map, which is subsequently refined with the actual images collected at the microscope. There are several methods to produce this first structure. Among them, Random Conical Tilt (RCT) plays a prominent role due to its unbiased nature (it can create an initial model based on experimental measurements). In this article, we revise the fundamental mathematical expressions supporting RCT, providing new expressions handling all key geometrical parameters without the need of intermediate operations, leading to improved automation and overall reliability, essential for the success of cryo-EM when analyzing new complexes. We show that the here proposed RCT workflow based on the new formulation performs very well in practical cases, requiring very few image pairs (as low as 13 image pairs in one of our examples) to obtain relevant 3D maps.We thank Dr. Llorca for his support during the acquisition of the C3b images and Dr. Shaikh for his support in the use of Spider for the RCT reconstructions. The authors would like to acknowledge economical support from the Spanish Ministry of Economy and Competitiveness through grants AIC-A-2011-0638 and BIO2013-44647-R, the Comunidad de Madrid through grant CAM (S2010/BMD-2305), as well as a postdoctoral Juan de la Cierva grant with reference JCI-2011-10185 to Javier Vargas. Vahid Abrishami is a holder of La Caixa scholarship and C.O.S. Sorzano is recipient of a Ramon y Cajal fellowship

Comparative antioxidant activity of cultivated and wild Vaccinium species investigated by EPR, human neutrophil burst and COMET assay

objectives: The Vaccinium (V.) spp. berries are considered a source of antioxidants, mainly belonging to polyphenols, specifically flavonoids and anthocyanins. Wild genotypes generally contain more antioxidants than cultivated counterparts. So, seven different antioxidants assays on extracts from cultivated and wild Vaccinium berries were performed, to evaluate their difference in terms of bioactivity on oxidative protection and minimum dosage to have a significant action. Materials and Methods: Four cell-free antioxidant assays (ABTS radical scavenging and electronic paramagnetic resonance using Fremy's salt, superoxide anion and hydroxyl radical), and three assays on human cells (two luminol amplified chemiluminescence, LACL, one on DNA damage, COMET) were used to measure the effects of cultivated blueberry (V. corymbosum) and wild bilberry (V. myrtillus) on the differently induced oxidative stress. Concentrations vs activity patterns were obtained by successive dilutions of extracts in order to identify both EC50 and minimum significant activity (MSA). Results: All the assays (except for the hydroxyl radical scavenging) showed a good relationship mainly with anthocyanin and polyphenol content and the significant greater activity of wild Vaccinium extracts. In fact, LACL data gave an EC50 of 11.8 and an MSA of 5.2 g were calculated as fresh weight dosage in cultivated berries, compared with lower doses in wild berries, EC50 of 5.7 g and MSA of 3.4 g. Conclusions: Wild Vaccinium extracts averaged 3.04 and 2.40 fold more activity than cultivated extracts by EC50 and MSA, respectively. COMET assay confirmed the stronger action on DNA protection in wild samples

XTEND: Extending the depth of field in cryo soft X-ray tomography

We have developed a new data collection method and processing framework in full field cryo soft X-ray tomography to computationally extend the depth of field (DOF) of a Fresnel zone plate lens. Structural features of 3D-reconstructed eukaryotic cells that are affected by DOF artifacts in standard reconstruction are now recovered. This approach, based on focal series projections, is easily applicable with closed expressions to select specific data acquisition parameters.This work was partially supported by MINECO grants BFU2014-54181 to JLC and AIC-A-2011-0638, BIO2013-44647-R and BIO2016-76400-R to JMC, Madrid. Regional government grants S2013/MIT-2850 to JLC and S2010/BMD-2305 to JMC, National Science Foundation grant DMS-1114901 to GTH, the European Union through BioStruct-X Project 283570 and Horizon 2020 through grant iNEXT (INFRAIA-1-2014-2015, Proposal: 653706).S