Detection and Automated Scoring of Dicentric Chromosomes in Nonstimulated Lymphocyte Prematurely Condensed Chromosomes After Telomere and Centromere Staining

PurposeTo combine telomere and centromere (TC) staining of premature chromosome condensation (PCC) fusions to identify dicentrics, centric rings, and acentric chromosomes, making possible the realization of a dose–response curve and automation of the process.Methods and MaterialsBlood samples from healthy donors were exposed to 60Co irradiation at varying doses up to 8 Gy, followed by a repair period of 8 hours. Premature chromosome condensation fusions were carried out, and TC staining using peptide nucleic acid probes was performed. Chromosomal aberration (CA) scoring was carried out manually and automatically using PCC-TCScore software, developed in our laboratory.ResultsWe successfully optimized the hybridization conditions and image capture parameters, to increase the sensitivity and effectiveness of CA scoring. Dicentrics, centric rings, and acentric chromosomes were rapidly and accurately detected, leading to a linear-quadratic dose–response curve by manual scoring at up to 8 Gy. Using PCC-TCScore software for automatic scoring, we were able to detect 95% of dicentrics and centric rings.ConclusionThe introduction of TC staining to the PCC fusion technique has made possible the rapid scoring of unstable CAs, including dicentrics, with a level of accuracy and ease not previously possible. This new approach can be used for biological dosimetry in radiation emergency medicine, where the rapid and accurate detection of dicentrics is a high priority using automated scoring. Because there is no culture time, this new approach can also be used for the follow-up of patients treated by genotoxic therapy, creating the possibility to perform the estimation of induced chromosomal aberrations immediately after the blood draw

Micelle-smectic phase coexistence: Origin of the maximum swelling of a mixed lamellar phase

We use the water corner of the model ternary system water/DDAB (a cationic double tailed surfactant)/LS (an uncharged single chain glycolipid) to investigate maximum swelling of a charged lamellar phase. The phase diagram is determined by visual observation and small angle neutron scattering. Upon the addition of the glycolipid in this mixed lamellar structure, a decrease of the maximum swelling is observed. A transition from vesicles to micelles is also evidenced in the dilute coexisting phase. By calculating explicitly the osmotic pressure in the micellar phase and in the lamellar phase through the Poisson-Boltzmann equation, we demonstrate that equilibrium of the osmotic pressure in the two phases and depletion of the lamellar phase by the micellar phase explain quantitatively the decrease of maximum swelling observed when the LS to DDAB ratio increases

Increased genomic alteration complexity and telomere shortening in B-CLL cells resistant to radiation-induced apoptosis

B-cell chronic lymphocytic leukemia (B-CLL) results in an accumulation of mature CD5+/CD23+ B cells due to an uncharacterized defect in apoptotic cell death. B-CLL is not characterized by a unique recurrent genomic alteration but rather by genomic instability giving rise frequently to several chromosomal aberrations. Besides we reported that ∼15% of B-CLL patients present malignant B-cells resistant to irradiation-induced apoptosis, contrary to ∼85% of patients and normal human lymphocytes. Telomere length shortening is observed in radioresistant B-CLL cells. Using fluorescence in situ hybridization (FISH) and multicolour FISH, we tested whether specific chromosomal aberrations might be associated with the radioresistance of a subset of B-CLL cells and whether they are correlated with telomere shortening. In a cohort of 30 B-CLL patients, all of the radioresistant B-CLL cell samples exhibited homozygous or heterozygous deletion of 13q14.3 in contrast to 52% of the radiosensitive samples. In addition to the 13q14.3 deletion, ten out of the 11 radioresistant B-cell samples had another clonal genomic alteration such as trisomy 12, deletion 17p13.1, mutation of the p53 gene or translocations in contrast to only three out of 19 radiosensitive samples. Telomere fusions and non-reciprocal translocations, hallmarks of telomere dysfunction, are not increased in radioresistant B-CLL cells. These findings suggest (i) that the 13q14.3 deletion accompanied by another chromosomal aberration is associated with radioresistance of B-CLL cells and (ii) that telomere shortening is not causative of increased clonal chromosomal aberrations in radioresistant B-CLL cells

Consequence of single break near telomere in human cells

Low dose radiation effects remain an open question, mainly due to the lack of models that permit to address it. Using human cells tagged with a plasmid integrated on end of a marker chromosome, we demonstrated that a single break near the telomere has dramatic consequences on the genome stability, inducing the panel of chromosomal instability detected in cancer cells including gene amplification and large chromosome imbalances (LOH up to 100 Mb), Sabatier et al, Mol Cancer Res 2005. One of the consequences of the chromosome instability induced by a single telomere loss is to let emerge cells with high proliferative advantages. An increase of the tumorigenicity of these cells is observed when transplanted on nude mice. Such cell models would be very informative to characterize the role of the modulation of DNA damage repair (NHEJ and HR) in the consequence of single chromosome break. First we tested the feasibility of using replicative small interfering vectors for efficient and long term silencing in human cells. We silenced NHEJ protein -DNAPK, LigIV, and XRCC4- and HR protein -Rad51/Rad52/Rad54- in Hela cells. Our data suggested that the major effect of NHEJ on telomere maintenance is indirect via misrepaired breaks in subtelomeric and telomeric whereas the major effect of HR on telomere maintenance is direct via telomere replication and telomere recombination. We are working on the invalidation of DNA repair genes in telomere tagged models. Having demonstrated that a single break near telomere could have such consequences we now address the question of radiation-induced chromosome breaks that will occur at low doses (25 mGy -> 1DSB per cell), stuying clonogenic survival and the emergence of resistant clones due to chromosome breakage

Comparison of Individual Radiosensitivity to γ-Rays and Carbon Ions.

International audienceCarbon ions are an up-and-coming ion species, currently being used in charged particle radiotherapy. As it is well established that there are considerable interindividual differences in radiosensitivity in the general population that can significantly influence clinical outcomes of radiotherapy, we evaluate the degree of these differences in the context of carbon ion therapy compared with conventional radiotherapy. In this study, we evaluate individual radiosensitivity following exposure to carbon-13 ions or γ-rays in peripheral blood lymphocytes of healthy individuals based on the frequency of ionizing radiation (IR)-induced DNA double strand breaks (DSBs) that was either misrepaired or left unrepaired to form chromosomal aberrations (CAs) (simply referred to here as DSBs for brevity). Levels of DSBs were estimated from the scoring of CAs visualized with telomere/centromere-fluorescence in situ hybridization (TC-FISH). We examine radiosensitivity at the dose of 2 Gy, a routinely administered dose during fractionated radiotherapy, and we determined that a wide range of DSBs were induced by the given dose among healthy individuals, with highly radiosensitive individuals harboring more IR-induced breaks in the genome than radioresistant individuals following exposure to the same dose. Furthermore, we determined the relative effectiveness of carbon irradiation in comparison to γ-irradiation in the induction of DSBs at each studied dose (isodose effect), a quality we term "relative dose effect" (RDE). This ratio is advantageous, as it allows for simple comparison of dose-response curves. At 2 Gy, carbon irradiation was three times more effective in inducing DSBs compared with γ-irradiation (RDE of 3); these results were confirmed using a second cytogenetic technique, multicolor-FISH. We also analyze radiosensitivity at other doses (0.2-15 Gy), to represent hypo- and hyperfractionation doses and determined that RDE is dose dependent: high ratios at low doses, and approaching 1 at high doses. These results could have clinical implications as IR-induced DNA damage and the ensuing CAs and genomic instability can have significant cellular consequences that could potentially have profound implications for long-term human health after IR exposure, such as the emergence of secondary cancers and other pathobiological conditions after radiotherapy

Characterization of telomere maintenance and chromosomal integrity in human fibroblasts and keratinocytes

In spite of the significant progress in cancer biology and modern radiation biology in the last decades, it is still an open question how exactly different human cell types respond to ionizing radiation. The majority of studies have been done on two types of human cells: skin fibroblasts and peripheral blood lymphocytes. The main reason for this is the relatively easy access to these cells in individuals. Other cell types, such as normal epithelial cells, have been used on a much smaller scale and consequently there is relatively little data on how normal epithelial cells respond to ionizing radiation in comparison with fibroblasts. The study of epithelial cells is highly relevant for the elucidation of radiation-induced carcinogenesis as the majority of human solid cancers will develop from epithelial cells. The work presented here is the characterization of karyotypes and telomere loss in non-irradiated fibroblast and keratinocyte populations from the same donor during the different stages of proliferation in vitro and results for the response of fibroblasts to five doses of γ-radiation spanning the range 10-2000 mGy with respect to cell proliferation and kinetics of γH2AX foci. An increase in the incidence of telomere loss with doublings in vitro was noted; however, it followed different kinetics in the two cell types. An increase in clonal and de novo chromosomal aberrations was observed in both fibroblasts and keratinocytes as proliferation progressed into senescent phases. Clonal emergence from the second senescent plateau characterized by progressive instability of the genome and different kinetics of telomere maintenance, was observed in keratinocytes. Fibroblasts and keratinocytes showed different kinetics of γH2AX foci, with apparent higher initial induction in fibroblasts, but faster disappearance of the induced foci. The radiosensitivity and the transmission of radiation-induced damage could largely differ according to the cell type status at irradiation (fibroblasts/ keratinocytes, young/ senescent) and their role in the long-term occurrence of radiation-induced tumours needs to be characterized

Preparation and characterization – including in situ Small Angle X-Ray Scattering – of gas chromatographic capillary columns with mesoporous silica thin films as stationary phases

International audienceIn this study the preparation of various mesoporous silica thin films as new stationary phases for gas chromatography (GC) columns is presented. The synthesis was performed inside capillaries via a sol gel process using a templating route. The as-obtained columns were found to be highly efficient for the fast separation of light n-alkanes (C1-C5) mixture; these columns exhibiting a normalized retention 30 times higher than that of a commercially available silica column used as standard. A particular effort was directed towards the characterization of the stationary phase physical features: thin film inspection by Scanning Electron Microscopy and, for the first time to our knowledge, in situ SAXS characterization using synchrotron radiation were used to study the impact of the pore-network structuration on the GC properties. Worm-like, cubic and hexagonal phases were observed for specific preparation conditions. Unexpectedly, the normalized retention relative to film thickness appeared higher with disordering of the pores network. (C) 2015 Elsevier B.V. All rights reserved

Effects of carbon monoxide, carbon dioxide, and methane on nickel/yttria-stabilized zirconia-based solid oxide fuel cells performance for direct coupling with a gasifier

International audienceThis work focuses on the role of CO and CO2 usually present in syngas generated by biomass gasification when they are flowed in a Ni-YSZ-based SOPC. Polarization, power versus intensity and electrochemical impedance spectroscopy measurements are performed and the theoretical aspect is enhanced with comparison with values of the Nernst potentials of the two H2O/H-2 and CO2/CO couples. The CO as electrochemical reactant is clearly highlighted, and also compared to H2. Indeed, power density values of 460 mW cm(-2) and 540 mW cm(-2) are obtained at 750 C when the cell is fueled with a 50% CO-50% N-2 mixture and a 50% H-2-50% N-2 mixture respectively, and the use of CO leads to higher power density values when the percentage of the fuel in the Fuel/N-2 mixture is lower than 27%, the use of higher amounts of CO inducing a large increase of the total resistance of the cell. The impact on the efficiency of the cell of the presence of CH4 is also examined using electrochemical measurements. Finally, power density values of 502 mW cm(-2) is obtained at 750 degrees C when the cell is fueled with a 50% N2-20% CO-17% H-2-10% CO2-3% CH4 mixture which appears to a be a realistic composition corresponding to the exhaust of the gasifier. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved

Lead Exposure Induces Telomere Instability in Human Cells

<div><p>Lead (Pb) is an important environmental contaminant due to its widespread use over many centuries. While it affects primarily every organ system of the body, the most pernicious effects of Pb are on the central nervous system leading to cognitive and behavioral modification. Despite decades of research, the mechanisms responsible for Pb toxicity remain poorly understood. Recent work has suggested that Pb exposure may have consequences on chromosomal integrity as it was shown that Pb exposure leads to the generation of γH2Ax foci, a well-established biomarker for DNA double stranded break (DSB formation). As the chromosomal localization of γH2Ax foci plays an important role in determining the molecular mechanism responsible for their formation, we examined the localization of Pb-induced foci with respect to telomeres. Indeed, short or dysfunctional telomeres (uncapped or damaged telomeres) may be recognized as DSB by the DNA repair machinery, leading to “telomere-Induced Foci” (TIFs). In the current study, we show that while Pb exposure did not increase intra-chromosomal foci, it significantly induced TIFs, leading in some cases, to chromosomal abnormalities including telomere loss. The evidence suggests that these chromosomal abnormalities are likely due to perturbation of telomere replication, in particular on the lagging DNA strand. We propose a mechanism by which Pb exposure leads to the loss of telomere maintenance. As numerous studies have demonstrated a role for telomere maintenance in brain development and tissue homeostasis, our results suggest a possible mechanism for lead-induced neurotoxicity.</p></div