Publications

Filter by Keyword

Filter by Application

Filter by Product/Solution


Tuberculosis (Edinburgh, Scotland), 125, 101993
September, 2020

Machine-assisted interpretation of auramine stains substantially increases through-put and sensitivity of microscopic tuberculosis diagnosis.

Horvath, L., Hänselmann, S., Mannsperger, H., Degenhardt, S., Last, K., Zimmermann, S., Burckhardt, I.

Of all bacterial infectious diseases, infection by Mycobacterium tuberculosis poses one of the highest morbidity and mortality burdens on humans throughout the world. Due to its speed and cost-efficiency, manual microscopy of auramine-stained sputum smears remains a crucial first-line detection method. However, it puts considerable workload on laboratory staff and suffers from a limited sensitivity. Here we validate a scanning and analysis system that combines fully-automated microscopy with deep-learning based image analysis. After automated scanning, the system summarizes diagnosis-relevant image information and presents it to the microbiologist in order to assist diagnosis. We tested the benefit of the automated scanning and analysis system using 531 slides from routine workflow, of which 56 were from culture positive specimen. Assistance by the scanning and analysis system allowed for a higher sensitivity (40/56 positive slides detected) than manual microscopy (34/56 positive slides detected), while greatly reducing manual slide-analysis time from a recommended 5-15 min to around 10 s per slide on average.

Digital object identifier (DOI): 10.1016/j.tube.2020.101993

International journal of radiation biology, 96, 214--219
February, 2020

An alternative approach for the induction of premature chromosome condensation in human peripheral blood lymphocytes using mitotic Akodon cells.

Selvan Gnana Sekaran, Tamizh, Ricoul, Michelle, Brochard, Patricia, Herate, Cecile, Sabatier, Laure

The premature chromosome condensation (PCC) technique is used to study exposure to external radiation through the determination of chromosome fragments observed in interphase cells. The presence of large telomeric signals in CHO cells interferes with the detection of PCC fragments and the identification of dicentric chromosomes. We present an improved method for the fusion of G0-lymphocytes with mitotic cells (few chromosomes and weakly-staining telomeric sequences) to induce PCC in combination with rapid quantification of dicentric chromosomes and centric rings as an alternative to the classical CHO cell fusion technique. Whole blood from three healthy volunteers was γ-irradiated with 0, 2, or 4 Gy. Following a 24 h incubation post-exposure at 37 °C, chromosome spreads of isolated lymphocytes were prepared by standard PCC procedures using mitotic cells. The percentage of scorable fusions, measured by telomere/centromere (T/C) staining, for -induced PCC was higher than that for CHO-induced PCC, irrespective of radiation exposure. Importantly, both techniques gave the same result for biodosimetry evaluation. The mitotic cell-induced PCC fusion assay, in combination with the scoring of dicentric chromosomes and rings by T/C staining of G0-lymphocytes is a suitable alternative for fast and reliable dose estimation after accidental radiation exposure.

Digital object identifier (DOI): 10.1080/09553002.2019.1625493

Scientific reports, 10, 2899
February, 2020

A High Throughput Approach to Reconstruct Partial-Body and Neutron Radiation Exposures on an Individual Basis.

Shuryak, Igor, Turner, Helen C., Perrier, Jay R., Cunha, Lydia, Canadell, Monica Pujol, Durrani, Mohammad H., Harken, Andrew, Bertucci, Antonella, Taveras, Maria, Garty, Guy, Brenner, David J.

Biodosimetry-based individualized reconstruction of complex irradiation scenarios (partial-body shielding and/or neutron + photon mixtures) can improve treatment decisions after mass-casualty radiation-related incidents. We used a high-throughput micronucleus assay with automated scanning and imaging software on ex-vivo irradiated human lymphocytes to: a) reconstruct partial-body and/or neutron exposure, and b) estimate separately the photon and neutron doses in a mixed exposure. The mechanistic background is that, compared with total-body photon irradiations, neutrons produce more heavily-damaged lymphocytes with multiple micronuclei/binucleated cell, whereas partial-body exposures produce fewer such lymphocytes. To utilize these differences for biodosimetry, we developed metrics that describe micronuclei distributions in binucleated cells and serve as predictors in machine learning or parametric analyses of the following scenarios: (A) Homogeneous gamma-irradiation, mimicking total-body exposures, vs. mixtures of irradiated blood with unirradiated blood, mimicking partial-body exposures. (B) X rays vs. various neutron + photon mixtures. The results showed high accuracies of scenario and dose reconstructions. Specifically, receiver operating characteristic curve areas (AUC) for sample classification by exposure type reached 0.931 and 0.916 in scenarios A and B, respectively. R for actual vs. reconstructed doses in these scenarios reached 0.87 and 0.77, respectively. These encouraging findings demonstrate a proof-of-principle for the proposed approach of high-throughput reconstruction of clinically-relevant complex radiation exposure scenarios.

Digital object identifier (DOI): 10.1038/s41598-020-59695-9

International journal of molecular sciences, 21
February, 2020

Micronucleus Assay: The State of Art, and Future Directions.

Sommer, Sylwester, Buraczewska, Iwona, Kruszewski, Marcin

During almost 40 years of use, the micronucleus assay (MN) has become one of the most popular methods to assess genotoxicity of different chemical and physical factors, including ionizing radiation-induced DNA damage. In this minireview, we focus on the position of MN among the other genotoxicity tests, its usefulness in different applications and visibility by international organizations, such as International Atomic Energy Agency, Organization for Economic Co-operation and Development and International Organization for Standardization. In addition, the mechanism of micronuclei formation is discussed. Finally, foreseen directions of the MN development are pointed, such as automation, buccal cells MN and chromothripsis phenomenon.

Digital object identifier (DOI): 10.3390/ijms21041534

Mutation research, 849, 503086
January, 2020

Analysis of historical negative control group data from the rat in vivo micronucleus assay.

Lovell, D. P., Fellows, M., Saul, J., Whitwell, J., Custer, L., Dertinger, S., Escobar, P., Fiedler, R., Hemmann, U., Kenny, J., Smith, R., van der Leede, B. M., Zeller, A.

A database of micronuclei counts for historical negative control data from rat in vivo micronuclei tests performed in 10 different laboratories was established. Data were available from over 4000 negative control rats from 10 laboratories. The mean frequency of micronucleated cells (MN)/1000 cells ranged from 0.44 to 2.22, a 5-fold range. Overall there were no major sex or strain differences in frequency, although there were some small but statistically significant differences within laboratories. There was appreciable variability between experiments compared with variability within experiments in some laboratories. No specific factor was identified which could explain this variability although it was noted that many different vehicles were used in the experiments. It is hoped that these data will help laboratories beginning studies with the rat micronucleus assay and those involved in the assessment of micronucleus assay results.

Digital object identifier (DOI): 10.1016/j.mrgentox.2019.503086

Mutation research, 849, 503141
January, 2020

Premature chromosome condensation assay to study influence of high-level natural radiation on the initial DNA double strand break repair in human G0 lymphocytes.

Vivek Kumar, P. R., Karuppasamy, C. V., Ramachandran, E. N., Anil Kumar, V., Jaikrishan, G., Das, Birajalaxmi

The inherent capacity of individuals to efficiently repair ionizing radiation induced DNA double strand breaks (DSBs) may be inherited, however, it is influenced by several epigenetic and environmental factors. A pilot study tested whether chronic low dose natural radiation exposure influences the rejoining of initial DNA DSBs induced by a 2 Gy γ-irradiation in 22 individuals from high (>1.5 mGy/year) and normal (≤1.5 mGy/year) level natural radiation areas (H&NLNRA) of Kerala. Rejoining of DSBs (during 1 h at 37 °C, immediately after irradiation) was evaluated at the chromosome level in the presence and absence of wortmannin (a potent inhibitor of DSB repair in normal human cells) using a cell fusion-induced premature chromosome condensation (PCC) assay. The PCC assay quantitates DSBs in the form of excess chromosome fragments in human G lymphocytes without the requirement for cell division. A quantitative difference was observed in the early rejoining of DNA DSBs between individuals from HLNRA and NLNRA, with HLNRA individuals showing a higher (P = 0.05) mean initial repair ratio. The results indicate an influence of chronic low dose natural radiation on initial DNA DSB repair in inhabitants of HLNRA of the Kerala coast.

Digital object identifier (DOI): 10.1016/j.mrgentox.2020.503141

Cancer letters, 469, 355--366
January, 2020

PD-L1+ aneuploid circulating tumor endothelial cells (CTECs) exhibit resistance to the checkpoint blockade immunotherapy in advanced NSCLC patients.

Zhang, Lina, Zhang, Xinyong, Liu, Yanxia, Zhang, Tongmei, Wang, Ziyu, Gu, Meng, Li, Yilin, Wang, Daisy Dandan, Li, Weiying, Lin, Peter Ping

Sustained angiogenesis and increased PD-L1 expression on endothelial and carcinoma cells contribute toward fostering an immunosuppressive microenvironment suitable for tumor growth. PD-L1 CTCs were reported to associate with poor prognosis in NSCLC patients. However, whether or not aneuploid circulating tumor endothelial cells (CTECs) express PD-L1, then serve as a surrogate biomarker to evaluate immunotherapy efficacy remains unknown. In this study, a novel SE-iFISH strategy was established to comprehensively quantify and characterize a full spectrum of aneuploid CTCs and CTECs in advanced NSCLC patients subjected to second-line anti-PD-1 (nivolumab) immunotherapy. In situ co-detection of diverse subtypes of aneuploid CTCs and CTECs expressing PD-L1 and Vimentin was performed. The present clinical study demonstrated that significant amounts of PD-L1 aneuploid CTCs and CTECs could be detected in histopathologic hPD-L1 patients. In contrast to decreased PD-L1 CTCs, the number of multiploid PD-L1 CTECs (≥tetrasomy 8) undergoing post-therapeutic karyotype shifting increased in patients along with tumor progression following anti-PD-1 treatment. Progressive disease (PD) lung cancer patients possessing multiploid PD-L1 CTECs had a significantly shorter PFS compared to those without PD-L1 CTECs. In carcinoma patients, aneuploid CTCs and CTECs may exhibit a functional interplay with respect to tumor angiogenesis, progression, metastasis, and response to immunotherapy.

Digital object identifier (DOI): 10.1016/j.canlet.2019.10.041

BMC plant biology, 19, 183
May, 2019

Development and characterisation of interspecific hybrid lines with genome-wide introgressions from Triticum timopheevii in a hexaploid wheat background.

Devi, Urmila, Grewal, Surbhi, Yang, Cai-Yun, Hubbart-Edwards, Stella, Scholefield, Duncan, Ashling, Stephen, Burridge, Amanda, King, Ian P, King, Julie

Triticum timopheevii (2n = 4x = 28; A<sup>t</sup>A<sup>t</sup>GG), is an important source for new genetic variation for wheat improvement with genes for potential disease resistance and salt tolerance. By generating a range of interspecific hybrid lines, T. timopheevii can contribute to wheat's narrow gene-pool and be practically utilised in wheat breeding programmes. Previous studies that have generated such introgression lines between wheat and its wild relatives have been unable to use high-throughput methods to detect the presence of wild relative segments in such lines. A whole genome introgression approach, exploiting homoeologous recombination in the absence of the Ph1 locus, has resulted in the transfer of different chromosome segments from both the A and G genomes of T. timopheevii into wheat. These introgressions have been detected and characterised using single nucleotide polymorphism (SNP) markers present on a high-throughput Axiom® Genotyping Array. The analysis of these interspecific hybrid lines has resulted in the detection of 276 putative unique introgressions from T. timopheevii, thereby allowing the generation of a genetic map of T. timopheevii containing 1582 SNP markers, spread across 14 linkage groups representing each of the seven chromosomes of the A and G genomes of T. timopheevii. The genotyping of the hybrid lines was validated through fluorescence in situ hybridisation (FISH). Comparative analysis of the genetic map of T. timopheevii and the physical map of the hexaploid wheat genome showed that synteny between the two species is highly conserved at the macro-level and confirmed the presence of inter- and intra-genomic translocations within the A and G genomes of T. timopheevii that have been previously only detected through cytological techniques. In this work, we report a set of SNP markers present on a high-throughput genotyping array, able to detect the presence of T. timopheevii in a hexaploid wheat background making it a potentially valuable tool for marker assisted selection (MAS) in wheat pre-breeding programs. These valuable resources of high-density molecular markers and wheat-T. timopheevii hybrid lines will greatly enhance the work being undertaken for wheat improvement through wild relative introgressions.

Digital object identifier (DOI): 10.1186/s12870-019-1785-z

Biochemical and biophysical research communications, 511, 658–664
April, 2019

Distinctive Krebs cycle remodeling in iPSC-derived neural and mesenchymal stem cells.

Benlamara, Sarah, Aubry, Laetitia, Fabregue, Julien, Bénit, Paule, Rustin, Pierre, Rak, Malgorzata

Mitochondria play a vital role in proliferation and differentiation and their remodeling in the course of differentiation is related to the variable energy and metabolic needs of the cell. In this work, we show a distinctive mitochondrial remodeling in human induced pluripotent stem cells differentiated into neural or mesenchymal progenitors. While leading to upregulation of the citrate synthase-α-ketoglutarate dehydrogenase segment of the Krebs cycle and increased respiratory chain activities and respiration in the mesenchymal stem cells, the remodeling in the neural stem cells resulted in downregulation of α-ketoglutarate dehydrogenase, upregulation of isocitrate dehydrogenase 2 and the accumulation of α-ketoglutarate. The distinct, lineage-specific changes indicate an involvement of these Krebs cycle enzymes in cell differentiation.

Digital object identifier (DOI): 10.1016/j.bbrc.2019.02.033

Microbiological research, 221, 28–35
April, 2019

Muscodor brasiliensis sp. nov. produces volatile organic compounds with activity against Penicillium digitatum.

Pena, Lorena C, Jungklaus, Gustavo H, Savi, Daiani C, Ferreira-Maba, Lisandra, Servienski, André, Maia, Beatriz H L N S, Annies, Vinicius, Galli-Terasawa, Lygia V, Glienke, Chirlei, Kava, Vanessa

Endophytic fungi belonging to Muscodor genus are considered as promising alternatives to be used in biological control due to the production of volatile organic compounds (VOCs). The strains LGMF1255 and LGMF1256 were isolated from the medicinal plant Schinus terebinthifolius and, by morphological data and phylogenetic analysis, identified as belonging to Muscodor genus. Phylogenetic analysis suggests that strain LGMF1256 is a new species, which is herein introduced as Muscodor brasiliensis sp. nov. The analysis of VOCs production revealed that compounds phenylethyl alcohol, α-curcumene, and E (β) farnesene until now has been reported only from M. brasiliensis, data that supports the classification of strain LGMF1256 as a new species. M. brasiliensis completely inhibited the phytopathogen P. digitatum in vitro. We also evaluated the ability of VOCs from LGMF1256 to inhibit the development of green mold symptoms by inoculation of P. digitatum in detached oranges. M. brasiliensis reduced the severity of diseases in 77%, and showed potential to be used for fruits storage and transportation to prevent the green mold symptoms development, eventually reducing the use of fungicides.

Digital object identifier (DOI): 10.1016/j.micres.2019.01.002

Journal of cell science, 132
March, 2019

Synthetic lethality of cytolytic HSV-1 in cancer cells with ATRX and PML deficiency.

Han, Mingqi, Napier, Christine E, Frölich, Sonja, Teber, Erdahl, Wong, Ted, Noble, Jane R, Choi, Eugene H Y, Everett, Roger D, Cesare, Anthony J, Reddel, Roger R

Cancers that utilize the alternative lengthening of telomeres (ALT) mechanism for telomere maintenance are often difficult to treat and have a poor prognosis. They are also commonly deficient for expression of ATRX protein, a repressor of ALT activity, and a component of promyelocytic leukemia nuclear bodies (PML NBs) that are required for intrinsic immunity to various viruses. Here, we asked whether ATRX deficiency creates a vulnerability in ALT cancer cells that could be exploited for therapeutic purposes. We showed in a range of cell types that a mutant herpes simplex virus type 1 (HSV-1) lacking ICP0, a protein that degrades PML NB components including ATRX, was ten- to one thousand-fold more effective in infecting ATRX-deficient cells than wild-type ATRX-expressing cells. Infection of co-cultured primary and ATRX-deficient cancer cells revealed that mutant HSV-1 selectively killed ATRX-deficient cells. Sensitivity to mutant HSV-1 infection also correlated inversely with PML protein levels, and we showed that ATRX upregulates PML expression at both the transcriptional and post-transcriptional levels. These data provide a basis for predicting, based on ATRX or PML levels, which tumors will respond to a selective oncolytic herpesvirus.

Digital object identifier (DOI): 10.1242/jcs.222349

European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences, 129, 181–189
March, 2019

Multi- and unilamellar liposomal encapsulation of ciprofloxacin as ways to modify its phototoxicity and photodegradation.

Zgadzaj, A, Giebułtowicz, J, Gubernator, J, Podbielska, M, Sommer, S, Zaremba-Czogalla, M, Nałęcz-Jaweckia, G

Liposomes are vesicular preparations that improve bioavailability of many pharmaceuticals, used even in ocular therapy. In addition, it is well documented that vesicular carriers could affect the photodegradation of molecules encapsulated inside, which is especially important for drugs that may exhibit phototoxicity when they are applied topically on sensitive light-exposed tissues. In this study, we investigated the effect of ciprofloxacin encapsulation into liposomes on its photodegradation, phototoxicity and photogenotoxicity in vitro at the concentration ranges applied in ophthalmology. We tested two variants of liposomes: large unilamellar vesicles (LUV) and multilamellar vesicles (MLV) in comparison to antibiotic solutions without phospholipids (CPX). On the basis of our research, the kinetics of ciprofloxacin photolysis was the fastest in formulations with vesicles with low drug-to-lipid ratio. Depending on vesicles type (drug-to-lipid ratio, MLV or LUV) and time of irradiation different degradants were produced. We proposed structures of the novel ciprofloxacin photolysis products characteristic for vesicles. We did not notice any photoprotective effect of application of ciprofloxacin encapsulation into liposomes, but it significantly affected the photodegradation product profile of the drug and the Photo-Irritation-Factor of the vesicular preparations. In the MTT and micronucleus assays impact of encapsulation was not as clearly visible.

Digital object identifier (DOI): 10.1016/j.ejps.2019.01.006

Journal of applied genetics, 60, 63–70
February, 2019

Structural and copy number chromosome abnormalities in canine cutaneous mast cell tumours.

Vozdova, Miluse, Kubickova, Svatava, Cernohorska, Halina, Fröhlich, Jan, Fictum, Petr, Rubes, Jiri

Mast cell tumours (MCTs) are the most common skin tumours in dogs. Their clinical behaviour is variable and their aetiology remains largely unknown. We performed a metaphase fluorescence in situ hybridisation (FISH) with whole chromosome painting probes, and interphase FISH with BAC probes for 14 cancer-related genes to reveal clonal structural chromosome rearrangements and copy number variants (CNVs) in canine cutaneous MCTs. The metaphase FISH performed in three MCTs revealed several clonal monosomies and trisomies and two different chromosome rearrangements. No centric fusions were detected. The interphase FISH showed a variety of low frequency CNVs for the individual cancer-related genes. The heterogeneous character of the detected abnormalities indicates increased chromosome instability in canine MCTs. The clonal gain of chromosome 11 was detected in 81% (13/16) of the MCTs. Further research is needed to evaluate the significance of this abnormality as prognostic factor for the survival time or recurrence risk assessments in canine cutaneous MCTs.

Digital object identifier (DOI): 10.1007/s13353-018-0471-4

Cell death & disease, 10, 186
February, 2019

Type 3 inositol 1,4,5-trisphosphate receptor has antiapoptotic and proliferative role in cancer cells.

Rezuchova, Ingeborg, Hudecova, Sona, Soltysova, Andrea, Matuskova, Miroslava, Durinikova, Erika, Chovancova, Barbora, Zuzcak, Michal, Cihova, Marina, Burikova, Monika, Penesova, Adela, Lencesova, Lubomira, Breza, Jan, Krizanova, Olga

Although the involvement of type 1 (IP R1) and type 2 (IP R2) inositol 1,4,5-trisphosphate receptors in apoptosis induction has been well documented in different cancer cells and tissues, the function of type 3 IP R (IP R3) is still elusive. Therefore, in this work we focused on the role of IP R3 in tumor cells in vitro and in vivo. We determined increased expression of this receptor in clear cell renal cell carcinoma compared to matched unaffected part of the kidney from the same patient. Thus, we hypothesized about different functions of IP R3 compared to IP R1 and IP R2 in tumor cells. Silencing of IP R1 prevented apoptosis induction in colorectal cancer DLD1 cells, ovarian cancer A2780 cells, and clear cell renal cell carcinoma RCC4 cells, compared to apoptosis in cells treated with scrambled siRNA. As expected, silencing of IP R3 and subsequent apoptosis induction resulted in increased levels of apoptosis in all these cells. Further, we prepared a DLD1/IP R3_del cell line using CRISPR/Cas9 gene editing method. These cells were injected into nude mice and tumor's volume was compared with tumors induced by DLD1 cells. Lower volume of tumors originated from DLD1/IP R3_del cells was observed after 12 days, compared to wild type DLD1 cells. Also, the migration of these cells was lesser compared to wild type DLD1 cells. Apoptosis under hypoxic conditions was more pronounced in DLD1/IP R3_del cells than in DLD1 cells. These results clearly show that IP R3 has proliferative and anti-apoptotic effect in tumor cells, on contrary to the pro-apoptotic effect of IP R1.

Digital object identifier (DOI): 10.1038/s41419-019-1433-4

The journal of pathology. Clinical research, 5, 63–78
January, 2019

Combined epithelial marker analysis of tumour budding in stage II colorectal cancer.

Slik, Khadija, Blom, Sami, Turkki, Riku, Välimäki, Katja, Kurki, Samu, Mustonen, Harri, Haglund, Caj, Carpén, Olli, Kallioniemi, Olli, Korkeila, Eija, Sundström, Jari, Pellinen, Teijo

Tumour budding predicts survival of stage II colorectal cancer (CRC) and has been suggested to be associated with epithelial-to-mesenchymal transition (EMT). However, the underlying molecular changes of tumour budding remain poorly understood. Here, we performed multiplex immunohistochemistry (mIHC) to phenotypically profile tumours using known EMT-associated markers: E-cadherin (adherence junctions), integrin β4 (ITGB4; basement membrane), ZO-1 (tight junctions), and pan-cytokeratin. A subpopulation of patients showed high ITGB4 expression in tumour buds, and this coincided with a switch of ITGB4 localisation from the basal membrane of intact epithelium to the cytoplasm of budding cells. Digital image analysis demonstrated that tumour budding with high ITGB4 expression in tissue microarray (TMA) cores correlated with tumour budding assessed from haematoxylin and eosin (H&amp;E) whole sections and independently predicted poor disease-specific survival in two independent stage II CRC cohorts (hazard ratio [HR] = 4.50 (95% confidence interval [CI] = 1.50-13.5), n = 232; HR = 3.52 (95% CI = 1.30-9.53), n = 72). Furthermore, digitally obtained ITGB4-high bud count in random TMA cores was better associated with survival outcome than visual tumour bud count in corresponding H&amp;E-stained samples. In summary, the mIHC-based phenotypic profiling of human tumour tissue shows strong potential for the molecular characterisation of tumour biology and for the discovery of novel prognostic biomarkers.

Digital object identifier (DOI): 10.1002/cjp2.119

Cells, 8
January, 2019

Oxidative Stress Induces Telomere Dysfunction and Senescence by Replication Fork Arrest.

Coluzzi, Elisa, Leone, Stefano, Sgura, Antonella

Oxidative DNA damage, particularly 8-oxoguanine, represents the most frequent DNA damage in human cells, especially at the telomeric level. The presence of oxidative lesions in the DNA can hinder the replication fork and is able to activate the DNA damage response. In this study, we wanted to understand the mechanisms by which oxidative damage causes telomere dysfunction and senescence in human primary fibroblasts. After acute oxidative stress at telomeres, our data demonstrated a reduction in TRF1 and TRF2, which are involved in proper telomere replication and T-loop formation, respectively. Furthermore, we observed a higher level of γH2AX with respect to 53BP1 at telomeres, suggesting a telomeric replication fork stall rather than double-strand breaks. To confirm this finding, we studied the replication of telomeres by Chromosome Orientation-FISH (CO-FISH). The data obtained show an increase in unreplicated telomeres after hydrogen peroxide treatment, corroborating the idea that the presence of 8-oxoG can induce replication fork arrest at telomeres. Lastly, we analyzed the H3K9me3 histone mark after oxidative stress at telomeres, and our results showed an increase of this marker, most likely inducing the heterochromatinization of telomeres. These results suggest that 8-oxoG is fundamental in oxidative stress-induced telomeric damage, principally causing replication fork arrest.

Digital object identifier (DOI): 10.3390/cells8010019

Annals of laboratory medicine, 39, 91–95
January, 2019

Dose Estimation Curves Following In Vitro X-ray Irradiation Using Blood From Four Healthy Korean Individuals.

Jang, Mi Ae, Han, Eun Ae, Lee, Jin Kyung, Cho, Kwang Hwan, Shin, Hee Bong, Lee, You Kyoung

Cytogenetic dosimetry is useful for evaluating the absorbed dose of ionizing radiation based on analysis of radiation-induced chromosomal aberrations. We created two types of dose-response calibration curves for dicentric chromosomes (DC) and translocations (TR) induced by X-ray irradiation, using an electron linear accelerator, which is the most frequently used medical device in radiotherapy. We irradiated samples from four healthy Korean individuals and compared the resultant curves between individuals. Aberration yields were studied in a total of 31,800 and 31,725 metaphases for DC and TR, respectively, obtained from 11 X-ray irradiation dose-points (0, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, and 5 Gy). The dose-response relationship followed a linear-quadratic equation, Y=C+αD+βD², with the coefficients C=0.0011 for DC and 0.0015 for TR, α =0.0119 for DC and 0.0048 for TR, and β=0.0617 for DC and 0.0237 for TR. Correlation coefficients between irradiation doses and chromosomal aberrations were 0.971 for DC and 0.6 for TR, indicating a very strong and a moderate correlation, respectively. This is the first study implementing cytogenetic dosimetry following exposure to ionizing X-radiation.

Digital object identifier (DOI): 10.3343/alm.2019.39.1.91

Chemosphere, 215, 703–709
January, 2019

Nanomaterials induce DNA-protein crosslink and DNA oxidation: A mechanistic study with RTG-2 fish cell line and Comet assay modifications.

Klingelfus, T, Disner, G R, Voigt, C L, Alle, L F, Cestari, M M, Leme, D M

Genotoxic effects of nanomaterials (NMs) have been controversially reported in literature, and the mode of action (MoA) via DNA oxidation is cited as the main damage caused by them. Evidence of nano-silver as a crosslinker has been previously reported by the present research team in an in vivo fish genotoxicity study. Thus, aiming to confirm the evidence about NMs as crosslinker agent, the present investigation elucidated the genotoxic potential of NMs and their genotoxic MoA through in vitro assay with RTG-2 cells line (rainbow trout gonadal) by exposure to nano-silver (PVP-coated) and nano-titanium. The types and levels of DNA damage were assessed by the Comet assay (standard alkaline, hOGG1-modified alkaline, and two crosslink-modified alkaline versions). It was demonstrated that the use of the standard alkaline Comet assay alone may inaccurately predict the genotoxicity of NMs since oxidative and crosslink DNA damages were also verified in RTG-2 cells when assessed by the modified versions of the alkaline protocol. More importantly, it was confirmed that both nano-silver and nano-titanium acted as DNA-protein crosslinkers through the Comet assay version with proteinase K. As both nano-silver and nano-titanium present a great risk to aquatic life, these findings reinforce the need of genotoxicity testing strategies that encompass the assessment of different types of DNA damage, in order to ensure an accurate prediction of the genotoxic potential of NMs.

Digital object identifier (DOI): 10.1016/j.chemosphere.2018.10.118

Cells, 8(7), 708
2019

X-rays Activate Telomeric Homologous Recombination Mediated Repair in Primary Cells

De Vitis, M., Berardinelli, F., Coluzzi, E, Marinaccio, J.and O’Sullivan, R.J.and Sgura, A.

Cancer cells need to acquire telomere maintenance mechanisms in order to counteract progressive telomere shortening due to multiple rounds of replication. Most human tumors maintain their telomeres expressing telomerase whereas the remaining 15%–20% utilize the alternative lengthening of telomeres (ALT) pathway. Previous studies have demonstrated that ionizing radiations (IR) are able to modulate telomere lengths and to transiently induce some of the ALT-pathway hallmarks in normal primary fibroblasts. In the present study, we investigated the telomere length modulation kinetics, telomeric DNA damage induction, and the principal hallmarks of ALT over a period of 13 days in X-ray-exposed primary cells. Our results show that X-ray-treated cells primarily display telomere shortening and telomeric damage caused by persistent IR-induced oxidative stress. After initial telomere erosion, we observed a telomere elongation that was associated to the transient activation of a homologous recombination (HR) based mechanism, sharing several features with the ALT pathway observed in cancer cells. Data indicate that telomeric damage activates telomeric HR-mediated repair in primary cells. The characterization of HR-mediated telomere repair in normal cells may contribute to the understanding of the ALT pathway and to the identification of novel strategies in the treatment of ALT-positive cancers.

Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 847, 503087
2019

Optimization and validation of automated dicentric chromosome analysis for radiological/nuclear triage applications

Ryana, Terri L., Escalonaa, Maria B., Smith, Tammy L., Albanese, Joseph, Iddinsa, Carol J., Balajee, Adayabalam S.

Dicentric Chromosome Assay (DCA) is the most preferred cytogenetic technique for absorbed radiation dose assessment in exposed humans. However, DCA is somewhat impractical for triage application owing to its labor intensive and time consuming nature. Although lymphocyte culture for 48 h <em>in vitro</em> is inevitable for DCA, manual scoring of dicentric chromosomes (DCs) requires an additional time of 24–48 h, making the overall turnaround time of 72–96 h for dose estimation. To accelerate the speed of DC analysis for dose estimation, an automated tool was optimized and validated for triage mode of scoring. Several image training files were created to improve the specificity of automated DC analysis algorithm. Accuracy and efficiency of the automated (unsupervised) DC scoring was compared with the semi-automated scoring that involved human verification and correction of DCs (elimination of false positives and inclusion of true positives). DC scoring was performed by both automated and semi-automated modes for different doses of X-rays and γ-rays (0 Gy–5 Gy). Biodoses estimated from the frequencies of DCs detected by both automated (unsupervised) and semi-automated (supervised) scoring modes were grossly similar to the actual delivered doses in the range of 0.5 to 3 Gy of low LET radiation. We suggest that the automated DC tool can be effectively used for large scale radiological/nuclear incidents where a rapid segregation is essential for prioritizing moderately or severely exposed humans to receive appropriate medical countermeasures.

Digital object identifier (DOI): https://doi.org/10.1016/j.mrgentox.2019.503087