Search Results (35)

The murine Alpha Mouse Liver 12 (AML12) cell line, established over four decades ago, is one of the most commonly used non-transformed hepatocyte models in basic and pre-clinical liver research. Despite its widespread use, a comprehensive and current molecular characterization has been lacking. In this study, we combined cytogenetics with high-resolution genomic technologies to establish a detailed genetic reference profile of AML12. Inverted DAPI banding and multicolor fluorescence in situ hybridization (m-FISH) revealed a complex yet stable, near-tetraploid karyotype featuring double X-chromosome deletions [del(X)(A3)×2], a recurrent derivative chromosome der(3)t(2;3)(A2;H4), biallelic deletions of 17D1, two dicentric chromosomes dic(X;17), and multiple whole-chromosome gains (e.g., +1, +6, +15, +19×4) and losses (e.g., −4, −12, −16, −18). Multicolor banding (mcb) further pinpointed cryptic inversions on chromosomes 7 and 11. Copy number imbalances were visualized as in silico array comparative genomic hybridization (aCGH)-style profiles inferred from these metaphase-based assays, and no independent array- or sequencing-based copy number variation (CNV) experiment was performed in this study. Short tandem repeat (STR) profiling created a unique 16-locus authentication barcode that unambiguously distinguishes AML12 from other murine cell lines in public databases. Bulk RNA sequencing (RNA-seq) further demonstrated a transcriptional profile in AML12 cells that is indicative of hepatocyte origin while also revealing partial de-differentiation and reduced expression of selected urea cycle, gluconeogenic, and xenobiotic-metabolizing transcripts, consistent with limited mature hepatocyte functions. These functional inferences are likely based on gene expression patterns rather than on direct physiological assays. In summary, our study provides (i) the first integrated cytogenetic, STR, and next-generation sequencing dataset for AML12, (ii) a practical authentication panel for routine laboratory use, and (iii) reference information that will enhance the interpretation, reproducibility, and translational relevance of future studies using this versatile hepatocyte model. Full article

Background: Chromosome 3p (chr3p) is frequently deleted in multiple cancers, indicating the presence of shared tumor suppressors. In aggressive uveal melanomas (UVM), this deletion often co-occurs with chr8q amplification (8q+), suggesting strong selection pressure during UVM evolution. Methods: To understand the pattern of genomic alterations mediated by chr3p deletion, we have developed an algorithm for detecting isochromosomes in 10,632 TCGA cancer patients. We further perform integrative genomics analysis to investigate how chr3p deletion could affect subsequent cancer genome evolution and synthetic lethality in UVM. Results: Analysis of genomic alterations in 33 different cancer types implicates the deletion or deleterious mutations of SET-domain-containing 2 (SETD2) at chr3p21 in significantly facilitating the formation of isochromosomes, thereby promoting genomic instability conducive to rapid cancer genome evolution. Fracturing of dicentric isochromosomes during cell division is pervasive and follows the dynamic fragmentation pattern of solids under impulse. In the most aggressive UVM subtype, chr3 deletion includes MITF, a master regulator of melanocyte survival and differentiation, and co-occurs with 8q+. We demonstrate that MITF is a master transcriptional regulator of GNAQ/GNA11 and associated synthetic-lethal genes in UVM. MITF maintains MAPK and calcium homeostasis in UVM, and its hemizygous deletion is thus accidental, likely creating an early crisis during oncogenesis. We further show that MITF, MYC, and GNAQ/GNA11 form coupled regulatory feedback loops in the melanocyte lineage, and MITF deletion in UVM creates acute dependency on MYC-mediated rescue via 8q+. The discovered feedback loops predict both overall and relapse-free patient survival within the most aggressive UVM subtype, explain sensitivity to therapeutic gene perturbations, and inform effective combinatorial therapies. Conclusions: SETD2 deletion potentiates isochromosome formation across diverse cancers. Combinatorial targeting of MITF together with a previously identified synthetic lethal gene may benefit UVM patients harboring both chr3 deletion and 8q+. Full article

Exposure to ionizing and non-ionizing radiation from environmental and clinical settings can significantly threaten genomic stability, especially when combined. This ex vivo study investigates the potential combined effects of gamma radiation and ultraviolet B (UVB) exposure on human peripheral blood mononuclear cells (PBMCs) from healthy donors by exposing whole blood and isolated PBMCs to 1 Gy of gamma rays, to an absolute dose of approximately 100 J/m² of UVB, or to their combination. Combined exposure resulted in significantly elevated γH2AX foci formation and chromosomal aberrations relative to individual stressors, with the most pronounced effects observed in isolated PBMCs. Notably, lymphocytes from some donors failed to proliferate after UVB or co-exposure. Based on our results, a predictive biophysical model derived from dicentric yield was developed to estimate the gamma-ray equivalent dose from co-exposure, indicating up to ~9% increase in lifetime cancer risk. Although this proof-of-concept study included only a small number of donors and focused on two endpoints (γH2AX and dicentric assays), it provides a controlled framework for investigating mechanisms of radiation-induced genomic instability. The results emphasize the importance of accounting for mixed radiation exposures in genotoxic risk assessment and radiation protection. Full article

The ozone layer in the Earth’s atmosphere filters solar radiation and limits the unwanted effects on humans. A depletion of this ozone shield would permit hazardous levels of UV solar radiation, especially in the UVB range, to bombard Earth’s surface, resulting in potentially significant effects on human health. The concern for these adverse effects intensifies if we consider that the UVB solar radiation is combined with secondary cosmic radiation (SCR) components, such as protons and muons, as well as terrestrial gamma rays. This research aims to delve into the intricate interplay between cosmic and solar radiation on earth at the cellular level, focusing on their synergistic effects on human cell biology. Through a multidisciplinary approach integrating radiobiology and physics, we aim to explore key aspects of biological responses, including cell viability, DNA damage, stress gene expression, and finally, genomic instability. To assess the impact of the combined exposure, normal i.e., non-malignant human cells (skin fibroblasts, keratinocytes, monocytes, and lymphocytes) were exposed to high-energy protons or gamma rays in combination with UVB. Cellular molecular and cytogenetic biomarkers of radiation exposure, such as DNA damage (γH2AΧ histone protein and dicentric chromosomes), as well as the expression pattern of various stress genes, were analyzed. In parallel, the MTS reduction and lactate dehydrogenase assays were used as indicators of cell viability, proliferation, and cytotoxicity. Results reveal remaining DNA damage for the co-exposed samples compared to samples exposed to only one type of radiation in all types of cells, accompanied by increased genomic instability and distinct stress gene expression patterns detected at 24–48 h post-exposure. Understanding the impact of combined radiation exposures is crucial for assessing the health risks posed to humans if the ozone layer is partially depleted, with structural and functional damages inflicted by combined cosmic and UVB exposure. Full article

The cell line Sarcoma 180, which is also called S-180 (or S180), was established about 110 years ago from a murine axillary sarcoma. It has been applied in >5000 studies but was never genetically characterized in detail; this study fills that gap. The cell line Sarcoma 180 was analyzed for its chromosomal constitution using molecular cytogenetic approaches, specifically murine multicolor banding (mcb). Additionally, array comparative genomic hybridization was performed to characterize copy number alterations. Sarcoma 180 has a near tetraploid karyotype without Y-chromosome material and only two X-chromosomes. The complex karyotype includes dicentrics and simple and complex rearrangements and shows a relatively high chromosomal instability. An in silico translation of the obtained results to the human genome indicated that Sarcoma 180 is suitable as a model for advanced human mesenchymal chondrosarcoma. Full article

Background: The Neuro-2a cell line, derived from a murine neuroblastoma (NB), was established as early as 1969 and originates from a transplantable tumor that arose spontaneously in an A/Jax male mouse in 1940. Since then, it has been applied in over 10,000 studies and is used by the World Organization for Animal Health for the routine diagnosis of rabies. Surprisingly, however, Neuro-2a has never been genetically characterized in detail; this study fills that gap. Methods: The Neuro-2a cell line and two of its derivatives, Neuro-2a TR-alpha and Neuro-2a TR-beta, were analyzed for their chromosomal constitution using molecular cytogenetic approaches. Array comparative genomic hybridization was performed to characterize copy number alterations. Results: Neuro-2A has a hyper-tetraploid karyotype with 70 to 97 chromosomes per cell, and the karyotypes of its two examined derivatives were quite similar. Neither of them had a Y-chromosome. The complex karyotype of Neuro-2a includes mitotically stable dicentres, neocentrics, and complex rearrangements resembling chromothripsis events. Although no amplification of euchromatin or oncogenes was detected, there are five derivative chromosomes with the amplification of centromere-near heterochromatic material and 1–5 additional derivatives consisting only of such material. Conclusions: Since satellite DNA amplification has recently been found in advanced human tumors, this finding may be the corresponding equivalent in mice. An in silico translation of the obtained results into the human genome indicated that Neuro-2A is suitable as a model for advanced human NB. Full article

The Dicentric Chromosome Assay (DCA) is widely used in biological dosimetry, where the number of dicentric chromosomes induced by ionizing radiation (IR) exposure is quantified to estimate the absorbed radiation dose an individual has received. Dicentric chromosome scoring is a laborious and time-consuming process which is performed manually in most cytogenetic biodosimetry laboratories. Further, dicentric chromosome scoring constitutes a bottleneck when several hundreds of samples need to be analyzed for dose estimation in the aftermath of large-scale radiological/nuclear incident(s). Recently, much interest has focused on automating dicentric chromosome scoring using Artificial Intelligence (AI) tools to reduce analysis time and improve the accuracy of dicentric chromosome detection. Our study aims to detect dicentric chromosomes in metaphase plate images using an ensemble of artificial neural network detectors suitable for datasets that present a low number of samples (in this work, only 50 images). In our approach, the input image is first processed by several operators, each producing a transformed image. Then, each transformed image is transferred to a specific detector trained with a training set processed by the same operator that transformed the image. Following this, the detectors provide their predictions about the detected chromosomes. Finally, all predictions are combined using a consensus function. Regarding the operators used, images were binarized separately applying Otsu and Spline techniques, while morphological opening and closing filters with different sizes were used to eliminate noise, isolate specific components, and enhance the structures of interest (chromosomes) within the image. Consensus-based decisions are typically more precise than those made by individual networks, as the consensus method can rectify certain misclassifications, assuming that individual network results are correct. The results indicate that our methodology worked satisfactorily in detecting a majority of chromosomes, with remarkable classification performance even with the low number of training samples utilized. AI-based dicentric chromosome detection will be beneficial for a rapid triage by improving the detection of dicentric chromosomes and thereby the dose prediction accuracy. Full article

The process of end-joining during nonhomologous repair of DNA double-strand breaks (DSBs) after radiation damage is considered. Experimental evidence has revealed that the dynamics of DSB ends exhibit subdiffusive motion rather than simple diffusion with rare directional movement. Traditional models often overlook the rare long-range directed motion. To address this limitation, we present a heterogeneous anomalous diffusion model consisting of subdiffusive fractional Brownian motion interchanged with short periods of long-range movement. Our model sheds light on the underlying mechanisms of heterogeneous diffusion in DSB repair and could be used to quantify the DSB dynamics on a time scale inaccessible to single particle tracking analysis. The model predicts that the long-range movement of DSB ends is responsible for the misrepair of DSBs in the form of dicentric chromosome lesions. Full article

Count data consists of both observed and unobserved events. The analysis of count data often encounters overdispersion, where traditional Poisson models may not be adequate. In this paper, we introduce a tractable one-parameter mixed Poisson distribution, which combines the Poisson distribution with the improved second-degree Lindley distribution. This distribution, called the Poisson-improved second-degree Lindley distribution, is capable of effectively modeling standard count data with overdispersion. However, if the frequency of the unobserved events is unknown, the proposed distribution cannot be directly used to describe the events. To address this limitation, we propose a modification by truncating the distribution to zero. This results in a tractable zero-truncated distribution that encompasses all types of dispersions. Due to the unknown frequency of unobserved events, the population size as a whole becomes unknown and requires estimation. To estimate the population size, we develop a Horvitz–Thompson-like estimator utilizing truncated distribution. Both the untruncated and truncated distributions exhibit desirable statistical properties. The estimators for both distributions, as well as the population size, are asymptotically unbiased and consistent. The current study demonstrates that both the truncated and untruncated distributions adequately explain the considered medical datasets, which are the number of dicentric chromosomes after being exposed to different doses of radiation and the number of positive Salmonella. Moreover, the proposed population size estimator yields reliable estimates. Full article

Dicentric chromosome assay (DCA) is one of the cytogenetic dosimetry methods where the absorbed dose is estimated by counting the number of dicentric chromosomes, which is a major radiation-induced change in DNA. However, DCA is a time-consuming task and requires technical expertise. In this study, a neural network was applied for automating the DCA. We used YOLOv5, a one-stage detection algorithm, to mitigate these limitations by automating the estimation of the number of dicentric chromosomes in chromosome metaphase images. YOLOv5 was pretrained on common object datasets. For training, 887 augmented chromosome images were used. We evaluated the model using validation and test datasets with 380 and 300 images, respectively. With pretrained parameters, the trained model detected chromosomes in the images with a maximum F1 score of 0.94 and a mean average precision (mAP) of 0.961. Conversely, when the model was randomly initialized, the training performance decreased, with a maximum F1 score and mAP of 0.82 and 0.873%, respectively. These results confirm that the model could effectively detect dicentric chromosomes in an image. Consequently, automatic DCA is expected to be conducted based on deep learning for object detection, requiring a relatively small amount of chromosome data for training using the pretrained network. Full article

Ionising radiation is an important form of treatment for human cancer; however, the side effects associated with oxidative damage caused by radiation compromise its effectiveness. This work aimed to quantify the major bioactive components of freeze-dried kiwifruit (KD) and strawberry (SD) extracts and assess their potential efficacy as radioprotective agents in human blood lymphocytes. Their possible genotoxic and cytotoxic effects were also evaluated. The study was conducted by pre-treating human lymphocytes with KD and SD (50, 400, and 800 µg/mL) before radiation at 2 Gy. The results showed that SD presented a higher antioxidant capacity (12.6 mmol Trolox equivalents/100 g db) and higher values of total phenolic compounds (2435 mg of gallic acid equivalents/100 g db), while KD had the highest vitamin C content (322 mg ascorbic acid/100 g db). Regarding phenolic compounds, pelargonidin-3-glucoside was the most abundant in SD (1439 mg/1000 g db) and quercetin-3-O-galactoside in KD (635 mg/1000 g db). None of the tested concentrations of both fruit extracts showed a genotoxic effect. SD (800 µg/mL) reduced the sister chromatid exchange frequency and mitotic index. The efficacy of KD (400 and 800 µg/mL) in lowering the dicentric chromosome frequency demonstrated its radioprotective activity. Full article

In the event of a radiological or nuclear accident, or when physical dosimetry is not available, the scoring of radiation-induced chromosomal aberrations in lymphocytes constitutes an essential tool for the estimation of the absorbed dose of the exposed individual and for effective triage. Cytogenetic biodosimetry employs different cytogenetic assays including the scoring of dicentrics, micronuclei, and translocations as well as analyses of induced premature chromosome condensation to define the frequency of chromosome aberrations. However, inherent challenges using these techniques include the considerable time span from sampling to result, the sensitivity and specificity of the various techniques, and the requirement of highly skilled personnel. Thus, techniques that obviate these challenges are needed. The introduction of telomere and centromere (TC) staining have successfully met these challenges and, in addition, greatly improved the efficiency of cytogenetic biodosimetry through the development of automated approaches, thus reducing the need for specialized personnel. Here, we review the role of the various cytogenetic dosimeters and their recent improvements in the management of populations exposed to genotoxic agents such as ionizing radiation. Finally, we discuss the emerging potentials to exploit these techniques in a wider spectrum of medical and biological applications, e.g., in cancer biology to identify prognostic biomarkers for the optimal triage and treatment of patients. Full article

Butterfly chromosomes are holocentric, i.e., lacking a localized centromere. Potentially, this can lead to rapid karyotypic evolution through chromosome fissions and fusions, since fragmented chromosomes retain kinetic activity, while fused chromosomes are not dicentric. However, the actual mechanisms of butterfly genome evolution are poorly understood. Here, we analyzed chromosome-scale genome assemblies to identify structural rearrangements between karyotypes of satyrine butterfly species. For the species pair Erebia ligea–Maniola jurtina, sharing the ancestral diploid karyotype 2n = 56 + ZW, we demonstrate a high level of chromosomal macrosynteny and nine inversions separating these species. We show that the formation of a karyotype with a low number of chromosomes (2n = 36 + ZW) in Erebia aethiops was based on ten fusions, including one autosome–sex chromosome fusion, resulting in a neo-Z chromosome. We also detected inversions on the Z sex chromosome that were differentially fixed between the species. We conclude that chromosomal evolution is dynamic in the satyrines, even in the lineage that preserves the ancestral chromosome number. We hypothesize that the exceptional role of Z chromosomes in speciation may be further enhanced by inversions and sex chromosome–autosome fusions. We argue that not only fusions/fissions but also inversions are drivers of the holocentromere-mediated mode of chromosomal speciation. Full article

Amongst the 460 karyotypes of Polyphagan Coleoptera that we studied, 50 (10.8%) were carriers of an X autosome rearrangement. In addition to mitotic metaphase analysis, the correct diagnosis was performed on meiotic cells, principally at the pachytene stage. The percentages of these inter-chromosomal rearrangements, principally fusions, varied in relation to the total diploid number of chromosomes: high (51%) below 19, null at 19, low (2.7%) at 20 (the ancestral and modal number), and slightly increasing from 7.1% to 16.7% from 22 to above 30. The involvement of the X in chromosome fusions appears to be more than seven-fold higher than expected for the average of the autosomes. Examples of karyotypes with X autosome rearrangements are shown, including insertion of the whole X in the autosome (ins(A;X)), which has never been reported before in animals. End-to-end fusions (Robertsonian translocations, terminal rearrangements, and pseudo-dicentrics) are the most frequent types of X autosome rearrangements. As in the 34 species with a 19,X formula, there was no trace of the Y chromosome in the 50 karyotypes with an X autosome rearrangement, which demonstrates the dispensability of this chromosome. In most instances, C-banded heterochromatin was present at the X autosome junction, which suggests that it insulates the gonosome from the autosome portions, whose genes are subjected to different levels of expression. Finally, it is proposed that the very preferential involvement of the X in inter-chromosome rearrangements is explained by: (1) the frequent acrocentric morphology of the X, thus the terminal position of constitutive heterochromatin, which can insulate the attached gonosomal and autosomal components; (2) the dispensability of the Y chromosome, which considerably minimizes the deleterious consequences of the heterozygous status in male meiosis, (3) following the rapid loss of the useless Y chromosome, the correct segregation of the X autosome–autosome trivalent, which ipso facto is ensured by a chiasma in its autosomal portion. Full article

Millions of people around the world are exposed to low doses of ionizing radiation from diagnostic computed tomography (CT) scans. Currently available data on the potential cancer risk after CT scans are contradictory and therefore demand further investigations. The aim of the current study was to obtain estimations of genome damage after CT scans in 42 non-cancer patients and to conduct a comparison of the results with 22 control subjects. The frequency of dicentric ring chromosomes and chromosome breaks was significantly increased in irradiated patients compared to the controls. The distribution of dicentrics among the cells demonstrated non-Poisson distribution that reflected non-uniform and partial-body radiation exposure. A fraction of patients followed Poisson distribution, which is typical for uniform whole-body exposures. Some patients demonstrated a level of dicentrics similar to the control subjects. The individual variations in the frequency and dicentric distribution suggested complex mechanisms of chromosome aberration induction and elimination that could be associated with individual radiosensitivity, as well as previous diagnostics that used ionizing radiation or the redistribution of small fractions of irradiated lymphocytes within the circulatory pull. In conclusion, CT scans may cause genome damage and possible increases in cancer risk. The introduction of a specific follow-up of such patients, especially in the case of repeated CT scans, is suggested. Full article