Metaphase Cells Research Articles

Study of the Toxic Manifestation of Etoposide the Concomitant Exposure of Acetyl Salicylic Acid As a Protector on the Example of C57Bl/6J Mice Line

The paper presents the results of evaluating the genetic and cytogenetic toxicity of etoposide, as well as the evaluation results of protective properties of aspirin against the toxicity of etoposide in C57Bl/6J mice. The cytogenetic activity and protective properties assessment was carried out using the chromosome aberration analysis in metaphase cells. Etoposide was tested at a concentration of 15 and 3 mg/kg and aspirin — at a concentration of 25 mg/kg of the animal. The results of metaphase analysis showed that etoposide exhibits cytogenetic toxicity in both doses, and aspirin exhibits protective properties against the toxicity of etoposide at a concentration of 15 mg/kg. The results of chromosome analysis regarding the protective properties of aspirin against etoposide at a concentration of 3 mg/kg are ambiguous and require further experiments.

Techniques and advances in mammalian chromosome identification

Cytogenetics, through the study of the karyotype, with its characteristics such as diploid number (2n), chromosomal morphology of autosomes (fundamental number – FN), and sexual system, constitutes an important tool that can help clarify the phylogenetic relationships and taxonomic status of several taxa, receiving the name cytotaxonomy. In addition to conventional staining, there are several classical techniques of chromosomal bandings, such as GTG, CBG, and Ag-NORs bandings, which help in species comparisons to search for homologies and understand chromosome structure, function, and behavior. Systematic and taxonomic problems, as well as chromosome structure and behavior, have been elucidated in some groups of mammals with these banding techniques. In this study, we present the procedures for direct chromosomal preparations from bone marrow and peripheral blood used in the field and laboratory to obtain suspended mitotic metaphase cells. We also describe the main techniques of conventional staining and chromosomal banding for analyzing karyotypes in mammals.

Radiation exposure determination in a secure, cloud-based online environment.

Rapid sample processing and interpretation of estimated exposures will be critical for triaging exposed individuals after a major radiation incident. The dicentric chromosome (DC) assay assesses absorbed radiation using metaphase cells from blood. The Automated Dicentric Chromosome Identifier and Dose Estimator System (ADCI) identifies DCs and determines radiation doses. This study aimed to broaden accessibility and speed of this system, while protecting data and software integrity. ADCI Online is a secure web-streaming platform accessible worldwide from local servers. Cloud-based systems containing data and software are separated until they are linked for radiation exposure estimation. Dose estimates are identical to ADCI on dedicated computer hardware. Image processing and selection, calibration curve generation, and dose estimation of 9 test samples completed in<2days. ADCI Online has the capacity to alleviate analytic bottlenecks in intermediate-to-large radiation incidents. Multiple cloned software instances configured on different cloud environments accelerated dose estimation to within clinically relevant time frames.

The implementation of artificial intelligence to the low-cost metaphase finder.

Biological dosimetry is used to estimate one's dose by biological phenomena. The most popular and 'gold standard' phenomenon is the appearance of dicentric chromosomes in metaphases. The metaphase finder is a tool for biological dosimetry that finds metaphase cells on glass slides. It consists of an automated microscope, auto-focus system, X-Y stage, camera and computer. It does the image analysis of the microscopic images of the glass slides and displays the positions of metaphase cells. In this paper, the author reported that the prototype of AI-implemented metaphase finder was combined with the microscope system by file sharing and image transfer program, and that the metaphase finder system's accuracy was compared with previous non-AI system, using the same samples.

Chromosome Detection in Metaphase Cell Images Using Morphological Priors.

Reliable chromosome detection in metaphase cell (MC) images can greatly alleviate the workload of cytogeneticists for karyotype analysis and the diagnosis of chromosomal disorders. However, it is still an extremely challenging task due to the complicated characteristics of chromosomes, e.g., dense distributions, arbitrary orientations, and various morphologies. In this paper, we propose a novel rotated-anchor-based detection framework, named DeepCHM, for fast and accurate chromosome detection in MC images. Our framework has three main innovations: 1) A deep saliency map representing chromosomal morphological features is learned end-to-end with semantic features. This not only enhances the feature representations for anchor classification and regression but also guides the anchor setting to significantly reduce redundant anchors. This accelerates the detection and improves the performance; 2) A hardness-aware loss weights the contribution of positive anchors, which effectively reinforces the model to identify hard chromosomes; 3) A model-driven sampling strategy addresses the anchor imbalance issue by adaptively selecting hard negative anchors for model training. In addition, a large-scale benchmark dataset with a total of 624 images and 27,763 chromosome instances was built for chromosome detection and segmentation. Extensive experimental results demonstrate that our method outperforms most state-of-the-art (SOTA) approaches and successfully handles chromosome detection, with an AP score of 93.53%. Code and dataset are available at: https://github.com/wangjuncongyu/DeepCHM.

Automated Analysis of Telomere Intensity from Quantitative Fluorescence In-Situ Hybridization using CellProfiler

Telomeres are noncoding DNA sequences that protect chromosome ends from degradation. Quantitative fluorescence in situ hybridization (Q-FISH) is the favored technique for measuring telomere length. It can be applied to individual chromosomes in metaphase cells. Much of the currently available software to analyze fluorescence signal intensity as a proxy for telomere length is only commercially available. This study sought to develop a pipeline for the automated analysis of telomere fluorescence intensities in metaphase Q-FISH using modules from the open-source, freely available CellProfiler software. Intensities telomere and centromere were stained with fluorophore-conjugated peptide nucleic acid probes then evaluated using our pipeline. Telomere and centromere intensities were measured in a total of 30 images, giving an average relative telomere intensity of 22.17 %. Overall, our study showed that the pipeline can automatically identify and measure fluorescence signals from telomeres and centromeres in approximately 36 sec/image. However, the pipeline requires high-quality metaphase chromosome images with minimal touching and overlapping of chromosomes. Nevertheless, CellProfiler provides a free and flexible platform for the automated analysis of Q-FISH images that can be easily optimized and shared between researchers

Morphological and Functional Characterization of Cow Oocytes for Assisted Reproduction Techniques

In in vitro production of embryos (IVP), oocytes from the ovaries of slaughterhouse animals are commonly used. However, the quality of these oocytes is highly variable due to unknown category and condition of the slaughtered females. As a result, a heterogeneous population of oocytes with varying capacities for maturation and supporting embryonic development is obtained. The aim of this study was to investigate the morphological and biological characteristics of ovaries and oocytes from slaughtered bovines in order to establish selection criteria for oocytes to be used in IVP. To achieve this, the ovaries were categorized based on the presence or absence of structures such as Corpus Luteum (CL), Dominant Follicle (DF), both (CL+DF), or none (NO). The number and diameter of the follicles were also determined and grouped into three categories: &lt;3 mm, 3-8 mm, or &gt;8 mm. The Cumulus-Oocyte Complexes (COCs) obtained through follicular aspiration were analyzed for several parameters. These included, oocyte coverage by the Cumulus Cells (CC), the number of layers of CC, compaction and thickness of the CC layers. In denuded oocytes, the area, perimeter and diameter of the ooplasm as well as the thickness of the Zona Pellucida (ZP) were measured. Additionally, staining with Hoechst 33342, propidium iodide, or Rhodamine 123 was performed to analyze nuclear and metaphase morphology, cell viability, and mitochondrial functionality, respectively. All these determinations were made on microphotographs captured using a camera attached to an inverted microscope and processed using the Fiji software. Statistical analysis was conducted using ANOVA and Bonferroni’s Test. The results revealed a predominance of ovaries with CL+DF, and in terms of follicular size, the &lt;3 mm category exhibited the highest frequency of follicles, followed by the 3-8 mm category. Approximately 87.83±6.51% of the COCs showed complete coverage by CC; with 69.66±7.86% of the CC exhibiting compaction and 21.92±7.86% being slightly relaxed. A correlation was observed between the variable of CC layers and CC diameter (quantitative), with 91.42% of COCs having maximum diameters. Approximately, 83.11±4.27% of the oocytes displayed homogenous ooplasm, with the following measurements: area 12±0.37µm2 , perimeter 387.5±10µm, diameter 123±1.1µm, and ZP thickness 12.2±0.17µm. Moreover, 97.61± 0.11% of the oocytes were found to be viable, with no alterations observed in nuclear morphology and 100% displayed mitochondrial and metaphase activity. In conclusion, the ovaries and their corresponding COCs obtained from slaughtered females exhibit characteristics indicative of good quality for use in in vitro procedures.

Terminal microdeletion of chromosome 18 in a Malaysian boy characterized with few features of typical 18q- deletion syndrome: a case report

BackgroundThe 18q- deletion syndrome is a rare congenital chromosomal disorder caused by a partial deletion of the long arm of chromosome 18. The diagnosis of a patient with this syndrome relies on the family medical history, physical examination, developmental assessment, and cytogenetic findings. However, the phenotype of patients with 18q- deletion syndrome can be highly variable, ranging from almost normal to severe malformations and intellectual disability, and normal cytogenetic findings are common, thus complicating the diagnosis. Interestingly, only few characteristic features of typical 18q- deletion syndrome were found in the patient, despite sharing the same critical region. To our knowledge, this is the first report of a Malaysian individual with 18q- terminal microdeletion diagnosed with microarray-based technology.Case presentationHere we report a 16-year-old Malaysian Chinese boy, a product of a non-consanguineous marriage, who presented with intellectual disability, facial dysmorphism, high arched palate, congenital talipes equinovarus (clubfoot), congenital scoliosis, congenital heart defect, and behavioral problems. A routine chromosome analysis on 20 metaphase cells showed a normal 46, XY G-banded karyotype. Array-based comparative genomic hybridization was performed using a commercially available 244 K 60-mer oligonucleotide microarray slide according to the manufacturer’s protocol. This platform allows genome-wide survey and molecular profiling of genomic aberrations with an average resolution of about 10 kB. In addition, multiplex ligation-dependent probe amplification analysis was carried out using SALSA MLPA kit P320 Telomere-13 to confirm the array-based comparative genomic hybridization finding. Array-based comparative genomic hybridization analysis revealed a 7.3 MB terminal deletion involving chromosome band 18q22.3-qter. This finding was confirmed by multiplex ligation-dependent probe amplification, where a deletion of ten probes mapping to the 18q22.3-q23 region was detected, and further multiplex ligation-dependent probe amplification analysis on his parents showed the deletion to be de novo.ConclusionThe findings from this study expand the phenotypic spectrum of the 18q- deletion syndrome by presenting a variation of typical 18q- deletion syndrome features to the literature. In addition, this case report demonstrated the ability of the molecular karyotyping method, such as array-based comparative genomic hybridization, to assist in the diagnosis of cases with a highly variable phenotype and variable aberrations, such as 18q- deletion syndrome.

Optical genome mapping for cytogenomic analysis of hematological neoplasms.

e19016 Background: Optical genome mapping (OGM) provides a genome-wide analysis for structural variants and copy number changes in one-assay. Compared with conventional G-banded karyotyping (GBK), OGM provides significantly higher resolution and does not require metaphase cells. In this study, we assessed the added value of OGM for cytogenomic analysis of hematological neoplasms. Methods: OGM was performed on blood or bone marrow using Saphyr from Bionano Genomics on 63 patients diagnosed with various types of hematological malignancy (see Table). Variant calling was made by the Rare Variant Analysis pipeline on Bionano Access after applying recommended filters. Each patient had karyotype and/or fluorescence in situ hybridization (FISH) results available for comparison and confirmation. Only Tier 1 (pathogenic) and Tier 2 (likely pathologic) abnormalities were included for comparison purposes in this study. Results: The results were completely concordant between GBK/FISH and OGM in 30 (47.6%) patients, including 10 patients who showed no cytogenetic abnormality (normal). Fully discordant results were found in 8 (12.7%) patients: 2 had an abnormal karyotype but normal OGM, the discrepancy was mainly due to the clonal size (&lt;20%) below the limitation of detection of OGM; 6 had a normal karyotype but cytogenetic abnormalities were detected by OGM, the discrepancy was mainly due to failure of tumor cells growth (n=5) or subtle cytogenetic abnormality undetectable by GBK (n=1). Partially concordant results between karyotype/FISH and OGM were observed in 25 (40%) patients, some abnormalities were detected by both, some were detected by GBK alone (n=4) or by OGM alone (n=25), the latter group included 10 cases with chromothripsis which only be detected by OGM. Overall, OGM provided clinically significant information in 14 (22%) patients regarding diagnosis, risk stratification, and/or identifying therapeutic targets. The added value was more common in lymphoid neoplasms (9 of 27 patients, 33%). Conclusions: OGM provides additional clinically relevant cytogenetic information in the workup of hematological neoplasms. This added value is more apparent in lymphoid neoplasms. These improvements are largely contributable to the higher resolution provided by OGM and analysis independent of metaphase cells. A major limitation of OGM usage is the need for a clonal size of at least 20%. [Table: see text]

Cytogenetic evidence and dmrt linkage indicate male heterogamety in a non-bilaterian animal.

The diversity of sex determination systems in animals suggests that sex chromosomes evolve independently across different lineages. However, the present data on these systems is largely limited and represented mainly by bilaterian animals. Sex chromosomes and sex determination system based on cytogenetic evidence remain a mystery among non-bilaterians, the most basal animals. Here, we investigated the sex determination system of a non-bilaterian (Goniopora djiboutiensis) based on karyotypic analysis and identification of locus of dmrt1, a known master sex-determining gene in many animals. Results showed that among the three isolated dmrt genes, GddmrtC was sperm-linked. Fluorescence in situ hybridization revealed that 47% of the observed metaphase cells contained the GddmrtC locus on the shorter chromosome of the heteromorphic pair, whereas the other 53% contained no GddmrtC locus and pairing of the longer chromosome of the heteromorphic pair was observed. These findings provided the cytogenetic evidence for the existence of the Y sex chromosome in a non-bilaterian animal and supports male heterogamety as previously reported in other non-bilaterian species using RAD sequencing. The Y chromosome-specific GddmrtC sequence was most homologous to the vertebrate dmrt1, which is known for its role in male sex determination and differentiation. Our result on identification of putative sex chromosomes for G. djiboutiensis may contribute into understanding of the possible genetic sex determination systems in non-bilaterian animals.

Application of fluorescence in situ hybridization technique to verify the clonalities of non-clonal cytogenetic abnormalities identified in Myelodysplastic syndrome

To assess the value of fluorescence in situ hybridization (FISH) technique for the verification of the clonalities of non-clonal cytogenetic abnormalities (n-CCA) identified by conventional chromosome banding analysis (CBA) in patients with Myelodysplastic syndrome (MDS). Clinical data and results of karyotyping and FISH assays for 91 patients of MDS with n-CCA identified by CBA were retrospectively analyzed. In total 94 non-clonal +8, 5q-, -7/7q- or 20q- were detected by CBA, among which 43 (45.7%) were verified to be clonal abnormalities by FISH. The detection rates for +8, 5q-, -7/7q- and 20q- by FISH were 47.6% (30/63), 25% (2/8), 41.7% (5/12), 40% (2/5) and 66.7% (4/6), respectively, with the positive cells accounting for 4% to 90% of all counted cells, with a median value of 7%. The 91 patients were divided into three groups including ≥ 20, 10 ~< 20 and < 10 based on the numbers of metaphase cells in CBA, and the detection rates by FISH for the three groups were 43.7% (31/71), 33.3% (3/9) and 63.6% (7/11), respectively, which showed no statistically difference (P > 0.05). Continuous CBA and FISH surveys were conducted for 26 patients who received supportive treatment, and the results revealed that 91.7% (11/12) of FISH-verified positive abnormalities had persisted, whereas 92.9% (13/14) of the n-CCA verified as negative by FISH was transient. Nearly half of the CBA identified n-CCA have been verified as clonal aberrations by FISH, and the FISH detection rate showed no correlation with the number of metaphase cells. FISH test is strongly recommended for verifying the clonalities of n-CCA detected by CBA, and continuous cytogenetic survey of the patients with MDS is necessary.

Effect of cypermethrin insecticide on root chromosome morphometry of scallion (Allium fistulosum)

Abstract. Haq FAN, Etikawati N, Solichatun. 2023. Effect of cypermethrin insecticide on root chromosome morphometry of scallion (Allium fistulosum). Cell Biol Dev 7: 14-21. Farmers in Tawangmangu, Karanganyar, Central Java, Indonesia, commonly use a pesticide known as cypermethrin insecticide whenever growing scallion (Allium fistulosum L.). If used at excessive levels, cypermethrin pesticide can be fatal to plants. Chromosome morphological changes are an indicator of cypermethrin insecticide's toxicity. Chromosomal morphometry, the study of chromosomal size and shape, can provide an overall picture of the quality of a plant's development. This research aimed to evaluate cypermethrin insecticide's impact on A. fistulosum root chromosomal morphometrics. Information gleaned from chromosomal morphometry includes chromosome shape, chromosome size, aberration type, chromosomal aberration index, relative asymmetry index, relative centromere index, and mitotic chromosome index. The concentration of cypermethrin was the sole independent variable in this study, which used a Completely Randomized Design (CRD) factorial arrangement. Long green onions were treated with varying concentrations of cypermethrin for up to four weeks: 0 mg/L, 0.05 mg/L, 0.10 mg/L, 0.15 mg/L, 0.20 mg/L, and 0.25 mg/L. The squash method was used to prepare the plant's apex roots. The root chromosomal visualization output was subjected to quantitative and qualitative descriptive analysis. The mitotic index decreased, and the chromosome aberration index increased after exposure to cypermethrin pesticides. The higher the concentration of the pesticide cypermethrin, the lower the mitotic index and the greater the chromosome aberration index. Long green onion root cells exposed to cypermethrin insecticide underwent chromosomal aberrations that could be identified qualitatively. Sticky chromosomes, chromosome bridge, chromosome agglutination, disrupted metaphase, disturbed anaphase, and hypoploid cell are examples of chromosomal aberrations.

Cytogenotoxic effects of 3-epicaryoptin in Allium cepa L. root apical meristem cells.

Diterpenoid 3-epicaryoptin (C26H36O9) is abundant in the leaves of Clerodendrum inerme, a traditionally used medicinal plant, and has insect antifeedant activities. Here, we aim to explore the cytogenotoxic effects of compound 3-epicaryoptin in Allium cepa root apical meristem cells. 3-epicaryoptin (concentrations of 100, 150, and 200µgmL-1) and the standard compound colchicine (200µgmL-1) were applied to A. cepa roots for 2, 4, and 4 + 16h (4-h treatment followed by 16-h recovery). Cytogenotoxicity was analyzed by studying the root growth retardation (RGR), mitotic index (MI), and chromosomal aberrations. The result showed statistically significant (p < 0.01), concentration-dependent RGR effects of 3-epicaryoptin treatment compared with the negative control. A study of cell frequency in different phases of cell division observed a significant (p < 0.001) increase in the metaphase cell percentage (66.2 ± 0.58%, 150µgmL-1), which subsequently caused an increase in the frequency of MI (12.29 ± 0.34%, 150µgmL-1) at 4h of 3-epicaryoptin treatment and that was comparable with the colchicine action. The cytological study revealed that the 3-epicaryoptin treatment could induce different types of chromosomal abnormalities, such as colchicine-like metaphase, vagrant chromosomes, sticky chromosomes, anaphase bridge, lagging chromosomes, multipolar anaphase-telophase, and an increased frequency of micronuclei and polyploid cells. These findings indicate that 3-epicaryoptin is cytogenotoxic, and thus, C. inerme should be used with caution in traditional medicine.

Fusion Gene-Based Classification of Variant Cytogenetic Rearrangements in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) represents a heterogeneous disease entity that is continuously moving to a more genetically defined classification. The classification of AML with recurrent chromosomal translocations, including those involving core binding factor subunits, plays a critical role in diagnosis, prognosis, treatment stratification, and residual disease evaluation. Accurate classification of variant cytogenetic rearrangements in AML contributes to effective clinical management. We report here the identification of four variant t(8;V;21) translocations in newly diagnosed AML patients. Two patients showed a t(8;14) and a t(8;10) variation, respectively, with a morphologically normal-appearing chromosome 21 in each initial karyotype. Subsequent fluorescence in situ hybridization (FISH) on metaphase cells revealed cryptic three-way translocations t(8;14;21) and t(8;10;21). Each resulted in RUNX1::RUNX1T1 fusion. The other two patients showed karyotypically visible three-way translocations t(8;16;21) and t(8;20;21), respectively. Each resulted in RUNX1::RUNX1T1 fusion. Our findings demonstrate the importance of recognizing variant forms of t(8;21) translocations and emphasize the value of applying RUNX1::RUNX1T1 FISH for the detection of cryptic and complex rearrangements when abnormalities involving chromosome band 8q22 are observed in patients with AML.

Telomere Fragility and MiDAS: Managing the Gaps at the End of the Road.

Telomeres present inherent difficulties to the DNA replication machinery due to their repetitive sequence content, formation of non-B DNA secondary structures, and the presence of the nucleo-protein t-loop. Especially in cancer cells, telomeres are hot spots for replication stress, which can result in a visible phenotype in metaphase cells termed "telomere fragility". A mechanism cells employ to mitigate replication stress, including at telomeres, is DNA synthesis in mitosis (MiDAS). While these phenomena are both observed in mitotic cells, the relationship between them is poorly understood; however, a common link is DNA replication stress. In this review, we will summarize what is known to regulate telomere fragility and telomere MiDAS, paying special attention to the proteins which play a role in these telomere phenotypes.

Piperine-Chlorogenic Acid Hybrid Inhibits the Proliferation of the SK-MEL-147 Melanoma Cells by Modulating Mitotic Kinases.

Melanoma is considered the most aggressive form of skin cancer, showing high metastatic potential and persistent high mortality rates despite the introduction of immunotherapy and targeted therapies. Thus, it is important to identify new drug candidates for melanoma. The design of hybrid molecules, with different pharmacophore fragments combined in the same scaffold, is an interesting strategy for obtaining new multi-target and more effective anticancer drugs. We designed nine hybrid compounds bearing piperine and chlorogenic acid pharmacophoric groups and evaluated their antitumoral potential on melanoma cells with distinct mutational profiles SK-MEL-147, CHL-1 and WM1366. We identified the compound named PQM-277 (3a) to be the most cytotoxic one, inhibiting mitosis progression and promoting an accumulation of cells in pro-metaphase and metaphase by altering the expression of genes that govern G2/M transition and mitosis onset. Compound 3a downregulated FOXM1, CCNB1, CDK1, AURKA, AURKB, and PLK1, and upregulated CDKN1A. Molecular docking showed that 3a could interact with the CUL1-RBX1 complex, which activity is necessary to trigger molecular events essential for FOXM1 transactivation and, in turn, G2/M gene expression. In addition, compound 3a effectively induced apoptosis by increasing BAX/BCL2 ratio. Our findings demonstrate that 3a is an important antitumor candidate prototype and support further investigations to evaluate its potential for melanoma treatment, especially for refractory cases to BRAF/MEK inhibitors.

P530: Utility of examining 100 metaphase cells in the evaluation of low-level mosaicism

P530: Utility of examining 100 metaphase cells in the evaluation of low-level mosaicism

The mitotic role of adenomatous polyposis coli requires its bilateral interaction with tubulin and microtubules.

Adenomatous polyposis coli (APC) is a scaffold protein with tumour suppressor properties. Mutations causing the loss of its C-terminal domain (APC-C), which bears cytoskeleton-regulating sequences, correlate with colorectal cancer. The cellular roles of APC in mitosis are widely studied, but the molecular mechanisms of its interaction with the cytoskeleton are poorly understood. Here, we investigated how APC-C regulates microtubule properties, and found that it promotes both microtubule growth and shrinkage. Strikingly, APC-C accumulates at shrinking microtubule extremities, a common characteristic of depolymerases. Cryo-electron microscopy revealed that APC-C adopts an extended conformation along the protofilament crest and showed the presence of ring-like tubulin oligomers around the microtubule wall, which required the presence of two APC-C sub-domains. A mutant of APC-C that was incapable of decorating microtubules with ring-like tubulin oligomers exhibited a reduced effect on microtubule dynamics. Finally, whereas native APC-C rescued defective chromosome alignment in metaphase cells silenced for APC, the ring-incompetent mutant failed to correct mitotic defects. Thus, the bilateral interaction of APC-C with tubulin and microtubules likely contributes to its mitotic functions.

Development and implementation of a metaphase DNA model for ionizing radiation induced DNA damage calculation

Objective. To develop a metaphase chromosome model representing the complete genome of a human lymphocyte cell to support microscopic Monte Carlo (MMC) simulation-based radiation-induced DNA damage studies. Approach. We first employed coarse-grained polymer physics simulation to obtain a rod-shaped chromatid segment of 730 nm in diameter and 460 nm in height to match Hi–C data. We then voxelized the segment with a voxel size of 11 nm per side and connected the chromatid with 30 types of pre-constructed nucleosomes and 6 types of linker DNAs in base pair (bp) resolutions. Afterward, we piled different numbers of voxelized chromatid segments to create 23 pairs of chromosomes of 1–5 μm long. Finally, we arranged the chromosomes at the cell metaphase plate of 5.5 μm in radius to create the complete set of metaphase chromosomes. We implemented the model in gMicroMC simulation by denoting the DNA structure in a four-level hierarchical tree: nucleotide pairs, nucleosomes and linker DNAs, chromatid segments, and chromosomes. We applied the model to compute DNA damage under different radiation conditions and compared the results to those obtained with G0/G1 model and experimental measurements. We also performed uncertainty analysis for relevant simulation parameters. Main results. The chromatid segment was successfully voxelized and connected in bps resolution, containing 26.8 mega bps (Mbps) of DNA. With 466 segments, we obtained the metaphase chromosome containing 12.5 Gbps of DNA. Applying it to compute the radiation-induced DNA damage, the obtained results were self-consistent and agreed with experimental measurements. Through the parameter uncertainty study, we found that the DNA damage ratio between metaphase and G0/G1 phase models was not sensitive to the chemical simulation time. The damage was also not sensitive to the specific parameter settings in the polymer physics simulation, as long as the produced metaphase model followed a similar contact map distribution. Significance. Experimental data reveal that ionizing radiation induced DNA damage is cell cycle dependent. Yet, DNA chromosome models, except for the G0/G1 phase, are not available in the state-of-the-art MMC simulation. For the first time, we successfully built a metaphase chromosome model and implemented it into MMC simulation for radiation-induced DNA damage computation.

Kinesin-5 Eg5 is essential for spindle assembly, chromosome stability and organogenesis in development

Chromosome stability relies on bipolar spindle assembly and faithful chromosome segregation during cell division. Kinesin-5 Eg5 is a plus-end-directed kinesin motor protein, which is essential for spindle pole separation and chromosome alignment in mitosis. Heterozygous Eg5 mutations cause autosomal-dominant microcephaly, primary lymphedema, and chorioretinal dysplasia syndrome in humans. However, the developmental roles and cellular mechanisms of Eg5 in organogenesis remain largely unknown. In this study, we have shown that Eg5 inhibition leads to the formation of the monopolar spindle, chromosome misalignment, polyploidy, and subsequent apoptosis. Strikingly, long-term inhibition of Eg5 stimulates the immune responses and the accumulation of lymphocytes in the mouse spleen through the innate and specific immunity pathways. Eg5 inhibition results in metaphase arrest and cell growth inhibition, and suppresses the formation of somite and retinal development in zebrafish embryos. Our data have revealed the essential roles of kinesin-5 Eg5 involved in cell proliferation, chromosome stability, and organogenesis during development. Our findings shed a light on the cellular basis and pathogenesis in microcephaly, primary lymphedema, and chorioretinal dysplasia syndrome of Eg5-mutation-positive patients.