Applicability of flow cytometry γH2AX assay in population studies: suitability of fresh and frozen whole blood samples

Phosphorylation of H2AX histone (γH2AX) represents an early event in the DNA damage response against double-strand breaks (DSB); hence, its measurement provides a surrogate biomarker of DSB. Recently, we reported initial steps in the standardization of γH2AX assay in peripheral blood leukocytes (PBL), addressing the possibility of using cryopreserved samples, and the need of phytohaemagglutinin (PHA) stimulation prior analysis (Toxicol Sci 2015, 144:406-13). Validating the use of whole blood samples as cell specimen for this assay would be particularly useful for human population studies. Hence, in the current study we determined for the first time the feasibility of whole blood samples, both fresh and frozen, to be used in the γH2AX assay, evaluated by flow cytometry, and the convenience of PHA stimulation. Freshly collected and cryopreserved whole blood samples were treated with bleomycin (BLM), actinomycin-D (Act-D) and mitomycin C (MMC); half of the samples were previously incubated with PHA. Results were compared with those from PBL. Negative responses in MMC treatments were probably due to the quiescence of unstimulated cells, or to the short treatment time in PHA stimulated cells. Fresh whole blood samples exhibited a more intense response to BLM and Act-D treatments in stimulated cells, probably due to DSB indirectly produced from other less relevant types of DNA damage. Results obtained in frozen whole blood samples indicate that PHA stimulation is not advisable. In conclusion, this study demonstrates that whole blood samples can be used to assess DSB-related genotoxicity by the flow cytometry γH2AX assay.This work was supported by Xunta de Galicia [ED431B 2019/02], Ministerio de Educación, Cultura y Deporte [BEAGAL18/00142 to V.V], and Deputación Provincial de A Coruña [to M.S.-F. and N.F.-B.]

Comparison between carbon dust produced in laboratory plasmas and in Tore Supra

12th International Congress on Plasma Physics, 25-29 October 2004, Nice (France)Laboratory experiments are proposed to understand the growth mechanisms of spherical carbonaceous dust observed in Tokamaks with inside wall elements in graphite materials. The sputtering process is used to form continuous carbon vapours. Their cooling in the plasmas gives rise to carbon clusters which size goes increasing with time. In the nanometer scale range, the obtained primary particles are spherical. They can also agglomerate in the plasma likely by coulomb attraction and form spherical aggregates of higher size. A comparison between the carbon structure of these dust grains and of some dust samples collected on the toroïdal pumped limiter surface of Tore Supra is also proposed. The differences are discussed

Exploring frailty-related biomarkers and potential influence of environmental factors

Recent evidence advocates that healthy ageing may be possible, with morbidity compressed to later years. One area of concern is the burden of environmentally induced disease in susceptible populations. Older adults are a well-recognized susceptible population due to the decline of immune defences and the burden of multiple chronic diseases. As a susceptible population, the burden of environmentally induced disease and lifestyle factors is an increasing concern. Frailty is an age-related syndrome expected to increase over the next decades given the observed demographic shift. This syndrome has been identified to be the most common condition leading to disability, institutionalization and death in the older adults and the risk factors associated with its development are yet to be clarified. The main aim of the present study is to investigate a relation between frailty status, biomarkers and environmental exposures

Suitability of salivary leucocytes to assess DNA repair ability in human biomonitoring studies by the challenge-comet assay

The challenge-comet assay is a simple but effective approach that provides a quantitative and functional determination of DNA repair ability, and allows to monitor the kinetics of repair process. Peripheral blood mononuclear cells (PBMC) are the cells most frequently employed in human biomonitoring studies using the challenge-comet assay, but having a validated alternative of non-invasive biomatrix would be highly convenient for certain population groups and circumstances. The objective of this study was to validate the use of salivary leucocytes in the challenge-comet assay. Leucocytes were isolated from saliva samples and challenged (either in fresh or after cryopreservation) with three genotoxic agents acting by different action mechanisms: bleomycin, methyl methanesulfonate, and ultraviolet radiation. Comet assay was performed just after treatment and at other three additional time points, in order to study repair kinetics. The results obtained demonstrated that saliva leucocytes were as suitable as PBMC for assessing DNA damage of different nature that was efficiently repaired over the evaluated time points, even after 5 months of cryopreservation (after a 24 h stimulation with PHA). Furthermore, a new parameter to determine the efficacy of the repair process, independent of the initial amount of damage induced, is proposed, and recommendations to perform the challenge-comet assay with salivary leucocytes depending on the type of DNA repair to be assessed are suggested. Validation studies are needed to verify whether the method is reproducible and results reliable and comparable among laboratories and studies. © 2022 The AuthorsFunding text 1: This work was funded by the Spanish Ministry of Science and Innovation : MCIN/AEI/10.13039/501100011033 (Grants PID2020-113788RB-I00 and PID2020-114908 GA-I00 ), NanoBioBarriers project (PTDC/MED-TOX/31162/2017), Xunta de Galicia (ED431B 2022/16), co-financed by the Operational Program for Competitiveness and Internationalization (POCI) through European Regional Development Funds ( FEDER / FNR ), Spanish Ministry of Education, Culture and Sport [ BEAGAL18/00142 to V.V.], and Spanish Ministry of Economy and Competitiveness , co-financed by the European Social Fund [ RYC-2015-18394 to L.L,-L,]. Funding for open access charge: Universidade da Coruña/CISUG. ; Funding text 2: This work was funded by the Spanish Ministry of Science and Innovation: MCIN/AEI/10.13039/501100011033 (Grants PID2020-113788RB-I00 and PID2020-114908 GA-I00), NanoBioBarriers project (PTDC/MED-TOX/31162/2017), Xunta de Galicia (ED431B 2022/16), co-financed by the Operational Program for Competitiveness and Internationalization (POCI) through European Regional Development Funds (FEDER/FNR), Spanish Ministry of Education, Culture and Sport [BEAGAL18/00142 to V.V.], and Spanish Ministry of Economy and Competitiveness, co-financed by the European Social Fund [RYC-2015-18394 to L.L,-L,]. Funding for open access charge: Universidade da Coruña/CISUG

Effects of Zinc Oxide Nanoparticle Exposure on Human Glial Cells and Zebrafish Embryos

Zinc oxide nanoparticles (ZnO NPs) are among the most widely used nanomaterials. They have multiple applications in cosmetics, textiles, paints, electronics and, recently, also in biomedicine. This extensive use of ZnO NPs notably increases the probability that both humans and wildlife are subjected to undesirable effects. Despite being among the most studied NPs from a toxicological point of view, much remains unknown about their ecotoxicological effects or how they may affect specific cell types, such as cells of the central nervous system. The main objective of this work was to investigate the effects of ZnO NPs on human glial cells and zebrafish embryo development and to explore the role of the released Zn2+ ions in these effects. The effects on cell viability on human A172 glial cells were assessed with an MTT assay and morphological analysis. The potential acute and developmental toxicity was assessed employing zebrafish (Danio rerio) embryos. To determine the role of Zn2+ ions in the in vitro and in vivo observed effects, we measured their release from ZnO NPs with flame atomic absorption spectrometry. Then, cells and zebrafish embryos were treated with a water-soluble salt (zinc sulfate) at concentrations that equal the number of Zn2+ ions released by the tested concentrations of ZnO NPs. Exposure to ZnO NPs induced morphological alterations and a significant decrease in cell viability depending on the concentration and duration of treatment, even after removing the overestimation due to NP interference. Although there were no signs of acute toxicity in zebrafish embryos, a decrease in hatching was detected after exposure to the highest ZnO NP concentrations tested. The ability of ZnO NPs to release Zn2+ ions into the medium in a concentration-dependent manner was confirmed. Zn2+ ions did not seem entirely responsible for the effects observed in the glial cells, but they were likely responsible for the decrease in zebrafish hatching rate. The results obtained in this work contribute to the knowledge of the toxicological potential of ZnO NPs.This research was funded by the Ministry of Science and Innovation: MCIN/AEI/10.13039/501100011033 (grant PID2020-114908GA-I00), Xunta de Galicia (ED431B 2022/16 and ED481A 2019/003 to A.A-G.), CICA-Disrupting Project 2021SEM-B2, and Ministry of Education, Culture and Sport (BEAGAL18/00142 to V.V.)

Toxoplasma gondii IgG Serointensity Is Positively Associated With Frailty

Background: Persistent inflammation related to aging (inflammaging) is exacerbated by chronic infections and contributes to frailty in older adults. We hypothesized associations between Toxoplasma gondii (T. gondii), a common parasite causing an oligosymptomatic unremitting infection, and frailty, and secondarily between T. gondii and previously reported markers of immune activation in frailty.Methods: We analyzed available demographic, social, and clinical data in Spanish and Portuguese older adults [N = 601; age: mean (SD) 77.3 (8.0); 61% women]. Plasma T. gondii immunoglobulin G (IgG) serointensity was measured with an enzyme-linked immunosorbent assay. The Fried criteria were used to define frailty status. Validated translations of Mini-Mental State Examination, Geriatric Depression Scale, and the Charlson Comorbidity Index were used to evaluate confounders. Previously analyzed biomarkers that were significantly associated with frailty in both prior reports and the current study, and also related to T. gondii serointensity, were further accounted for in multivariable logistic models with frailty as outcome.Results: In T. gondii-seropositives, there was a significant positive association between T. gondii IgG serointensity and frailty, accounting for age (p = .0002), and resisting adjustment for multiple successive confounders. Among biomarkers linked with frailty, kynurenine/tryptophan and soluble tumor necrosis factor receptor II were positively associated with T. gondii serointensity in seropositives (p < .05). Associations with other biomarkers were not significant.Conclusions: This first reported association between T. gondii and frailty is limited by a cross-sectional design and warrants replication. While certain biomarkers of inflammaging were associated with both T. gondii IgG serointensity and frailty, they did not fully mediate the T. gondii-frailty association.This work was supported in part by the Spanish Ministry of Science and Innovation: MCIN/AEI/10.13039/501100011033(grant PID2020-113788RB-I00); Xunta de Galicia (grant ED431B 2022/16); Ministry of Education, Culture and Sport (grant BEAGAL18/00142 to V.V.); and Ministry of Economy and Competitiveness, cofinanced by the European Social Fund (grant RYC-2015-18394 to L.L.-L.). Additionally supported, in part, by the University of Maryland School of Medicine Center for Research on Aging in Baltimore, Maryland; a Clinical Science Research & Development Service Merit Award, Office of Research and Development, U.S. Department of Veterans Affairs, Washington, District of Columbia (grant 1 I01 CX001310-01 to T.T.P.); a R01 grant from the National Institute on Aging, National Institutes of Health, Bethesda, Maryland (grant NIA R01 AG018859 to E.J.K.); and by the Military and Veteran Microbiome: Consortium for Research and Education in Aurora, Colorado (L.A.B., A.J.H., C.A.L., T.T.P.). The opinions expressed in the article belong to the authors and cannot be construed as official positions or opinions of the funders, including the U.S. Veterans Affairs Administration and the National Institutes of Health. Data collected and used for the analyses reported in this article are not available because the initial consent did not include this sharing and because other primary analyses have not been completed. Funding for open access charge: Universidade da Coruna/CISUG

Detailed spectral and morphological analysis of the shell type SNR RCW 86

Aims: We aim for an understanding of the morphological and spectral properties of the supernova remnant RCW~86 and for insights into the production mechanism leading to the RCW~86 very high-energy gamma-ray emission. Methods: We analyzed High Energy Spectroscopic System data that had increased sensitivity compared to the observations presented in the RCW~86 H.E.S.S. discovery publication. Studies of the morphological correlation between the 0.5-1~keV X-ray band, the 2-5~keV X-ray band, radio, and gamma-ray emissions have been performed as well as broadband modeling of the spectral energy distribution with two different emission models. Results:We present the first conclusive evidence that the TeV gamma-ray emission region is shell-like based on our morphological studies. The comparison with 2-5~keV X-ray data reveals a correlation with the 0.4-50~TeV gamma-ray emission.The spectrum of RCW~86 is best described by a power law with an exponential cutoff at $E_{cut}=(3.5\pm 1.2_{stat})$ TeV and a spectral index of $\Gamma$~$1.6\pm 0.2$. A static leptonic one-zone model adequately describes the measured spectral energy distribution of RCW~86, with the resultant total kinetic energy of the electrons above 1 GeV being equivalent to $\sim$0.1\% of the initial kinetic energy of a Type I a supernova explosion. When using a hadronic model, a magnetic field of $B$~100$\mu$G is needed to represent the measured data. Although this is comparable to formerly published estimates, a standard E$^{-2}$ spectrum for the proton distribution cannot describe the gamma-ray data. Instead, a spectral index of $\Gamma_p$~1.7 would be required, which implies that ~$7\times 10^{49}/n_{cm^{-3}}$erg has been transferred into high-energy protons with the effective density $n_{cm^{-3}}=n/ 1$ cm^-3. This is about 10\% of the kinetic energy of a typical Type Ia supernova under the assumption of a density of 1~cm^-3.Comment: accepted for publication by A&

The exceptionally powerful TeV gamma-ray emitters in the Large Magellanic Cloud

The Large Magellanic Cloud, a satellite galaxy of the Milky Way, has been observed with the High Energy Stereoscopic System (H.E.S.S.) above an energy of 100 billion electron volts for a deep exposure of 210 hours. Three sources of different types were detected: the pulsar wind nebula of the most energetic pulsar known N 157B, the radio-loud supernova remnant N 132D and the largest non-thermal X-ray shell - the superbubble 30 Dor C. The unique object SN 1987A is, surprisingly, not detected, which constrains the theoretical framework of particle acceleration in very young supernova remnants. These detections reveal the most energetic tip of a gamma-ray source population in an external galaxy, and provide via 30 Dor C the unambiguous detection of gamma-ray emission from a superbubble.Comment: Published in Science Magazine (Jan. 23, 2015). This ArXiv version has the supplementary online material incorporated as an appendix to the main pape

Characterizing the gamma-ray long-term variability of PKS 2155-304 with H.E.S.S. and Fermi-LAT

Studying the temporal variability of BL Lac objects at the highest energies provides unique insights into the extreme physical processes occurring in relativistic jets and in the vicinity of super-massive black holes. To this end, the long-term variability of the BL Lac object PKS 2155-304 is analyzed in the high (HE, 100 MeV 200 GeV) gamma-ray domain. Over the course of ~9 yr of H.E.S.S observations the VHE light curve in the quiescent state is consistent with a log-normal behavior. The VHE variability in this state is well described by flicker noise (power-spectral-density index {\ss}_VHE = 1.10 +0.10 -0.13) on time scales larger than one day. An analysis of 5.5 yr of HE Fermi LAT data gives consistent results ({\ss}_HE = 1.20 +0.21 -0.23, on time scales larger than 10 days) compatible with the VHE findings. The HE and VHE power spectral densities show a scale invariance across the probed time ranges. A direct linear correlation between the VHE and HE fluxes could neither be excluded nor firmly established. These long-term-variability properties are discussed and compared to the red noise behavior ({\ss} ~ 2) seen on shorter time scales during VHE-flaring states. The difference in power spectral noise behavior at VHE energies during quiescent and flaring states provides evidence that these states are influenced by different physical processes, while the compatibility of the HE and VHE long-term results is suggestive of a common physical link as it might be introduced by an underlying jet-disk connection.Comment: 11 pages, 16 figure

Detection of variable VHE gamma-ray emission from the extra-galactic gamma-ray binary LMC P3

Context. Recently, the high-energy (HE, 0.1-100 GeV) $\gamma$-ray emission from the object LMC P3 in the Large Magellanic Cloud (LMC) has been discovered to be modulated with a 10.3-day period, making it the first extra-galactic $\gamma$-ray binary. Aims. This work aims at the detection of very-high-energy (VHE, >100 GeV) $\gamma$-ray emission and the search for modulation of the VHE signal with the orbital period of the binary system. Methods. LMC P3 has been observed with the High Energy Stereoscopic System (H.E.S.S.); the acceptance-corrected exposure time is 100 h. The data set has been folded with the known orbital period of the system in order to test for variability of the emission. Energy spectra are obtained for the orbit-averaged data set, and for the orbital phase bin around the VHE maximum. Results. VHE $\gamma$-ray emission is detected with a statistical significance of 6.4 $\sigma$. The data clearly show variability which is phase-locked to the orbital period of the system. Periodicity cannot be deduced from the H.E.S.S. data set alone. The orbit-averaged luminosity in the $1-10$ TeV energy range is $(1.4 \pm 0.2) \times 10^{35}$ erg/s. A luminosity of $(5 \pm 1) \times 10^{35}$ erg/s is reached during 20% of the orbit. HE and VHE $\gamma$-ray emissions are anti-correlated. LMC P3 is the most luminous $\gamma$-ray binary known so far.Comment: 5 pages, 3 figures, 1 table, accepted for publication in A&