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Do all birds' sex chromosomes follow the same canonical one-way direction of evolution? We combined cytogenetic and genomic approaches to analyze the process of the W chromosomal differentiation in two selected Passeriform species, named the Pale-breasted Thrush Turdus leucomelas and the Rufous-bellied thrush T. rufiventris. We characterized the full catalog of satellite DNAs (satellitome) of T. leucomelas, and the 10 TleSatDNA classes obtained together with 16 microsatellite motifs were in situ mapped in both species. Additionally, using Comparative Genomic Hybridization (CGH) assays, we investigated their intragenomic variations. The W chromosomes of both species did not accumulate higher amounts of both heterochromatin and repetitive sequences. However, while T. leucomelas showed a heterochromatin-poor W chromosome with a very complex evolutionary history, T. rufiventris showed a small and partially heterochromatic W chromosome that represents a differentiated version of its original autosomal complement (Z chromosome). The combined approach of CGH and sequential satDNA mapping suggest the occurrence of a former W-autosomal translocation event in T. leucomelas, which had an impact on the W chromosome in terms of sequence gains and losses. At the same time, an autosome, which is present in both males and females in a polymorphic state, lost sequences and integrated previously W-specific ones. This putative W-autosomal translocation, however, did not result in the emergence of a multiple-sex chromosome system. Instead, the generation of a neo-W chromosome suggests an unexpected evolutionary trajectory that deviates from the standard canonical model of sex chromosome evolution.

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Utilization of crop wild relatives of wheat can be very effective in building the genetic diversity to cater to the evolving strains of disease pathogens. Aegilops speltoides is a rich source of rust resistance genes however transferring those to wheat genome can be tedious due to co-transfer and preferential transmission of undesirable genes causing gametocidal activity. Such an unholy association was observed in Triticum aestivum-Ae. speltoides derivative line Sel. 2427 which possess the broad-spectrum leaf rust seedling resistance gene (LrS2427). Molecular analysis based on 35 K wheat breeder's array revealed the maximum percentage of Ae. speltoides genome introgression on homoeologous group 2. In situ hybridization studies revealed the presence of S genome in Sel. 2427, showing six translocations on four chromosomes. Karyotyping using repetitive probe (AAG)6 revealed that the two chromosomes involved are 2D and 2B. Genic regions causing gametocidal activity were identified by dissecting it into component traits and QTLs on 2D and 2B chromosomes were revealed in case of the trait seed shrivelling index. To break the inadvertent association of LrS2427 with gametocidal genes, F1(Agra Local X Sel. 2427) seeds were irradiated with gamma rays and stable leaf rust resistant mutants lacking gametocidal activity were developed. These mutants showed resistance to different races of leaf rust pathogen and showed superior agronomic performance as well. These mutants could be a great resource in wheat improvement for utilization of the leaf rust resistance gene LrS2427 without any yield penalty. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-024-01491-8.

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Genetic toxicology, strategically located at the intersection of genetics and toxicology, aims to demystify the complex interplay between exogenous agents and our genetic blueprint. Telomeres, the protective termini of chromosomes, play instrumental roles in cellular longevity and genetic stability. Traditionally karyotyping and fluorescence in situ hybridisation (FISH), have been indispensable tools for chromosomal analysis following exposure to genotoxic agents. However, their scope in discerning nuanced molecular dynamics is limited. Peptide Nucleic Acids (PNAs) are synthetic entities that embody characteristics of both proteins and nucleic acids and have emerged as potential game-changers. This perspective report comprehensively examines the vast potential of PNAs in genetic toxicology, with a specific emphasis on telomere research. PNAs' superior resolution and precision make them a favourable choice for genetic toxicological assessments. The integration of PNAs in contemporary analytical workflows heralds a promising evolution in genetic toxicology, potentially revolutionizing diagnostics, prognostics, and therapeutic avenues. In this timely review, we attempted to assess the limitations of current PNA-FISH methodology and recommend refinements.

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Recently, hypotheses regarding the evolutionary patterns of ribosomal genes in ant chromosomes have been under discussion. One of these hypotheses proposes a relationship between chromosomal location and the number of rDNA sites, suggesting that terminal locations facilitate the dispersion of rDNA clusters through ectopic recombination during meiosis, while intrachromosomal locations restrict them to a single chromosome pair. Another hypothesis suggests that the multiplication of rDNA sites could be associated with an increase in the chromosome number in Hymenoptera due to chromosomal fissions. In this study, we physically mapped rDNA sites in 15 new ant species and also reviewed data on rDNA available since the revision by Teixeira et al. (2021a). Our objectives were to investigate whether the new data confirm the relationship between chromosomal location and the number of rDNA sites, and whether the increase in the chromosome number is significant in the dispersion of rDNA clusters in ant karyotypes. Combining our new data with all information on ant cytogenetics published after 2021, 40 new species and nine new genera were assembled. Most species exhibited intrachromosomal rDNA sites on a single chromosome pair, while three species showed these genes in terminal regions of multiple chromosome pairs. On one hand, the hypothesis that the chromosomal location of rDNA clusters may facilitate the dispersion of rDNA sites in the ant genome, as previously discussed, was strengthened, but, on the other hand, the hypothesis of chromosomal fission as the main mechanism for dispersion of ribosomal genes in ants is likely to be refuted. Furthermore, in certain genera, the location of rDNA sites remained similar among the species studied, whereas in others, the distribution of these genes showed significant variation between species, suggesting a more dynamic chromosomal evolution.

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Somatic chromosomal mosaicism, chromosome instability, and cancer are intimately linked together. Addressing the role of somatic genome variations (encompassing chromosomal mosaicism and instability) in cancer yields paradoxical results. Firstly, somatic mosaicism for specific chromosomal rearrangement causes cancer per se. Secondly, chromosomal mosaicism and instability are associated with a variety of diseases (chromosomal disorders demonstrating less severe phenotypes, complex diseases), which exhibit cancer predisposition. Chromosome instability syndromes may be considered the best examples of these diseases. Thirdly, chromosomal mosaicism and instability are able to result not only in cancerous diseases but also in non-cancerous disorders (brain diseases, autoimmune diseases, etc.). Currently, the molecular basis for these three outcomes of somatic chromosomal mosaicism and chromosome instability remains incompletely understood. Here, we address possible mechanisms for the aforementioned scenarios using a system analysis model. A number of theoretical models based on studies dedicated to chromosomal mosaicism and chromosome instability seem to be valuable for disentangling and understanding molecular pathways to cancer-causing genome chaos. In addition, technological aspects of uncovering causes and consequences of somatic chromosomal mosaicism and chromosome instability are discussed. In total, molecular cytogenetics, cytogenomics, and system analysis are likely to form a powerful technological alliance for successful research against cancer.

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BACKGROUND: Unreduced gamete formation during meiosis plays a critical role in natural polyploidization. However, the unreduced gamete formation mechanisms in Triticum turgidum-Aegilops umbellulata triploid F1 hybrid crosses and the chromsome numbers and compostions in T. turgidum-Ae. umbellulata F2 still not known. RESULTS: In this study, 11 T.turgidum-Ae. umbellulata triploid F1 hybrid crosses were produced by distant hybridization. All of the triploid F1 hybrids had 21 chromosomes and two basic pathways of meiotic restitution, namely first-division restitution (FDR) and single-division meiosis (SDM). Only FDR was found in six of the 11 crosses, while both FDR and SDM occurred in the remaining five crosses. The chromosome numbers in the 127 selfed F2 seeds from the triploid F1 hybrid plants of 10 crosses (no F2 seeds for STU 16) varied from 35 to 43, and the proportions of euploid and aneuploid F2 plants were 49.61% and 50.39%, respectively. In the aneuploid F2 plants, the frequency of chromosome loss/gain varied among genomes. The chromosome loss of the U genome was the highest (26.77%) among the three genomes, followed by that of the B (22.83%) and A (11.81%) genomes, and the chromosome gain for the A, B, and U genomes was 3.94%, 3.94%, and 1.57%, respectively. Of the 21 chromosomes, 7U (16.54%), 5 A (3.94%), and 1B (9.45%) had the highest loss frequency among the U, A, and B genomes. In addition to chromosome loss, seven chromosomes, namely 1 A, 3 A, 5 A, 6 A, 1B, 1U, and 6U, were gained in the aneuploids. CONCLUSION: In the aneuploid F2 plants, the frequency of chromosome loss/gain varied among genomes, chromsomes, and crosses. In addition to variations in chromosome numbers, three types of chromosome translocations including 3UL·2AS, 6UL·1AL, and 4US·6AL were identified in the F2 plants. Furthermore, polymorphic fluorescence in situ hybridization karyotypes for all the U chromosomes were also identified in the F2 plants when compared with the Ae. umbellulata parents. These results provide useful information for our understanding the naturally occurred T. turgidum-Ae. umbellulata amphidiploids.

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BACKGROUND: Different patterns of sex chromosome differentiation are seen in Palaeognathae birds, a lineage that includes the ratites (Struthioniformes, Rheiformes, Apterygiformes, Casuariiformes, and the sister group Tinamiformes). While some Tinamiform species have well-differentiated W chromosomes, both Z and W of all the flightless ratites are still morphologically undifferentiated. Here, we conducted a comprehensive analysis of the ZW differentiation in birds using a combination of cytogenetic, genomic, and bioinformatic approaches. The whole set of satDNAs from the emu (Dromaius novaehollandiae) was described and characterized. Furthermore, we examined the in situ locations of these satDNAs alongside several microsatellite repeats and carried out Comparative Genomic Hybridizations in two related species: the greater rhea (Rhea americana) and the tataupa tinamou (Crypturellus tataupa). RESULTS: From the 24 satDNA families identified (which represent the greatest diversity of satDNAs ever uncovered in any bird species), only three of them were found to accumulate on the emu's sex chromosomes, with no discernible accumulation observed on the W chromosome. The W chromosomes of both the greater rhea and the emu did not exhibit a significant buildup of either C-positive heterochromatin or repetitive DNAs, indicating their large undifferentiation both at morphological and molecular levels. In contrast, the tataupa tinamou has a highly differentiated W chromosome that accumulates several DNA repeats. CONCLUSION: The findings provide new information on the architecture of the avian genome and an inside look at the starting points of sex chromosome differentiation in birds.

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BACKGROUND: Acute myeloid leukemia (AML) is characterized by clonal heterogeneity, leading to frequent relapses and drug resistance despite intensive clinical therapy. Although AML's clonal architecture has been addressed in many studies, practical monitoring of dynamic changes in those subclones during relapse and treatment is still understudied. METHOD: Fifteen longitudinal bone marrow (BM) samples were collected from three relapsed and refractory (R/R) AML patients. Using droplet digital polymerase chain reaction (ddPCR), the frequencies of patient's leukemic variants were assessed in seven cell populations that were isolated from each BM sample based on cellular phenotypes. By quantifying mutant clones at the diagnosis, remission, and relapse stages, the distribution of AML subclones was sequentially monitored. RESULTS: Minimal residual (MR) leukemic subclones exhibit heterogeneous distribution among BM cell populations, including mature leukocyte populations. During AML progression, these subclones undergo active phenotypic transitions and repopulate into distinct cell population regardless of normal hematopoiesis hierarchic order. Of these, MR subclones in progenitor populations of patient BM predominantly carry MR leukemic properties, leading to more robust expansion and stubborn persistence than those in mature populations. Moreover, a minor subset of MR leukemic subclones could be sustained at an extremely low frequency without clonal expansion during relapse. CONCLUSIONS: In this study, we observed treatment persistent MR leukemic subclones and their phenotypic changes during the treatment process of R/R AML patients. This underscores the importance of preemptive inhibition of clonal promiscuity in R/R AML, proposing a practical method for monitoring AML MR subclones.

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Chromosomal rearrangements are often associated with playing a role in the speciation process. However, the underlying mechanism that favors the genetic isolation associated with chromosomal changes remains elusive. In this sense, the genus Mazama is recognized by its high level of karyotype diversity among species with similar morphology. A cryptic species complex has been identified within the genus, with the red brocket deer (Mazama americana and Mazama rufa) being the most impressive example. The chromosome variation was clustered in cytotypes with diploid numbers ranging from 42 to 53 and was correlated with geographical location. We conducted an analysis of chromosome evolution of the red brocket deer complex using comparative chromosome painting and Bacterial Artificial Chromosome (BAC) clones among different cytotypes. The aim was to deepen our understanding of the karyotypic relationships within the red brocket, thereby elucidating the significant chromosome variation among closely related species. This underscores the significance of chromosome changes as a key evolutionary process shaping their genomes. The results revealed the presence of three distinct cytogenetic lineages characterized by significant karyotypic divergence, suggesting the existence of efficient post-zygotic barriers. Tandem fusions constitute the main mechanism driving karyotype evolution, following a few centric fusions, inversion X-autosomal fusions. The BAC mapping has improved our comprehension of the karyotypic relationships within the red brocket deer complex, prompting questions regarding the role of these changes in the speciation process. We propose the red brocket as a model group to investigate how chromosomal changes contribute to isolation and explore the implications of these changes in taxonomy and conservation.

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Introduction: The majority of small supernumerary marker chromosomes (sSMCs) are derived from one single chromosome. Complex sSMCs, on the other hand, consist of genetic material derived from more than one, normally two chromosomes. Complex sSMCs involving chromosomes 8 and 14 are rarely encountered. Case presentation: We present here a 14-month-old boy born from an unrelated couple. At birth, the baby was hypotonic and had a cleft lip and palate, as well as ocular involvement. Throughout the course of development, the baby experienced feeding difficulties, stunted growth, and delayed psychomotor development. Banding together with molecular cytogenetics revealed a balanced maternal translocation t(8;14)(p22.3;q21)mat, leading due to meiotic 3:1 segregation to a partial trisomy of chromosomes 8 and 14 in the affected boy. Discussion/Conclusion: This report highlights the importance of cytogenetics in diagnosis of rare genetic disorders, with impact on genetic counselling of patients and their families. There are three comparable cases in the literature involving both chromosomes 8 and 14, but with different breakpoints; the complex sSMC derived from chromosomes 8 and 14 in this case, characterized as der(14)t(8;14) (p22.3;q21)mat.

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The genus Vigna (Leguminosae) comprises about 150 species grouped into five subgenera. The present study aimed to improve the understanding of karyotype diversity and evolution in Vigna, using new and previously published data through different cytogenetic and DNA content approaches. In the Vigna subgenera, we observed a random distribution of rDNA patterns. The 35S rDNA varied in position, from terminal to proximal, and in number, ranging from one (V. aconitifolia, V. subg. Ceratotropis) to seven pairs (V. unguiculata subsp. unguiculata, V. subg. Vigna). On the other hand, the number of 5S rDNA was conserved (one or two pairs), except for V. radiata (V. subg. Ceratotropis), which had three pairs. Genome size was relatively conserved within the genus, ranging from 1C = 0.43 to 0.70 pg in V. oblongifolia and V. unguiculata subsp. unguiculata, respectively, both belonging to V. subg. Vigna. However, we observed a positive correlation between DNA content and the number of 35S rDNA sites. In addition, data from chromosome-specific BAC-FISH suggest that the ancestral 35S rDNA locus is conserved on chromosome 6 within Vigna. Considering the rapid diversification in the number and position of rDNA sites, such conservation is surprising and suggests that additional sites may have spread out from this ancestral locus.

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Fluorescence in situ hybridization (FISH) technique has been widely used to detect and localize specific DNA and RNA sequences in interphase nuclei and chromosomes in animals and plants. Here, we present a protocol for localization of genomic loci in nuclei of the model plant Arabidopsis thaliana. This protocol includes several advances and adaptations to A. thaliana, including preparation of nuclei and chromosomes without the use of liquid nitrogen, and an in situ hybridization procedure that preserves chromatin structure without the use of paraformaldehyde and formamide. Simultaneous denaturation of the BAC (bacterial artificial chromosome) probe and nuclei followed by annealing at high temperature allows hybridization in less than an hour. These hybridization conditions also provide high signal to noise ratio by a small number of washes. Thus, this simplified in situ hybridization procedure is completed in one working day.

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Although both are salient features of genomes, at first glance ribosomal DNAs and transposable elements are genetic elements with not much in common: whereas ribosomal DNAs are mainly viewed as housekeeping genes that uphold all prime genome functions, transposable elements are generally portrayed as selfish and disruptive. These opposing characteristics are also mirrored in other attributes: organization in tandem (ribosomal DNAs) versus organization in a dispersed manner (transposable elements); evolution in a concerted manner (ribosomal DNAs) versus evolution by diversification (transposable elements); and activity that prolongs genomic stability (ribosomal DNAs) versus activity that shortens it (transposable elements). Re-visiting relevant instances in which ribosomal DNA-transposable element interactions have been reported, we note that both repeat types share at least four structural and functional hallmarks: (1) they are repetitive DNAs that shape genomes in evolutionary timescales, (2) they exchange structural motifs and can enter co-evolution processes, (3) they are tightly controlled genomic stress sensors playing key roles in senescence/aging, and (4) they share common epigenetic marks such as DNA methylation and histone modification. Here, we give an overview of the structural, functional, and evolutionary characteristics of both ribosomal DNAs and transposable elements, discuss their roles and interactions, and highlight trends and future directions as we move forward in understanding ribosomal DNA-transposable element associations.

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Charadriiformes, which comprises shorebirds and their relatives, is one of the most diverse avian orders, with over 390 species showing a wide range of karyotypes. Here, we isolated and characterized the whole collection of satellite DNAs (satDNAs) at both molecular and cytogenetic levels of one of its representative species, named the wattled jacana (Jacana jacana), a species that contains a typical ZZ/ZW sex chromosome system and a highly rearranged karyotype. In addition, we also investigate the in situ location of telomeric and microsatellite repeats. A small catalog of 11 satDNAs was identified that typically accumulated on microchromosomes and on the W chromosome. The latter also showed a significant accumulation of telomeric signals, being (GA)10 the only microsatellite with positive hybridization signals among all the 16 tested ones. These current findings contribute to our understanding of the genomic organization of repetitive DNAs in a bird species with high degree of chromosomal reorganization contrary to the majority of bird species that have stable karyotypes.

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Small nuclear DNA (snDNA) are valuable cytogenetic markers for comparative studies in chromosome evolution because different distribution patterns were found among species. Parodontidae, a Neotropical fish family, is known to have female heterogametic sex chromosome systems in some species. The U2 and U4 snDNA sites have been found to be involved in Z and W chromosome differentiation in Apareiodon sp., Apareiodon affinis, and Parodon hilarii. However, few studies have evaluated snDNA sites as propulsors of chromosome diversification among closely related fish species. In this study, we investigated the distribution of U2 and U4 snDNA clusters in the chromosomes of 10 populations/species belonging to Apareiodon and Parodon, aiming to identify chromosomal homeologies or diversification. In situ localization data revealed a submetacentric pair carrying the U2 snDNA site among the populations/species analyzed. Furthermore, all studied species demonstrated homeology in the location of U4 snDNA cluster in the proximal region of metacentric pair 1, besides an additional signal showing up with a divergence in Apareiodon. Comparative chromosomal mapping of U4 snDNA also helped to reinforce the proposal of the ZZ/ZW1W2 sex chromosome system origin in an A. affinis population. According to cytogenetic data, the study corroborates the diversification in Parodontidae paired species with uncertain taxonomy.

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Fluorescence in situ hybridization (FISH) is an indispensable technique for studying chromosomes in plants. However, traditional FISH methods, such as BAC, rDNA, tandem repeats, and distributed repetitive sequence probe-based FISH, have certain limitations, including difficulties in probe synthesis, low sensitivity, cross-hybridization, and limited resolution. In contrast, oligo-based FISH represents a more efficient method for chromosomal studies in plants. Oligo probes are computationally designed and synthesized for any plant species with a sequenced genome and are suitable for single and repetitive DNA sequences, entire chromosomes, or chromosomal segments. Furthermore, oligo probes used in the FISH experiment provide high specificity, resolution, and multiplexing. Moreover, oligo probes made from one species are applicable for studying other genetically and taxonomically related species whose genome has not been sequenced yet, facilitating molecular cytogenetic studies of non-model plants. However, there are some limitations of oligo probes that should be considered, such as requiring prior knowledge of the probe design process and FISH signal issues with shorter probes of background noises during oligo-FISH experiments. This review comprehensively discusses de novo oligo probe synthesis with more focus on single-copy DNA sequences, preparation, improvement, and factors that affect oligo-FISH efficiency. Furthermore, this review highlights recent applications of oligo-FISH in a wide range of plant chromosomal studies.

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Crocodilians have maintained very similar karyotype structures and diploid chromosome numbers for around 100 million years, with only minor variations in collinearity. Why this karyotype structure has largely stayed unaltered for so long is unclear. In this study, we analyzed the karyotypes of six species belonging to the genera Crocodylus and Osteolaemus (Crocodylidae, true crocodiles), among which the Congolian endemic O. osborni was included and investigated. We utilized various techniques (differential staining, fluorescence in situ hybridization with repetitive DNA and rDNA probes, whole chromosome painting, and comparative genomic hybridization) to better understand how crocodile chromosomes evolved. We studied representatives of three of the four main diploid chromosome numbers found in crocodiles (2n = 30/32/38). Our data provided new information about the species studied, including the identification of four major chromosomal rearrangements that occurred during the karyotype diversification process in crocodiles. These changes led to the current diploid chromosome numbers of 2n = 30 (fusion) and 2n = 38 (fissions), derived from the ancestral state of 2n = 32. The conserved cytogenetic tendency in crocodilians, where extant species keep near-ancestral state, contrasts with the more dynamic karyotype evolution seen in other major reptile groups.

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The quality of chromosome preparation influences all downstream analyses and is therefore crucial. Hence, numerous protocols exist to produce microscopic slides with mitotic chromosomes. Nevertheless, due to the high content of fibers in and around a plant cell, preparation of plant chromosomes is still far from trivial and needs to be fine-tuned for each species and tissue type. Here, we outline the "dropping method," a straightforward and efficient protocol to prepare multiple slides with uniform quality from a single chromosome preparation. In this method, nuclei are extracted and cleaned to produce a nuclei suspension. In a drop-by-drop manner, this suspension is then applied from a certain height onto the slides, causing the nuclei to rupture and the chromosomes to spread. Due to the physical forces that accompany the dropping and spreading process, this method is best suited for species with small- to medium-sized chromosomes.

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Fluorescence in situ hybridization (FISH) has been widely used to visualize target DNA sequences in fixed chromosome samples by denaturing the dsDNA to allow complementary probe hybridization, thus damaging the chromatin structure by harsh treatments. To overcome this limitation, a CRISPR/Cas9-based in situ labeling method was developed, termed CRISPR-FISH. This method is also known as RNA-guided endonuclease-in situ labeling (RGEN-ISL). Here we present different protocols for the application of CRISPR-FISH on acetic acid: ethanol or formaldehyde-fixed nuclei and chromosomes as well as tissue sections for labeling repetitive sequences in a range of plant species. In addition, methods on how immunostaining can be combined with CRISPR-FISH are provided.

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