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eLife, 6
February, 2017

Epigenetic regulation of lateralized fetal spinal gene expression underlies hemispheric asymmetries.

Ocklenburg, Sebastian, Schmitz, Judith, Moinfar, Zahra, Moser, Dirk, Klose, Rena, Lor, Stephanie, Kunz, Georg, Tegenthoff, Martin, Faustmann, Pedro, Francks, Clyde, Epplen, Jörg T, Kumsta, Robert, Güntürkün, Onur

Lateralization is a fundamental principle of nervous system organization but its molecular determinants are mostly unknown. In humans, asymmetric gene expression in the fetal cortex has been suggested as the molecular basis of handedness. However, human fetuses already show considerable asymmetries in arm movements before the motor cortex is functionally linked to the spinal cord, making it more likely that spinal gene expression asymmetries form the molecular basis of handedness. We analyzed genome-wide mRNA expression and DNA methylation in cervical and anterior thoracal spinal cord segments of five human fetuses and show development-dependent gene expression asymmetries. These gene expression asymmetries were epigenetically regulated by miRNA expression asymmetries in the TGF-β signaling pathway and lateralized methylation of CpG islands. Our findings suggest that molecular mechanisms for epigenetic regulation within the spinal cord constitute the starting point for handedness, implying a fundamental shift in our understanding of the ontogenesis of hemispheric asymmetries in humans.

Digital object identifier (DOI): 10.7554/eLife.22784

International journal of radiation biology, 93, 36–47
January, 2017

RENEB intercomparison exercises analyzing micronuclei (Cytokinesis-block Micronucleus Assay).

Depuydt, Julie, Baeyens, Ans, Barnard, Stephen, Beinke, Christina, Benedek, Anett, Beukes, Philip, Buraczewska, Iwona, Darroudi, Firouz, De Sanctis, Stefania, Dominguez, Inmaculada, Monteiro Gil, Octávia, Hadjidekova, Valeria, Kis, Enikö, Kulka, Ulrike, Lista, Florigio, Lumniczky, Katalin, M'kacher, Radhia, Moquet, Jayne, Obreja, Doina, Oestreicher, Ursula, Pajic, Jelena, Pastor, Nuria, Popova, Ljubomira, Regalbuto, Elisa, Ricoul, Michelle, Sabatier, Laure, Slabbert, Jacobus, Sommer, Sylwester, Testa, Antonella, Thierens, Hubert, Wojcik, Andrzej, Vral, Anne

In the framework of the 'Realizing the European Network of Biodosimetry' (RENEB) project, two intercomparison exercises were conducted to assess the suitability of an optimized version of the cytokinesis-block micronucleus assay, and to evaluate the capacity of a large laboratory network performing biodosimetry for radiation emergency triages. Twelve European institutions participated in the first exercise, and four non-RENEB labs were added in the second one. Irradiated blood samples were shipped to participating labs, whose task was to culture these samples and provide a blind dose estimate. Micronucleus analysis was performed by automated, semi-automated and manual procedures. The dose estimates provided by network laboratories were in good agreement with true administered doses. The most accurate estimates were reported for low dose points (≤ 0.94 Gy). For higher dose points (≥ 2.7 Gy) a larger variation in estimates was observed, though in the second exercise the number of acceptable estimates increased satisfactorily. Higher accuracy was achieved with the semi-automated method. The results of the two exercises performed by our network demonstrate that the micronucleus assay is a useful tool for large-scale radiation emergencies, and can be successfully implemented within a large network of laboratories.

Digital object identifier (DOI): 10.1080/09553002.2016.1206231

International journal of radiation biology, 93, 48–57
January, 2017

Dose assessment intercomparisons within the RENEB network using G0-lymphocyte prematurely condensed chromosomes (PCC assay).

Terzoudi, Georgia I, Pantelias, Gabriel, Darroudi, Firouz, Barszczewska, Katarzyna, Buraczewska, Iwona, Depuydt, Julie, Georgieva, Dimka, Hadjidekova, Valeria, Hatzi, Vasiliki I, Karachristou, Ioanna, Karakosta, Maria, Meschini, Roberta, M'Kacher, Radhia, Montoro, Alegria, Palitti, Fabrizio, Pantelias, Antonio, Pepe, Gaetano, Ricoul, Michelle, Sabatier, Laure, Sebastià , Natividad, Sommer, Sylwester, Vral, Anne, Zafiropoulos, Demetre, Wojcik, Andrzej

Dose assessment intercomparisons within the RENEB network were performed for triage biodosimetry analyzing G0-lymphocyte PCC for harmonization, standardization and optimization of the PCC assay. Comparative analysis among different partners for dose assessment included shipment of PCC-slides and captured images to construct dose-response curves for up to Gy γ-rays. Accident simulation exercises were performed to assess the suitability of the PCC assay by detecting speed of analysis and minimum number of cells required for categorization of potentially exposed individuals. Calibration data based on Giemsa-stained fragments in excess of 46 PCC were obtained by different partners using galleries of PCC images for each dose-point. Mean values derived from all scores yielded a linear dose-response with approximately 4 excess-fragments/cell/Gy. To unify scoring criteria, exercises were carried out using coded PCC-slides and/or coded irradiated blood samples. Analysis of samples received 24 h post-exposure was successfully performed using Giemsa staining (1 excess-fragment/cell/Gy) or centromere/telomere FISH-staining for dicentrics. Dose assessments by RENEB partners using appropriate calibration curves were mostly in good agreement. The PCC assay is quick and reliable for whole- or partial-body triage biodosimetry by scoring excess-fragments or dicentrics in G0-lymphocytes. Particularly, analysis of Giemsa-stained excess PCC-fragments is simple, inexpensive and its automation could increase throughput and scoring objectivity of the PCC assay.

Digital object identifier (DOI): 10.1080/09553002.2016.1234725

International journal of radiation biology, 93, 58–64
January, 2017

The second gamma-H2AX assay inter-comparison exercise carried out in the framework of the European biodosimetry network (RENEB).

Moquet, Jayne, Barnard, Stephen, Staynova, Albena, Lindholm, Carita, Monteiro Gil, Octávia, Martins, Vanda, Rößler, Ute, Vral, Anne, Vandevoorde, Charlot, Wojewódzka, Maria, Rothkamm, Kai

Within the EU RENEB project, seven laboratories have taken part in training and harmonisation activities to strengthen triage gamma-H2AX-based radiation exposure assessment. This has culminated in a second triage biodosimetry exercise. Whole blood and separated lymphocyte samples were homogenously irradiated with (60)Co gamma rays at 0.5, 2.5 (blind samples), 0 and 2 Gy (reference samples). Following post-exposure incubations of 4 and 24 h, 16 samples were shipped on ice packs to each partner. The samples were stained and scored for gamma-H2AX foci, using manual and/or automated fluorescence microscope scoring strategies. Dose estimates were obtained and used to assign triage categories to the samples. Average dose estimates across all the laboratories correlated well with true doses. The most accurate assignment of triage category was achieved by manual scoring of the 4-h blood and lymphocyte samples. Only three samples out of a total of 46 were miscategorized in a way that could have adversely effected the clinical management of a radiation casualty. This inter-comparison exercise has demonstrated that following a recent acute radiation exposure, the gamma-H2AX assay could be a useful triage tool that can be successfully applied across a network of laboratories.

Digital object identifier (DOI): 10.1080/09553002.2016.1207822

International journal of radiation biology, 93, 99–109
January, 2017

Integration of new biological and physical retrospective dosimetry methods into EU emergency response plans - joint RENEB and EURADOS inter-laboratory comparisons.

Ainsbury, Elizabeth, Badie, Christophe, Barnard, Stephen, Manning, Grainne, Moquet, Jayne, Abend, Michael, Antunes, Ana Catarina, Barrios, Lleonard, Bassinet, Celine, Beinke, Christina, Bortolin, Emanuela, Bossin, Lily, Bricknell, Clare, Brzoska, Kamil, Buraczewska, Iwona, Castaño, Carlos Huertas, Čemusová, Zina, Christiansson, Maria, Cordero, Santiago Mateos, Cosler, Guillaume, Monaca, Sara Della, Desangles, François, Discher, Michael, Dominguez, Inmaculada, Doucha-Senf, Sven, Eakins, Jon, Fattibene, Paola, Filippi, Silvia, Frenzel, Monika, Georgieva, Dimka, Gregoire, Eric, Guogyte, Kamile, Hadjidekova, Valeria, Hadjiiska, Ljubomira, Hristova, Rositsa, Karakosta, Maria, Kis, Enikő, Kriehuber, Ralf, Lee, Jungil, Lloyd, David, Lumniczky, Katalin, Lyng, Fiona, Macaeva, Ellina, Majewski, Matthaeus, Vanda Martins, S, McKeever, Stephen W S, Meade, Aidan, Medipally, Dinesh, Meschini, Roberta, M'kacher, Radhia, Gil, Octávia Monteiro, Montero, Alegria, Moreno, Mercedes, Noditi, Mihaela, Oestreicher, Ursula, Oskamp, Dominik, Palitti, Fabrizio, Palma, Valentina, Pantelias, Gabriel, Pateux, Jerome, Patrono, Clarice, Pepe, Gaetano, Port, Matthias, Prieto, María Jesús, Quattrini, Maria Cristina, Quintens, Roel, Ricoul, Michelle, Roy, Laurence, Sabatier, Laure, Sebastià , Natividad, Sholom, Sergey, Sommer, Sylwester, Staynova, Albena, Strunz, Sonja, Terzoudi, Georgia, Testa, Antonella, Trompier, Francois, Valente, Marco, Hoey, Olivier Van, Veronese, Ivan, Wojcik, Andrzej, Woda, Clemens

RENEB, 'Realising the European Network of Biodosimetry and Physical Retrospective Dosimetry,' is a network for research and emergency response mutual assistance in biodosimetry within the EU. Within this extremely active network, a number of new dosimetry methods have recently been proposed or developed. There is a requirement to test and/or validate these candidate techniques and inter-comparison exercises are a well-established method for such validation. The authors present details of inter-comparisons of four such new methods: dicentric chromosome analysis including telomere and centromere staining; the gene expression assay carried out in whole blood; Raman spectroscopy on blood lymphocytes, and detection of radiation-induced thermoluminescent signals in glass screens taken from mobile phones. In general the results show good agreement between the laboratories and methods within the expected levels of uncertainty, and thus demonstrate that there is a lot of potential for each of the candidate techniques. Further work is required before the new methods can be included within the suite of reliable dosimetry methods for use by RENEB partners and others in routine and emergency response scenarios.

Digital object identifier (DOI): 10.1080/09553002.2016.1206233

Molecular cell
January, 2017

DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination.

Biehs, Ronja, Steinlage, Monika, Barton, Olivia, Juhász, Szilvia, Künzel, Julia, Spies, Julian, Shibata, Atsushi, Jeggo, Penny A, Löbrich, Markus

Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.

Digital object identifier (DOI): 10.1016/j.molcel.2016.12.016

Animal science journal = Nihon chikusan Gakkaiho, 88, 27–32
January, 2017

Comparative genomic hybridization in detection of DNA changes in canine lymphomas.

Drážovská, Monika, Šiviková, Katarína, Dianovský, Ján, Horňák, Miroslav

In this study, chromosomal imbalances in tumor tissues (lymphomas) and nucleotide changes in tumor suppressor TP53 were studied in a Bernese Mountain dog bitch and a cross breed bitch. Using comparative genomic hybridization, numerous chromosomal rearrangements were detected, which indicated the heterogeneity in tumor growth: in the cross breed bitch, a deletion on the chromosome 9, and duplications on chromosomes 5, 8 and 17 have been found. In the Bernese Mountain Dog bitch, losses on chromosomes 1, 5, 8, 12, 18, 22, 27, 29 and gains on chromosomes 1, 2, 9, 11, 15, 16, 18, 20, 23, 24, 25, 28, 29, 30, 34, 36, 37 and 38 were identified. With the sequencing of the TP53 gene, one silent mutation, transition A/G at position 138 in exon 5 was detected, without changing the amino acid.

Digital object identifier (DOI): 10.1111/asj.12582

Appl. Sci., 7, 330
2017

Large-Scale Permanent Slide Imaging and Image Analysis for Diatom Morphometrics

Michael Kloster, Oliver Esper, Gerhard Kauer, Bánk Beszteri

Light microscopy analysis of diatom frustules is widely used in basic and applied research, notably taxonomy, morphometrics, water quality monitoring and paleo-environmental studies. Although there is a need for automation in these applications, various developments in image processing and analysis methodology supporting these tasks have not become widespread in diatom-based analyses. We have addressed this issue by combining our automated diatom image analysis software SHERPA with a commercial slide-scanning microscope. The resulting workflow enables mass-analyses of a broad range of morphometric features from individual frustules mounted on permanent slides. Extensive automation and internal quality control of the results helps to minimize user intervention, but care was taken to allow the user to stay in control of the most critical steps (exact segmentation of valve outlines and selection of objects of interest) using interactive functions for reviewing and revising results. In this contribution, we describe our workflow and give an overview of factors critical for success, ranging from preparation and mounting through slide scanning and autofocus finding to final morphometric data extraction. To demonstrate the usability of our methods we finally provide an example application by analysing Fragilariopsis kerguelensis valves originating from a sediment core, which substantially extends the size range reported in the literature.

Digital object identifier (DOI): 10.3390

PloS one, 12, e0178877
2017

Depletion of ATP and glucose in advanced human atherosclerotic plaques.

Ekstrand, Matias, Widell, Emma, Hammar, Anna, Akyürek, Levent M, Johansson, Martin, Fagerberg, Björn, Bergström, Göran, Levin, Malin C, Fogelstrand, Per, Borén, Jan, Levin, Max

Severe hypoxia develops close to the necrotic core of advanced human atherosclerotic plaques, but the energy metabolic consequences of this hypoxia are not known. In animal models, plaque hypoxia is also associated with depletion of glucose and ATP. ATP depletion may impair healing of plaques and promote necrotic core expansion. To investigate if ATP depletion is present in human plaques, we analyzed the distribution of energy metabolites (ATP, glucose, glycogen and lactate) in intermediate and advanced human plaques. Snap frozen carotid endarterectomies from 6 symptomatic patients were analyzed. Each endarterectomy included a large plaque ranging from the common carotid artery (CCA) to the internal carotid artery (ICA). ATP, glucose, and glycogen concentrations were lower in advanced (ICA) compared to intermediate plaques (CCA), whereas lactate concentrations were higher. The lowest concentrations of ATP, glucose and glycogen were detected in the perinecrotic zone of advanced plaques. Our study demonstrates severe ATP depletion and glucose deficiency in the perinecrotic zone of human advanced atherosclerotic plaques. ATP depletion may impair healing of plaques and promote disease progression.

Digital object identifier (DOI): 10.1371/journal.pone.0178877

Nature Scientific Reports, 7(9789), 2-10
2017

Comprehensive in situ co-detection of aneuploid circulating endothelial and tumor cells

Peter Ping Lin, Olivier Gires, Daisy Dandan Wang, Linda, Li, Hongxia Wang

Conventional circulating tumor cell (CTC) detection strategies rely on cell surface marker EpCAM and intracellular cytokeratins (CKs) for isolation and identification, respectively. Application of such methods is considerably limited by inherent heterogeneous and dynamic expression or absence of EpCAM and/or CKs in CTCs. Here, we report a novel strategy, integrating antigen-independent subtraction enrichment and immunostaining-FISH (SE-iFISH), to detect a variety of aneuploid circulating rare cells (CRCs), including CTCs and circulating tumor endothelial cells (CECs). Enriched CRCs, maintained at high viability and suitable for primary tumor cell culture, are comprehensively characterized by in situ co-examination of chromosome aneuploidy by FISH and immunostaining of multiple biomarkers displayed in diverse fluorescence channels. We described and quantified for the first time the existence of individual aneuploid CD31+ CECs and co-existence of "fusion clusters" of endothelial-epithelial aneuploid tumor cells among enriched non-hematopoietic CRCs. Hence, SE-iFISH is feasible for efficient co-detection and in situ phenotypic and karyotypic characterization as well as quantification of various CRCs, allowing for their classification into diverse subtypes upon biomarker expression and chromosome ploidy. Enhanced SE-iFISH technology, assisted by the Metafer-iFISH automated CRC imaging system, provides a platform for the analysis of potential contributions of each subtype of CRCs to distinct clinical outcome.

Journal of applied toxicology : JAT
December, 2016

Genotoxic risk of ethyl-paraben could be related to telomere shortening.

Finot, F, Kaddour, A, Morat, L, Mouche, I, Zaguia, N, Cuceu, C, Souverville, D, Négrault, S, Cariou, O, Essahli, A, Prigent, N, Saul, J, Paillard, F, Heidingsfelder, L, Lafouge, P, Al Jawhari, M, Hempel, W M, El May, M, Colicchio, B, Dieterlen, A, Jeandidier, E, Sabatier, L, Clements, J, M'Kacher, R

<p>The ability of parabens to promote the appearance of multiple cancer hallmarks in breast epithelium cells provides grounds for regulatory review of the implication of the presence of parabens in human breast tissue. It is well documented that telomere dysfunction plays a significant role in the initiation of genomic instability during carcinogenesis in human breast cancer. In the present study, we evaluated the genotoxic effect of ethyl 4-hydroxybenzoate (ethyl-paraben), with and without metabolic activation (S9), in studies following OECD guidelines. We observed a significant increase in genotoxic damage using the Mouse Lymphoma Assay and in vitro micronucleus (MN) tests in the L5178Y cell line in the presence of S9 only after a short exposure. A high frequency of MN was observed in the TK6 cells after a short exposure (3 h) in the presence of S9 and a long exposure (26 h) without S9. We found significant increases in the MN frequency and induced chromosomal aberrations in the lymphocytes of only one donor after ethyl-paraben exposure in the presence of S9 after a short exposure. Cytogenetic characterization of the paraben-treated cells demonstrated telomere shortening associated with telomere loss and telomere deletions in L5178Y and TK6 cells and lymphocytes of the paraben sensitive-donor. In a control cohort of 68 human lymphocytes, telomere length and telomere aberrations were age-dependent and showed high inter-individual variation. This study is the first to link telomere shortening and the genotoxic effect of ethyl paraben in the presence of S9 and raises the possibility that telomere shortening may be a proxy for underlying inter-individual sensitivity to ethyl-paraben. Copyright © 2016 John Wiley &amp; Sons, Ltd.</p>

Digital object identifier (DOI): 10.1002/jat.3425

Oncotarget, 7, 75996–76005
November, 2016

The prognostic value of DNA damage level in peripheral blood lymphocytes of chemotherapy-naïve patients with germ cell cancer

Sestakova, Zuzana, Kalavska, Katarina, Hurbanova, Lenka, Jurkovicova, Dana, Gursky, Jan, Chovanec, Michal, Svetlovska, Daniela, Miskovska, Vera, Obertova, Jana, Palacka, Patrik, Rejlekova, Katarina, Sycova-Mila, Zuzana, Cingelova, Silvia, Spanik, Stanislav, Mardiak, Jozef, Chovanec, Miroslav, Mego, Michal

<p>Germ cell tumors (GCTs) are extraordinarily sensitive to cisplatin (CDDP)-based chemotherapy. DNA damage represents one of the most important factors contributing to toxic effects of CDDP-based chemotherapy. This study was aimed to evaluate the prognostic value of DNA damage level in peripheral blood lymphocytes (PBLs) from chemo-naïve GCT patients. PBLs isolated from 59 chemo-naïve GCT patients were included into this prospective study. DNA damage levels in PBLs were evaluated by the Comet assay and scored as percentage tail DNA by the Metafer-MetaCyte analyzing software. The mean ± SEM (standard error of the mean) of endogenous DNA damage level was 5.25 ± 0.64. Patients with DNA damage levels lower than mean had significantly better progression free survival (hazard ratio [HR] = 0.19, 95% CI (0.04 - 0.96), P = 0.01) and overall survival (HR = 0.00, 95% CI (0.00 - 0.0), P &lt; 0.001) compared to patients with DNA damage levels higher than mean. Moreover, there was significant correlation between the DNA damage level and presence of mediastinal lymph nodes metastases, IGCCCG (International Germ Cell Cancer Collaborative Group) risk group, and serum tumor markers level. These data suggest that DNA damage levels in PBLs of GCT patients may serve as an important prognostic marker early identifying patients with poor outcome.</p>

Digital object identifier (DOI): 10.18632/oncotarget.12515

Endocr Relat Cancer, 23(8), 635–650
August, 2016

Effect of low doses of estradiol and tamoxifen on breast cancer cell karyotypes.

Rondón-Lagos, Milena, Rangel, Nelson, Di Cantogno, Ludovica Verdun, Annaratone, Laura, Castellano, Isabella, Russo, Rosalia, Manetta, Tilde, Marchiò, Caterina, Sapino, Anna

Evidence supports a role of 17&-estradiol (E2) in carcinogenesis and the large majority of breast carcinomas are dependent on estrogen. The anti-estrogen tamoxifen (TAM) is widely used for both treatment and prevention of breast cancer; however, it is also carcinogenic in human uterus and rat liver, highlighting the profound complexity of its actions. The nature of E2- or TAM-induced chromosomal damage has been explored using relatively high concentrations of these agents, and only some numerical aberrations and chromosomal breaks have been analyzed. This study aimed to determine the effects of low doses of E2 and TAM (10(&8 )mol L(&1) and 10(&6 )mol L(&1) respectively) on karyotypes of MCF7, T47D, BT474, and SKBR3 breast cancer cells by comparing the results of conventional karyotyping and multi-FISH painting with cell proliferation. Estrogen receptor (ER)-positive (+) cells showed an increase in cell proliferation after E2 treatment (MCF7, T47D, and BT474) and a decrease after TAM treatment (MCF7 and T47D), whereas in ER& cells (SKBR3), no alterations in cell proliferation were observed, except for a small increase at 96 h. Karyotypes of both ER+ and ER& breast cancer cells increased in complexity after treatments with E2 and TAM leading to specific chromosomal abnormalities, some of which were consistent throughout the treatment duration. This genotoxic effect was higher in HER2+ cells. The ER&/HER2+ SKBR3 cells were found to be sensitive to TAM, exhibiting an increase in chromosomal aberrations. These in vitro results provide insights into the potential role of low doses of E2 and TAM in inducing chromosomal rearrangements in breast cancer cells.

Digital object identifier (DOI): 10.1530/ERC-16-0078

Mol Med Rep, 14(1), 103–110
July, 2016

A semi‑automated FISH‑based micronucleus‑centromere assay for biomonitoring of hospital workers exposed to low doses of ionizing radiation.

Vral, Anne, Decorte, Veerle, Depuydt, Julie, Wambersie, André, Thierens, Hubert

The aim of the present study was to perform cytogenetic analysis by means of a semi‑automated micronucleus‑centromere assay in lymphocytes from medical radiation workers. Two groups of workers receiving the highest occupational doses were selected: 10 nuclear medicine technicians and 10 interventional radiologists/cardiologists. Centromere‑negative micronucleus (MNCM‑) data, obtained from these two groups of medical radiation workers were compared with those obtained in matched controls. The blood samples of the matched controls were additionally used to construct a 'low‑dose' (0‑100 mGy) MNCM‑ dose‑response curve to evaluate the sensitivity and suitability of the micronucleus‑centromere assay as an 'effect' biomarker in medical surveillance programs. The physical dosimetry data of the 3 years preceding the blood sampling, based on single or double dosimetry practices, were collected for the interpretation of the micronucleus data. The in vitro radiation results showed that for small sized groups, semi‑automated scoring of MNCM‑ enables the detection of a dose of 50 mGy. The comparison of MNCM‑ yields in medical radiation workers and control individuals showed enhanced MNCM‑ scores in the medical radiation workers group (P=0.15). The highest MNCM‑ scores were obtained in the interventional radiologists/cardiologists group, and these scores were significantly higher compared with those obtained from the matched control group (P=0.05). The higher MNCM‑ scores observed in interventional radiologists/cardiologists compared with nuclear medicine technicians were not in agreement with the personal dosimetry records in both groups, which may point to the limitation of 'double dosimetry' procedures used in interventional radiology/cardiology. In conclusion, the data obtained in the present study supports the importance of cytogenetic analysis, in addition to physical dosimetry, as a routine biomonitoring method in medical radiation workers receiving the highest occupational radiation burdens.

Digital object identifier (DOI): 10.3892/mmr.2016.5265

Radiat Prot Dosimetry
July, 2016

A New Cytogenetic Biodosimetry Image Repository for the Dicentric Assay.

Romm, Horst, Beinke, Christina, Garcia, Omar, Di Giorgio, Marina, Gregoire, Eric, Livingston, Gordon, Lloyd, David, Martinez-Lopez, Wilner, Moquet, Jayne E., Sugarman, Stephen L., Wilkins, Ruth C., Ainsbury, Elizabeth A.

The BioDoseNet was founded by the World Health Organization as a global network of biodosimetry laboratories for building biodosimetry laboratory capacities in countries. The newly established BioDoseNet image repository is a databank of ~25 000 electronically captured images of metaphases from the dicentric assay, which have been previously analysed by international experts. The detailed scoring results and dose estimations have, in most cases, already been published. The compilation of these images into one image repository provides a valuable tool for training and research purposes in biological dosimetry. No special software is needed to view and score the image galleries. For those new to the dicentric assay, the BioDoseNet Image Repository provides an introduction to and training for the dicentric assay. It is an excellent instrument for intra-laboratory training purposes or inter-comparisons between laboratories, as recommended by the International Organization for Standardisation standards. In the event of a radiation accident, the repository can also increase the surge capacity and reduce the turnaround time for dose estimations. Finally, it provides a mechanism for the discussion of scoring discrepancies in difficult cases.

Digital object identifier (DOI): 10.1093/rpd/ncw158

Nucleic Acids Res
June, 2016

Chromosome thripsis by DNA double strand break clusters causes enhanced cell lethality, chromosomal translocations and 53BP1-recruitment.

Schipler, Agnes, Mladenova, Veronika, Soni, Aashish, Nikolov, Vladimir, Saha, Janapriya, Mladenov, Emil, Iliakis, George

Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs. Translocation-formation from DSB-clusters utilizes Parp1 activity, implicating alt-EJ in their formation. Immunofluorescence experiments show that single-DSBs and DSB-clusters uniformly provoke the formation of single γ-H2AX foci, suggesting similar activation of early DNA damage response (DDR). Live-cell imaging also shows similar single-focus recruitment of the early-response protein MDC1, to single-DSBs and DSB-clusters. Notably, the late DDR protein, 53BP1 shows in live-cell imaging strikingly stronger recruitment to DSB-clusters as compared to single-DSBs. This is the first report that chromatin thripsis, in the form of engineered DSB-clusters, compromises first-line DSB-repair pathways, allowing alt-EJ to function as rescuing-backup. DSB-cluster-formation is indirectly linked to the increased biological effectiveness of high ionization-density radiations, such as the alpha-particles emitted by radon gas or the heavy-ions utilized in cancer therapy. Our observations provide the first direct mechanistic explanation for this long-known effect.

Digital object identifier (DOI): 10.1093/nar/gkw487

J Radiat Res, 57(3), 220–226
June, 2016

Analysis of chromosome translocation frequency after a single CT scan in adults.

Abe, Yu, Miura, Tomisato, Yoshida, Mitsuaki A., Ujiie, Risa, Kurosu, Yumiko, Kato, Nagisa, Katafuchi, Atsushi, Tsuyama, Naohiro, Kawamura, Fumihiko, Ohba, Takashi, Inamasu, Tomoko, Shishido, Fumio, Noji, Hideyoshi, Ogawa, Kazuei, Yokouchi, Hiroshi, Kanazawa, Kenya, Ishida, Takashi, Muto, Satoshi, Ohsugi, Jun, Suzuki, Hiroyuki, Ishikawa, Tetsuo, Kamiya, Kenji, Sakai, Akira

We recently reported an increase in dicentric chromosome (DIC) formation after a single computed tomography (CT) scan (5.78-60.27 mSv: mean 24.24 mSv) and we recommended analysis of 2000 metaphase cells stained with Giemsa and centromere-FISH for dicentric chromosome assay (DCA) in cases of low-dose radiation exposure. In the present study, we analyzed the frequency of chromosome translocations using stored Carnoy's-fixed lymphocyte specimens from the previous study; these specimens were from 12 patients who were subject to chromosome painting of Chromosomes 1, 2 and 4. Chromosomes 1, 2 and 4 were analyzed in ∼5000 cells, which is equivalent to the whole-genome analysis of almost 2000 cells. The frequency of chromosome translocation was higher than the number of DICs formed, both before and after CT scanning. The frequency of chromosome translocations tended to be higher, but not significantly higher, in patients with a treatment history compared with patients without such a history. However, in contrast to the results for DIC formation, the frequency of translocations detected before and after the CT scan did not differ significantly. Therefore, analysis of chromosome translocation may not be a suitable assay for detecting chromosome aberrations in cases of low-dose radiation exposure from a CT scan. A significant increase in the frequency of chromosome translocations was not likely to be detected due to the high baseline before the CT scan; the high and variable frequency of translocations was probably due to multiple confounding factors in adults.

Digital object identifier (DOI): 10.1093/jrr/rrv090

Indian J Hematol Blood Transfus, 32(2), 154–161
June, 2016

Evaluation of ETV6/RUNX1 Fusion and Additional Abnormalities Involving ETV6 and/or RUNX1 Genes Using FISH Technique in Patients with Childhood Acute Lymphoblastic Leukemia.

Aydin, Cigdem, Cetin, Zafer, Manguoglu, Ayse Esra, Tayfun, Funda, Clark, Ozden Altiok, Kupesiz, Alphan, Akkaya, Bahar, Karauzum, Sibel Berker

<p>Childhood acute lymphoblastic leukemia (ALL) is the most common type of childhood leukemia. Specifically, ALL is a malignant disorder of the lymphoid progenitor cells, with a peak incidence among children aged 2-5 years. The t(12;21)(p13;q22) translocation occurs in 25 \% of childhood B cell precursor ALL. In this study, bone marrow samples were obtained from 165 patients with childhood ALL. We analyzed the t(12;21) translocation and other related abnormalities using the fluorescent in situ hybridization (FISH) technique with the ETV6(TEL)/RUNX1(AML1) ES dual color translocation probe. Conventional cytogenetic analyses were also performed. ETV6 and RUNX1 related chromosomal abnormalities were found in 42 (25.5 %) of the 165 patients with childhood ALL. Among these 42 patients, structural changes were detected in 33 (78.6 %) and numerical abnormalities in 9 (21.4 %). The frequency of FISH abnormalities in pediatric ALL cases were as follows: 8.5 % for t(12;21)(p13;q22) ETV6/RUNX1 fusion, 6.0 % for RUNX1 amplification, 3.0 % for tetrasomy/trisomy 21, 1.8 % for ETV6 deletion, 1.21 % for ETV6 deletion with RUNX1 amplification, 1.21 % for ETV6 amplification with RUNX1 amplification, 0.6 % for polyploidy, 0.6 % for RUNX1 deletion, and 0.6 % for diminished ETV6 signal. The most common structural abnormality was the t(12;21) translocation, followed by RUNX1 amplification and ETV6 deletion, while the most commonly observed numerical abnormality was trisomy 21.</p>

Digital object identifier (DOI): 10.1007/s12288-015-0557-7

Radiation research, 185, 658–667
June, 2016

Telomere Length in Aged Mayak PA Nuclear Workers Chronically Exposed to Internal Alpha and External Gamma Radiation.

Scherthan, Harry, Sotnik, Natalia, Peper, Michel, Schrock, Gerrit, Azizova, Tamara, Abend, Michael

<p>Telomeres consist of GC-rich DNA repeats and the "shelterin" protein complex that together protect chromosome ends from fusion and degradation. Telomeres shorten with age due to incomplete end replication and upon exposure to environmental and intrinsic stressors. Exposure to ionizing radiation is known to modulate telomere length. However, the response of telomere length in humans chronically exposed to radiation is poorly understood. Here, we studied relative telomere length (RTL) by IQ-FISH to leukocyte nuclei in a group of 100 workers from the plutonium production facility at the Mayak Production Association (PA) who were chronically exposed to alpha-emitting ((239)Pu) radiation and/or gamma (photon) radiation, and 51 local residents serving as controls, with a similar mean age of about 80 years. We applied generalized linear statistical models adjusted for age at biosampling and the second exposure type on a linear scale and observed an age-dependent telomere length reduction. In those individuals with the lowest exposure, a significant reduction of about 20% RTL was observed, both for external gamma radiation (≤1 Gy) and internal alpha radiation (≤0.05-0.1 Gy to the red bone marrow). In highly exposed individuals (&gt;0.1 Gy alpha, 1-1.5 Gy gamma), the RTL was similar to control. Stratification by gender revealed a significant (∼30%) telomere reduction in low-dose-exposed males, which was absent in females. While the gender differences in RTL may reflect different working conditions, lifestyle and/or telomere biology, absence of a dose response in the highly exposed individuals may reflect selection against cells with short telomeres or induction of telomere-protective effects. Our observations suggest that chronic systemic exposure to radiation leads to variable dose-dependent effects on telomere length.</p>

Digital object identifier (DOI): 10.1667/RR14271.1