Unstable Chromosome Research Articles

Establishment and Characterization of a New Cell Line from Enzootic Nasal Adenocarcinoma in Goats: ENA-1.

Enzootic nasal adenocarcinoma (ENA) is a contagious tumor disease of goats and sheep, which is caused by enzootic nasal tumor virus (ENTV). To better understand the pathogenesis of ENA, this study aimed to establish a goat ENA cell line (ENA-1). The cells have been characterized with regard to morphology, growth rate, ultrastructural features, chromosome number, expression of CK7 and CK18, tumorigenicity, species, and mycoplasma contamination. ENA-1 had an epithelioid cell morphology with an unstable chromosome number under a light microscope. Under an electron microscope, the cell nuclear heterogeneity was not obvious, and there were more intermediate filaments and a small number of immature retrovirus-like particles in the cytoplasm. ENA-1 had strong proliferative potential, and the cell multiplication time was about 36 h, which could make BALB/c nude mice develop tumors. CK7 and CK18 were expressed in the cytoplasm of primary goat tumors, in transplanted tumors from nude mice, and un ENA-1 cells with the same intensity. PCR revealed that ENA-1 continuously carried ENTV-2 up to the 17th generation with no germline contamination or mycoplasma contamination. In conclusion, using a serum-containing culture system, ENA-1 cells were successfully isolated, cultured, and purified from goat tumor tissues. The isolated ENA-1 cells retained robust proliferation potential and maintained their phenotype, indicating the potential application of the ENA-1 cell line as an in vitro model of ENA.

Влияние хронического облучения на показатели цитогенетических маркеров старения у жителей прибрежных сел реки Теча

The understanding of the exposure effects on the human health could be improved by analyzing the influence of the chronic low dose rate exposure on the senescence of the immune system cells. It will also help to develop the measures aimed at the mitigation of the adverse effects. The objective of the study is to investigate the influence of the chronic low dose rate exposure on the senescence of the immune system cells using the cytogenetic markers. In the course of the research the authors evaluated the cellular senescence markers — genome instability and telomere depletion — in T-lymphocytes of the individuals exposed in the Southern Urals (exposure doses were 0.001 Gy — 4.7 Gy, the age of examined people was 40–89 years). The data analysis has demonstrated that the effect of chronic exposure on the T-cell senescence was indirect. Unstable chromosome aberrations occurred statistically significantly more frequently in exposed people aged 40–59 years (p = 0.012). Frequency of lymphocytes with micronuclei in exposed individuals differed in men and women (p = 0.001). Statistically significant decrease in the telomere length was revealed (for the chromosome arms 1q, 3p, 3q, 20p, 20q, 13q, 15p, 22q (p < 0.05); 19p, 21q (p < 0.01)).

The genomes of seven economic Caesalpinioideae trees provide insights into polyploidization history and secondary metabolite biosynthesis

The Caesalpinioideae subfamily contains many well-known trees that are important for economic sustainability and human health, but a lack of genomic resources has hindered their breeding and utilization. Here, we present chromosome-level reference genomes for the two food and industrial trees Gleditsia sinensis (921 Mb) and Biancaea sappan (872 Mb), the three shade and ornamental trees Albizia julibrissin (705 Mb), Delonix regia (580 Mb), and Acacia confusa (566 Mb), and the two pioneer and hedgerow trees Leucaena leucocephala (1338 Mb) and Mimosa bimucronata (641 Mb). Phylogenetic inference shows that the mimosoid clade has a much higher evolutionary rate than the other clades of Caesalpinioideae. Macrosynteny comparison suggests that the fusion and breakage of an unstable chromosome are responsible for the difference in basic chromosome number (13 or 14) for Caesalpinioideae. After an ancient whole-genome duplication (WGD) shared by all Caesalpinioideae species (CWGD, ∼72.0 million years ago [MYA]), there were two recent successive WGD events, LWGD-1 (16.2–19.5 MYA) and LWGD-2 (7.1–9.5 MYA), in L. leucocephala. Thereafter, ∼40% gene loss and genome-size contraction have occurred during the diploidization process in L. leucocephala. To investigate secondary metabolites, we identified all gene copies involved in mimosine metabolism in these species and found that the abundance of mimosine biosynthesis genes in L. leucocephala largely explains its high mimosine production. We also identified the set of all potential genes involved in triterpenoid saponin biosynthesis in G. sinensis, which is more complete than that based on previous transcriptome-derived unigenes. Our results and genomic resources will facilitate biological studies of Caesalpinioideae and promote the utilization of valuable secondary metabolites.

Abstract A001: Genomic signatures of past and present chromosomal instability in Barrett’s esophagus and early esophageal adenocarcinoma

Abstract Abstract: The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations but how these alterations arise is poorly understood. Here we perform haplotype-specific analysis of chromosomal copy-number evolution in the progression of Barrett’s esophagus (BE) to esophageal adenocarcinoma (EAC) on multiregional whole-genome sequencing data of BE with dysplasia and microscopic EAC foci. We identify distinct patterns of copy-number evolution indicating multigenerational chromosomal instability that is initiated by cell division errors but propagated only after p53 loss. While abnormal mitosis, including whole-genome duplication, underlies chromosomal copy-number changes, segmental alterations display signatures of successive breakage-fusion-bridge cycles and chromothripsis of unstable dicentric chromosomes. Our analysis elucidates how multigenerational chromosomal instability generates copy-number variation in BE cells, precipitates complex alterations including DNA amplifications, and promotes their independent clonal expansion and transformation. In particular, we suggest sloping copy-number variation as a signature of ongoing chromosomal instability that precedes copy-number complexity. These findings suggest copy-number heterogeneity in advanced cancers originates from chromosomal instability in precancerous cells and such instability may be identified from the presence of sloping copy-number variation in bulk sequencing data. Citation Format: Chunyang Bao. Genomic signatures of past and present chromosomal instability in Barrett’s esophagus and early esophageal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr A001.

Integrative Multi-Omic Analysis for Prognosis Stratification in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy with a dismal prognosis. European LeukemiaNet (ELN) is crucial for tailoring AML treatment individually. Various AML models correlated survival and clinical drug response with immune cell differentiation state by deconvoluting transcriptomics. However, few comprehensive molecular subtyping models integrate multi-omic profiles for prognostic and drug response prediction. Thus, this study aimed to integrate DNA methylation, genomics, transcriptomics and ex vivo drug sensitivity screening of AML patients and explore their roles in facilitating AML molecular stratification and redefining prognosis. Firstly，data on 90 patients with AML(non-promyelocytic leukemia) were retrieved from The Cancer Genome Atlas (TCGA) for consensus clustering by 10 advanced consensus clustering algorithms. The optimal three distinct subtypes, UAMOCS1, UAMOCS2, and UAMCOS3, were identified to achieve the best clustering effect. Multi-omics were orchestrated together for UAMOCS shaping. The UAMOCS can significantly distinguish overall survival (median 18 vs. 27 vs. 45 months, P= 0.0048) among the three instinct subtypes with the integrated contribution of transcriptomic profiles and DNA methylation and mutation data. Also, the activity of AML-specific transcription factors and chromatin remodeling makers showed a diverse pattern among the three subtypes, epigenetically contributing to UAMOCS shaping. Next, the nearest template prediction, a model-free method, was applied to further validate this predictive model. Similar subtyping under this model is generated robustly by imitating 50 subtype-specific genes and capable of identifying survival in both the “ihCAMs-AML” (Figure 1) and GSE37642. The main validation cohort was a real-world de novo AML cohort (acute promyelocytic leukemia excluded), “ihCAMs-AML”（N=98）, aged 14-70 years, with a median follow-up of 28 (1-55) months. The samples were simultaneously available for methylome (N=31, 850K Methylation Chip), DNA mutation (N=96, targeted deep sequencing), transcriptome (N=98, bulk RNA-seq), and ex vivo drug screening (N=66, 17 drugs under 100% peak plasma concentration) profiling analysis. Clinical relevance showed that traditional cytogenetic stratification was strongly correlated with our system. UAMOCS1, acting as an “immune activator”, is characterized by enriching of AML myelodysplasia-related (AML-MR) gene mutations (especially RUNX1 mutation, P = 0.00839), unstable chromosome (deletions on 7q), increased abundance of immune cells with immune escaping marker and poor risk. UAMOCS2 was defined as an intermediate immune burden and a “monocytic-like” phenotype with intermediate survival, corresponding to its inclining result toward M4/M5. UAMOCS3 was an “immune desert” group supposed to be the most favorable subset among the three groups. The ex vivo drug screening result implied that UAMOCS also provide values in distinguishing drug response. UAMOCS1 hardly benefited from commonly used chemotherapeutic drugs compared with the other two distinct subtypes. Pharmacogenomics database GDSC (the Genomics of Drug Sensitivity in Cancer) among AML cell lines was used to evaluate potential drug targets for overcoming drug resistance. Receptor tyrosine kinase signaling inhibitors (SB505124 and EphB4) might be sensitive to UAMOCS1. Taken together, UAMOCS model was generated that can classify AML patients independently and help develop precise chemotherapeutic strategy for patients with AML in the future.

Genomic signatures of past and present chromosomal instability in Barrett’s esophagus and early esophageal adenocarcinoma

The progression of precancerous lesions to malignancy is often accompanied by increasing complexity of chromosomal alterations but how these alterations arise is poorly understood. Here we perform haplotype-specific analysis of chromosomal copy-number evolution in the progression of Barrett’s esophagus (BE) to esophageal adenocarcinoma (EAC) on multiregional whole-genome sequencing data of BE with dysplasia and microscopic EAC foci. We identify distinct patterns of copy-number evolution indicating multigenerational chromosomal instability that is initiated by cell division errors but propagated only after p53 loss. While abnormal mitosis, including whole-genome duplication, underlies chromosomal copy-number changes, segmental alterations display signatures of successive breakage-fusion-bridge cycles and chromothripsis of unstable dicentric chromosomes. Our analysis elucidates how multigenerational chromosomal instability generates copy-number variation in BE cells, precipitates complex alterations including DNA amplifications, and promotes their independent clonal expansion and transformation. In particular, we suggest sloping copy-number variation as a signature of ongoing chromosomal instability that precedes copy-number complexity. These findings suggest copy-number heterogeneity in advanced cancers originates from chromosomal instability in precancerous cells and such instability may be identified from the presence of sloping copy-number variation in bulk sequencing data.

Preliminary study of chromosome aberrations using Giemsa, two-colour fish, and micronucleus assays in lymphocytes of individuals living in elevated radon concentration areas.

The frequencies of unstable and stable chromosome aberrations and micronuclei were examined in peripheral blood samples from 10 individuals living in elevated radon concentration areas (Takandeang Village, Mamuju, Indonesia). Blood samples from 10 people living in Topoyo Village were used as a control group. For unstable chromosome aberration analysis, a dicentric chromosome assay was conducted using conventional Giemsa staining. Chromosomal painting of chromosomes 1 and 4 using the fluorescence in situ hybridisation technique was also applied to four subjects to assess the stable chromosome aberration. Our study showed no significant increases across all groups in dicentric and other unstable chromosome aberrations, such as rings and acentric fragments. Translocations were found in one person from Takandeang Village and two Topoyo Village inhabitants. The translocations found in the subjects from Takandeang Village were due more to aging factors than to radon exposure. The number of micronuclei per 1000 binucleus cells in Takandeang Village inhabitants was not significantly different than that in the control group (p=0.943). A more comprehensive analysis should be conducted in a subsequent study by increasing the number of study donors and the number of metaphases to be analysed in both dicentric chromosome assay and fluorescence in situ hybridisation assays. Such research could provide valid information on the cytogenetic effects of elevated indoor radon exposure.

Chromosome damage in regions with different levels of air pollution.

Air pollution is an important environmental factor influencing human health. In this study, we compared chromosome damage in city policemen from three cities in the Czech Republic: industrial Ostrava characterized by high levels of benzo[a]pyrene, Prague with heavy traffic emitting nitrogen oxides, and relatively clean Ceske Budejovice located in an area with predominantly agricultural activity. Chromosomal aberrations in lymphocytes were evaluated by fluorescence in situ hybridization with painting probes for chromosomes 1, 2, 3, and 4 in spring and autumn. An increase in the frequency of unstable chromosome aberrations, that is, dicentric chromosomes and acentric fragments, was observed in spring samples from Ostrava (p = .014 and p = .044, respectively) and Prague (p = .002 and p = .006, respectively) in comparison with Ceske Budejovice. The difference was significant only for samples taken after the winter period, when the concentration of pollutants in the air increases due to poor dispersion conditions. An increased frequency of dicentric chromosomes was observed in spring compared to autumn in both Ostrava and Prague (p = .017 and p = .023, respectively), but not in Ceske Budejovice. More breakpoints were observed on chromosome 1 than on the other chromosomes examined (p < .001). The number of breakpoints in the heterochromatin region 1p11-q12 was lower than in other parts of chromosome 1 (p < .001), suggesting a protective function of heterochromatin against damage. Our study showed, that air pollution increased the frequency of unstable chromosome aberrations, especially dicentric chromosomes. However, we did not show an effect on stable chromosome rearrangements.

Оценка влияния хронического облучения на преждевременное старение Т-лимфоцитов человека на основе нестабильных хромосомных аберраций

For more than 60 years, residents of the villages on the Techa River have been chronically exposed to combined radiation, receiving a wide range of doses. Red bone marrow (RBM) is the critical system in the exposure conditions. This study aimed to assess the effect of chronic exposure on premature aging of T-lymphocytes based on the frequency of unstable chromosome aberrations; the subjects were the residents of the Southern Urals that have been chronically exposed to radiation. The increased frequency of occurrence of dicentrics and rings in T-cells of the exposed persons was the marker of cellular aging, with the associated doses to the red bone marrow (RBM dose) at 0.5–2.5 Gy. The participants (RBM donors), both exposed and non-exposed, were divided into three age subgroups: 40–59 years old, 60–69 years old, 70–79 years old. The differences in the RBM dose among the exposed individuals were insignificant. In the exposed group, unstable chromosome aberrations (UCA) were recorded significantly more often than in the control group (p = 0.04). The age group of 40–59 years was the one where the exposed donors had significantly more frequently occurring chromosome aberrations compared to the non-exposed participants. There were no such differences registered in other age groups. The age-associated increase of the amount of chromosome aberrations was registered in the non-exposed group only. Chronic exposure to radiation indirectly promotes premature aging of T-lymphocytes: 1) in the long term, the exposed individuals had UCA significantly more often; 2) compared to the control group, the 40–59 years age subgroup of the exposed group had increased cytogenetic index. In the context of this study, the number of dicentrics and rings was not registered as increasing in the older age subgroups of exposed individuals, which may be due to the specifics of the donor inclusion criteria, which, for the elderly, may favor radioresistant individuals.

Homologous recombination suppresses transgenerational DNA end resection and chromosomal instability in fission yeast.

Chromosomal instability (CIN) drives cell-to-cell heterogeneity, and the development of genetic diseases, including cancer. Impaired homologous recombination (HR) has been implicated as a major driver of CIN, however, the underlying mechanism remains unclear. Using a fission yeast model system, we establish a common role for HR genes in suppressing DNA double-strand break (DSB)-induced CIN. Further, we show that an unrepaired single-ended DSB arising from failed HR repair or telomere loss is a potent driver of widespread CIN. Inherited chromosomes carrying a single-ended DSB are subject to cycles of DNA replication and extensive end-processing across successive cell divisions. These cycles are enabled by Cullin 3-mediated Chk1 loss and checkpoint adaptation. Subsequent propagation of unstable chromosomes carrying a single-ended DSB continues until transgenerational end-resection leads to fold-back inversion of single-stranded centromeric repeats and to stable chromosomal rearrangements, typically isochromosomes, or to chromosomal loss. These findings reveal a mechanism by which HR genes suppress CIN and how DNA breaks that persist through mitotic divisions propagate cell-to-cell heterogeneity in the resultant progeny.

Chromosome aberrations, micronucleus frequency, and catalase concentration in a population chronically exposed to high levels of radon

Purpose To deepen our knowledge on the effects of high levels of indoor radon exposure, we assessed the frequencies of unstable and stable chromosome aberrations and micronucleus (MN), as well as the concentration of an endogenous antioxidant (catalase, CAT), in blood samples of individuals chronically exposed to high indoor radon concentrations in Indonesia (Tande-Tande sub-village, Mamuju, West Sulawesi). Moreover, we also investigated the occurrence of a radio-adaptive response (RAR) in Tande-Tande sub-village inhabitants using the G2 MN assay. Materials and Methods The frequencies of dicentric (DC), acentric (AF), ring (R), and translocation (Tr) chromosomes in Tande-Tande inhabitants were compared to those in people living in a reference area with low levels of indoor radon levels (Topoyo village, Indonesia). The number of MN per 1000 binucleated cells (BNC) and CAT concentration per total protein was quantified and compared between groups. Lastly, we irradiated (2 Gy) phytohemagglutinin-stimulated samples in vitro and measured the frequency of MN to verify the occurrence of a RAR in Tande-Tande sub-village inhabitants. Results and Conclusion The frequencies of DC, AF, and Tr did not differ between Tande-Tande inhabitants and control subjects (p = 0.350, 0.521, 0.597). The frequency of MN in Tande-Tande inhabitants was significantly lower than that in the control group (p = 0.006). Similarly, CAT concentration in Tande-Tande inhabitants was also significantly lower than that in the control population (p < 0.001). Significant negative correlations were identified for MN number and CAT concentration versus indoor radon concentration, annual effective dose, or cumulative dose both within groups and when all data were analyzed together. Our findings indicate that, despite the high indoor radon levels, Tande-Tande inhabitants are not under oxidative stress, since this group had lower CAT concentration and MN frequency than those in the control group. The negative correlation between MN frequency and indoor radon concentration, annual effective dose, and cumulative dose suggests the occurrence of an RAR phenomenon in Tande-Tande sub-village inhabitants. This interpretation is also supported by the results of the G2 MN assay, which revealed lower MN frequencies after in vitro irradiation of samples from Tande-Tande sub-village inhabitants than those in samples from the control group (p = 0.0069, for cumulative MN frequency; p = 0.0146, for radiation-induced MN only).

Background Level of Unstable Chromosome Aberrations in the Kazakhstan Population: A Human Biomonitoring Study.

Kazakhstan is known as a country with a complex radioecological situation resulting from different sources such as a natural radiation background, extensive activities of the industrial system of the former Soviet Union and a well-known testing of nuclear power weapons occurred in the Semipalatinsk Test Site (STS) area. The present study focuses on the assessment of the background of dicentric chromosomes in Kazakhstan’s population, which is the starting point in the dose assessment of irradiated people, since the baseline level of spontaneous dicentrics can vary significantly in different populations. In this context, aiming to determine the background frequency of chromosome aberrations in the population of Kazakhstan, considering the heterogeneity of natural radiation background levels of its large territory, a selection of 40 control subjects living in four cities of North, South, West and East Kazakhstan was performed. The cytogenetic study on the selected groups showed fairly low background frequency values of chromosome aberrations (0.84 ± 0.83 per 1000 cells), comparable with other data in the literature on general populations, reporting background frequency values between 0.54 and 2.99 per 1000 cells. The obtained results should be taken into account when constructing the dose–effect calibration curve used in cytogenetic biodosimetry, as a “zero” dose point, which will reduce the uncertainty in quantifying the individual absorbed dose in emergency radiological situations.

Evolving Exploration of the Pathogenesis of CNS Germ Cell Tumors with Regard to Precision Medicine

Genomic and epigenomic analyses have progressed the exploration of the pathogenesis of CNS germ cell tumors(GCTs)in the past decade. GCTs are characterized by mutations in MAPK or PI3K pathways(55%)and unstable chromosomes, especially 12p gain(45%), as well as global hypomethylation in germinoma. Highly specific microRNA, miR-371a-3p, can be a diagnostic marker in serum and cerebrospinal fluid. Tumor cell content examined in H-E specimens has a prognostic value in germinoma: cases with higher tumor cell content show a worse prognosis. 12p gain in non-germinomatous GCTs(NGGCTs)has an unfavorable prognostic significance. PD-L1 and PD-1 are highly expressed in germinomas and the tumor cell microenvironment, respectively, highlighting the potential effectiveness of immune checkpoint inhibitors. Clinical trials from the Children's Oncology Group(COG)in the US and the Society for Paediatric Oncology(SIOP)in Europe and Japan have shown that whole ventricular irradiation is the most appropriate for germinomas, and that radiation fields can be reduced to the whole ventricle or a local area for localized NGGCTs. Toward personalized medicine, investigations into the structural abnormalities and variants in non-coding regions are needed to develop targeted therapy. A stratified treatment regimen is expected by incorporating newly-found biomarkers to reduce the treatment burden for generally young patients and circumvent late toxicity and sequelae. Establishing effective treatments is crucial for relapsed GCT that has a dismal prognosis.

Cytogenetic follow-up studies on humans with internal and external exposure to ionizing radiation

Cells exposed to ionizing radiation have a wide spectrum of DNA lesions that include DNA single-strand breaks, DNA double-strand breaks (DSBs), oxidative base damage and DNA-protein crosslinks. Among them, DSB is the most critical lesion, which when mis-repaired leads to unstable and stable chromosome aberrations. Currently, chromosome aberration analysis is the preferred method for biological monitoring of radiation-exposed humans. Stable chromosome aberrations, such as inversions and balanced translocations, persist in the peripheral blood lymphocytes of radiation-exposed humans for several years and, therefore, are potentially useful tools to prognosticate the health risks of radiation exposure, particularly in the hematopoietic system. In this review, we summarize the cytogenetic follow-up studies performed by REAC/TS (Radiation Emergency Assistance Center/Training site, Oak Ridge, USA) on humans exposed to internal and external radiation. In the light of our observations as well as the data existing in the literature, this review attempts to highlight the importance of follow-up studies for predicting the extent of genomic instability and its impact on delayed health risks in radiation-exposed victims.

Alpha-Particle Exposure Induces Mainly Unstable Complex Chromosome Aberrations which do not Contribute to Radiation-Associated Cytogenetic Risk.

The mechanism underlying the carcinogenic potential of α radiation is not fully understood, considering that cell inactivation (e.g., mitotic cell death) as a main consequence of exposure efficiently counteracts the spreading of heritable DNA damage. The aim of this study is to improve our understanding of the effectiveness of α particles in inducing different types of chromosomal aberrations, to determine the respective values of the relative biological effectiveness (RBE) and to interpret the results with respect to exposure risk. Human peripheral blood lymphocytes (PBLs) from a single donor were exposed ex vivo to doses of 0-6 Gy X rays or 0-2 Gy α particles. Cells were harvested at two different times after irradiation to account for the mitotic delay of heavily damaged cells, which is known to occur after exposure to high-LET radiation (including α particles). Analysis of the kinetics of cells reaching first or second (and higher) mitosis after irradiation and aberration data obtained by the multiplex fluorescence in situ hybridization (mFISH) technique are used to determine of the cytogenetic risk, i.e., the probability for transmissible aberrations in surviving lymphocytes. The analysis shows that the cytogenetic risk after α exposure is lower than after X rays. This indicates that the actually observed higher carcinogenic effect of α radiation is likely to stem from small scale mutations that are induced effectively by high-LET radiation but cannot be resolved by mFISH analysis.

O-154 First mitotic spindle formation led by sperm centrosome-dependent microtubule organising centres may cause high incidence of zygotic division errors in humans

Abstract Study question Why do multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans? Summary answer Failure in microtubule assembly during the first mitotic spindle body formation by sperm centrosome-dependent microtubule organising centres (MTOCs) may lead to chromosomal instability. What is known already Unlike that in mice, multinucleated blastomeres appear at high frequency in two-cell-stage embryos in humans. However, the underlying mechanism remains elusive. In mice, multiple acentriolar MTOCs appear around the male and female pronuclei after pronuclear disappearance and contribute to dual-spindle formation, engulfing each parental chromosome. This spindle formation may ensure an error-free division, keeping the chromosomes stable during the first cleavage, as observed in mice, but it is unclear whether a similar mechanism exists in humans. Study design, size, duration To examine how sperm centrosomes contribute to MTOC formation in humans, two types of 3PN zygotes derived fromeither conventional in vitro fertilization (c-IVF, n = 30) or intracytoplasmic sperm injection (ICSI, n = 10) were used. The zygotes were collected from October 2018 to January 2020. MTOC and mitotic spindle formation at consecutive stages of development during the first cleavage were analysed under static and dynamic conditions using immunofluorescence assay and fluorescent live-cell imaging. Participants/materials, setting, methods Under ethics approval, 3PN zygotes were donated by infertile couples undergoing c-IVF or ICSI cycles at the Yamashita Shonan Yume Clinic in Japan. All participants provided informed consent. Immunofluorescence assay was performed using antibodies against α-tubulin, pericentrin, and H3K9me3 after fixation with MTSB-XF solution. Fluorescent live-cell imaging was performed using TagGFP2-H2B mRNA (chromosome marker) and FusionRed-MAP4 mRNA (microtubule marker). Main results and the role of chance Immunofluorescence revealed that while 3PN zygotes derived from c-IVF showed four pericentrin dots, those derived from ICSI exhibited two pericentrin dots. In pro-metaphase, an independent group of chromosomes derived from each pronucleus and MTOCs were formed by the sperm centrosome at the core. Microtubules from each MTOC extended toward the chromosomes in the early metaphase; a quadrupolar spindle was formed in the c-IVF-derived zygotes, and a bipolar spindle was formed in the ICSI-derived zygotes by the MTOCs at the zygote apex after chromosome alignment. In pro-metaphase, the microtubules extended from the MTOCs to the nearest chromosome. Since microtubule assembly was found on oocyte-derived chromosomes, we hypothesised that whether a chromosome is surrounded by microtubules depends on the location of the MTOCs, irrespective of its origin. Live-cell imaging of histone H2B and MAP4 revealed that four MTOCs appeared around the three pronuclei just before the disappearance of the pronuclear membrane; microtubules then extended from the MTOCs toward the chromosomes, beginning to form a mitotic spindle as the chromosomes moved to the centre of the oocyte. Interestingly, one of the three assembled chromosome groups showed no microtubule assembly in the pro-metaphase. Similar results were obtained in all six 3PN zygotes subjected. Limitations, reasons for caution We demonstrated the high risk of developing bare chromosomes not surrounded by microtubules during the formation of the first mitotic spindle, using human tripronuclear zygotes. However, owing to unavailability of normal fertilized oocytes for this study because of the clinical use, we were unable to confirm this in normal zygotes. Wider implications of the findings Although two sperm centrosome-dependent MTOCs are expected to be formed in normal fertilized oocytes, these MTOCs are not sufficient to completely enclose physically separated female and male chromosomes with the microtubules. This explains the high frequency of zygotic division errors that lead to unstable human chromosomes. Trial registration number not applicable

Evaluating prognostic value and relevant gene signatures of tumor microenvironment characterization in esophageal carcinoma.

Tumor microenvironment (TME) cells are an important part of tumor tissues. There is increasing evidence that the TME plays a vital role in tumor prognosis, and is associated with patient survival in various kinds of malignances. To date, very little research has been conducted on how to effectively use TME to better evaluate the prognosis of patients with esophageal carcinoma (EC). The concept of a "TME score" was introduced to better distinguish the prognosis of patients. We employed bioinformatic methods to investigate the TME infiltration patterns of 160 patients with EC from the Cancer Genome Atlas (TCGA) cohort. TME clusters were identified using k-means clustering methods with 1,000 resampling times. The significance of the survival difference among patients belonging to different TME clusters was assessed by the log-rank test and Kaplan-Meier survival curves. Correlations between immune cell types and survival were calculated by a Cox regression, and the Pearson correlation coefficient (PCC) was used to measure the relationship among different immune cell types. We classified patient into 2 subtypes based on the optimal breakpoint of TME score determined by R package maxstat. Two TME phenotypes were defined based on the immune cell type fractions, and patients with a high TME score phenotype had a better prognosis than those with a low TME score phenotype. Kaplan-Meier analysis for differentially expressed micro ribonucleic acids (RNAs) and messenger RNAs also showed that different TME score subtypes were significantly associated with the prognosis of EC. Just as tumor mutational burden can predict the efficacy of immunotherapy, the TME score can predict the efficacy of immune checkpoint inhibitors (ICIs). The genomic alterations of 2 TME score subtypes of EC further revealed that genomic instability is prevalent in TMEs, and patients with a low TME score subtype have a more unstable chromosome status than those with a high subtype. Thus, TME score is an emerging prognostic biomarker for predicting the efficacy of ICIs.

Interspecific potato somatic hybrids between Solanum malmeanum and S. tuberosum provide valuable resources for freezing-tolerance breeding

Freezing stress affects the geographic distribution, growth, and development of potato, resulting in yield loss. Solanum malmeanum, a diploid wild species with strong freezing tolerance, was fused with the freezing sensitive dihaploid S. tuberosum by somatic hybridization. In our study, 980 calli were obtained, and 248 differentiated shoots were obtained from the calli. Parental-specific SSR markers were used to analyse the chromosome composition of the 80 randomly selected regenerated plants, obtaining 51 somatic hybrids. Among them, 44 somatic hybrids were tested with ploidy analysis in the years 2016 and 2020. During subculture, the genomic ploidy levels changed due to the composition of the unstable chromosome in 56.82% of the somatic hybrids. The somatic hybrids showed better freezing tolerance than the cultivated parent. Then, freezing-tolerant somatic hybrids were selected to backcross with cultivars, and we obtained valuable breeding resources with enhanced freezing tolerance and tuberization capacity similar to that of cultivars. The correlation analysis showed that freezing tolerance has no relation with tuberization capacity, which indicates that they are controlled by independent genetic loci.

A new assay capturing chromosome fusions shows a protection trade-off at telomeres and NHEJ vulnerability to low-density ionizing radiation.

Chromosome fusions threaten genome integrity and promote cancer by engaging catastrophic mutational processes, namely chromosome breakage–fusion–bridge cycles and chromothripsis. Chromosome fusions are frequent in cells incurring telomere dysfunctions or those exposed to DNA breakage. Their occurrence and therefore their contribution to genome instability in unchallenged cells is unknown. To address this issue, we constructed a genetic assay able to capture and quantify rare chromosome fusions in budding yeast. This chromosome fusion capture (CFC) assay relies on the controlled inactivation of one centromere to rescue unstable dicentric chromosome fusions. It is sensitive enough to quantify the basal rate of end-to-end chromosome fusions occurring in wild-type cells. These fusions depend on canonical nonhomologous end joining (NHEJ). Our results show that chromosome end protection results from a trade-off at telomeres between positive effectors (Rif2, Sir4, telomerase) and a negative effector partially antagonizing them (Rif1). The CFC assay also captures NHEJ-dependent chromosome fusions induced by ionizing radiation. It provides evidence for chromosomal rearrangements stemming from a single photon–matter interaction.

Chinese hamster ovary cell line DXB-11: chromosomal instability and karyotype heterogeneity

BackgroundChinese hamster ovary cell lines, also known as CHO cells, represent a large family of related, yet quite different, cell lines which are metabolic mutants derived from the original cell line, CHO-ori. Dihydrofolate reductase-deficient DXB-11 cell line, one of the first CHO derivatives, serves as the host cell line for the production of therapeutic proteins. It is generally assumed that DXB-11 is identical to DUKX or CHO-DUK cell lines, but, to our knowledge, DXB-11 karyotype has not been described yet.ResultsUsing differential staining approaches (G-, C-banding and Ag-staining), we presented DXB-11 karyotype and revealed that karyotypes of DXB-11 and CHO-DUK cells have a number of differences. Although the number of chromosomes is equal—20 in each cell line—DXB-11 has normal chromosomes of the 1st and 5th pairs as well as an intact chromosome 8. Besides, in DXB-11 line, chromosome der(Z9) includes the material of chromosomes X and 6, whereas in CHO-DUK it results from the translocation of chromosomes 1 and 6. Ag-positive nucleolar organizer regions were revealed in the long arms of chromosome del(4)(q11q12) and both chromosome 5 homologues, as well as in the short arms of chromosomes 8 and add(8)(q11). Only 19 from 112 (16.96%) DXB-11 cells display identical chromosome complement accepted as the main structural variant of karyotype. The karyotype heterogeneity of all the rest of cells (93, 83.04%) occurs due to clonal and nonclonal additional structural rearrangements of chromosomes. Estimation of the frequency of chromosome involvement in these rearrangements allowed us to reveal that chromosomes 9, der(X)t(X;3;4), del(2)(p21p23), del(2)(q11q22) /Z2, der(4) /Z7, add(6)(p11) /Z8 are the most stable, whereas mar2, probably der(10), is the most unstable chromosome. A comparative analysis of our own and literary data on CHO karyotypes allowed to designate conservative chromosomes, both normal and rearranged, that remain unchanged in different CHO cell lines, as well as variable chromosomes that determine the individuality of karyotypes of CHO derivatives.ConclusionDXB-11and CHO-DUK cell lines differ in karyotypes. The revealed differential instability of DXB-11 chromosomes is likely not incidental and results in karyotype heterogeneity of cell population.