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Hodgkin's disease: diagnosis by fine-needle aspiration biopsy. Analysis of cytologic criteria from a selected series.

Two hundred thirteen cases in which a lymph node aspirate and subsequent surgical biopsy had been performed were studied independently by four observers. Each observer attempted to discriminate involvement by Hodgkin's disease from involvement by other processes. The material included 18 cases of histologically confirmed Hodgkin's disease. The remaining 195 cases included a wide range of pathologic processes. All four observers were able to distinguish Hodgkin's disease from other processes in the great majority of cases. Two observers reviewed the aspirate material from the 18 cases of Hodgkin's disease to determine the presence and significance of elements known to characterize this disease, i.e., multinuclear and mononuclear Reed-Sternberg cells, polyploidal cells, granulomatous elements, metachromatic material, necrosis, eosinophils, neutrophils, and plasma cells. Reed-Sternberg cells were present in most but not all cases. Reed-Sternberg-like cells also were occasionally encountered in other processes. Polyploidal cells were invariably present and were useful in recognizing the disease. Granulomatous elements, metachromatic material, necrosis, eosinophils, and neutrophils were frequently present and, while not specific for the process, helped to draw attention to or substantiate the diagnosis of Hodgkin's disease.

Biopsy, Needle↗

Flow cytometric analysis of DNA content in Hürthle cell adenomas and carcinomas of the thyroid.

Paraffin-embedded surgical biopsy material from 17 Hürthle cell tumors of the thyroid was examined for DNA content by flow cytometry to assess the diagnostic and prognostic utility of ploidy determinations in these rare tumors. Both adenomas (11 cases) and carcinomas (6 cases) were studied. As a control for methods, ten randomly selected normal autopsy thyroids were analyzed, all of which demonstrated normal diploid DNA content. Among the Hürthle cell tumors, however, aneuploid peaks were present in six adenomas (55%) and in four carcinomas (67%). Similarly, polyploid DNA peaks in the absence of other aneuploid peaks were present in two adenomas and two carcinomas (18% and 33%, respectively). These findings demonstrate the limited value of aneuploidy or polyploidy as diagnostic features for malignancy in Hürthle cell tumors of the thyroid. As for prognosis, there does not appear to be any unfavorable prognostic significance for abnormal DNA content in histologically benign Hürthle cell tumors treated by surgical excision because no metastases or recurrences occurred in this group at a mean disease-free follow-up of 50 +/- 19 months for six aneuploid lesions and 19 +/- 7 months for two polyploid adenomas. Preliminary data suggest that aneuploidy may, however, have an important prognostic value for histologically defined Hürthle cell carcinomas, because the only patient to die from the tumor in this series had an aneuploid Hürthle carcinoma. Thus, the authors' data indicate that the diagnostic utility of DNA content in Hürthle cell tumors is extremely limited and that there does not appear to be any negative prognostic significance for aneuploidy in histologically defined Hürthle cell adenomas.

Adenoma↗

Phylogenomics reveals persistent gene-tree discordance in the Chenopodium album aggregate.

BACKGROUND AND AIMS: Complex genomic histories shaped by hybridisation and polyploidy can influence traits related to plant defence, stress tolerance and toxicity, particularly in Amaranthaceae, which includes crops such as quinoa and spinach. Within this family, white goosefoot (Chenopodium album), a widespread agricultural weed and traditional food resource, belongs to a diploid-polyploid aggregate with extensive phylogenetic discordance. Clarifying its evolutionary history provides context for interpreting ecologically and agronomically relevant trait variation across the aggregate. Building on the established genome-lineage framework, we tested whether discordance persists when constituent genome-lineage components are represented separately and whether the remaining signal is compatible with reticulate evolution. METHODS: We analysed 2,298 conserved nuclear BUSCO families across 27 assembly-level terminals using tree- and network-based approaches. Genome-lineage-aware analyses used 2,156 families after separating polyploid Chenopodium into A-H components, with Dysphania ambrosioides as outgroup. HyDe tested site-pattern asymmetry under global false-discovery-rate correction. KEY RESULTS: Assembly-level analyses grouped the Danish C. album aggregate accession Ca6-1 with hexaploid C. album sensu stricto dcCheAlbu1.1, whereas relationships among surrounding Chenopodium taxa were less stable. Genome-lineage-aware analyses recovered the expected B-, C- and D-affinity relationships, but substantial gene-family heterogeneity persisted. Reticulate network models fitted the assembly-level data better than bifurcating models, although inferred patterns differed between methods. HyDe detected significant site-pattern asymmetry in a small subset of loci, with most retained signal shared between the focal assemblies. CONCLUSIONS: Gene-tree discordance persists in the C. album aggregate after genome-lineage separation. The established genome-lineage framework captures the dominant phylogenomic structure, while residual heterogeneity is compatible with both tree-like and reticulate processes without identifying direct progenitors or a unique hybridisation history. This framework supports future analyses of lineage-specific and trait-associated loci related to plant defence, food quality and toxicity in C. album and related Amaranthaceae.

Chenopodium album↗

Comparative analysis of rDNA distribution in chromosomes of various species of Brassicaceae.

BACKGROUND AND AIMS: The Brassicaceae family encompasses numerous species of great agronomic importance, belonging to such genera, as Brassica, Raphanus, Sinapis and Armoracia. Many of them are characterized by extensive intraspecific diversity of phenotypes. The present study focuses on the polymorphism of number, appearance and chromosomal localization of ribosomal DNA (rDNA) sites and, when possible, in relation to polyploidy, in 42 accessions of Brassica species and ten accessions of Diplotaxis, Eruca, Raphanus and Sinapis species. METHODS: Chromosomal localization of ribosomal DNA was carried out using dual colour fluorescence in situ hybridization (FISH) with 5S rDNA and 25S rDNA sequences as probes on enzymatically digested root-tip meristematic cells. KEY RESULTS: Loci for 5S and 18S-5.8S-25S rDNA were determined for the first time in six taxa, and previously unreported rDNA constellations were described in an additional 12 accessions. FISH revealed frequent polymorphism in number, appearance and chromosomal localization of both 5S and 25S rDNA sites. This phenomenon was most commonly observed in the A genome of Brassica, where it involves exclusively pericentromeric sites of 5S and 25S rRNA genes. The intraspecific polymorphism was between subspecies/varieties or within a variety or cultivar (i.e. interindividual). CONCLUSIONS: The number of rDNA sites can differ up to 5-fold in species with the same chromosome number. In addition to the eight previously reported chromosomal types with ribosomal genes, three new variant types are described. The extent of polymorphism is genome dependent. Comparing the A, B and C genomes revealed the highest rDNA polymorphism in the A genome. The loci carrying presumably inactive ribosomal RNA genes are particularly prone to polymorphism. It can also be concluded that there is no obvious polyploidization-related tendency to reduce the number of ribosomal DNA loci in the allotetraploid species, when compared with their putative diploid progenitors. The observed differences are rather caused by the prevailing polymorphism within the diploids and allotetraploids. This would make it difficult to predict expected numbers of rDNA loci in natural polyploids.

Brassicaceae↗

Nuclear DNA content variation among Central European Koeleria taxa.

BACKGROUND AND AIMS: Polyploidization plays an important role in the evolution of many plant genera, including Koeleria. The knowledge of ploidy, chromosome number and genome size may enable correct taxonomic treatment when other features are insufficient as in Koeleria. Therefore, these characteristics and their variability were determined for populations of six central European Koeleria taxa. METHODS: Chromosome number analysis was performed by squashing root meristems, and ploidy and 2C nuclear DNA content were estimated by flow cytometry. KEY RESULTS: Three diploids (K. glauca, K. macrantha var. macrantha and var. pseudoglauca), one tetraploid (K. macrantha var. majoriflora), one decaploid (K. pyramidata) and one dodecaploid (K. tristis) were found. The 2C nuclear DNA content of the diploids ranged from 4.85 to 5.20 pg. The 2C DNA contents of tetraploid, decaploid and dodecaploid taxa were 9.31 pg, 22.89 pg and 29.23 pg, respectively. The DNA content of polyploids within the K. macrantha aggregate (i.e. within K. macrantha and K. pyramidata) was smaller than the expected multiple of the diploid genome (K. macrantha var. macrantha). Geography-correlated variation of DNA content was found for some taxa. Czech populations of K. macrantha var. majoriflora had a 5.06% smaller genome than the Slovak ones. An isolated eastern Slovakian population of K. tristis revealed 8.04% less DNA than populations from central Slovakia. In central and north-west Bohemia, diploid and tetraploid cytotypes of K. macrantha were sympatric; east from this region diploid populations, and towards the west tetraploid populations were dominant. CONCLUSIONS: Remarkable intra-specific inter-population differences in nuclear DNA content were found between Bohemian and Pannonian populations of Koeleria macrantha var. majoriflora and between geographically isolated central and eastern Slovakian populations of K. tristis. These differences occur over a relatively small geographical scale.

Cell Nucleus↗

Stimulation of DNA synthesis in carcinogen-induced diploid hepatocytes in vitro.

Diploid hepatocytes induced by a combination of diethylnitrosamine and 2-acetylaminofluorene were isolated and separated from polyploid hepatocytes by centrifugal elutriation. The diploid and polyploid cell fractions were approximately 90% pure and contained between 1 and 1.5 x 10(7) cells. When kept in monolayer cultures both cell populations responded to the mitogenic effect of EGF and insulin. However, the percentage of labelled nuclei was higher in predominantly diploid compared to predominantly tetraploid hepatocyte cultures at all epidermal growth factor (EGF) concentrations used in this study. At 10 ng EGF/ml and 10 mU insulin/ml the labelling index was twice as high in the diploid liver cells. Further work is required to show the relevance of the stronger response of the diploid cell fraction to mitogens in the process of carcinogenesis.

2-Acetylaminofluorene↗

A genetic linkage map of Saccharum spontaneum L. 'SES 208'.

The arbitrarily primed polymerase chain reaction was used to detect single-dose polymorphisms that, in turn, were used to generate a linkage map of a polyploid relative of cultivated sugarcane, Saccharum spontaneum 'SES 208' (2n = 64). The mapping population was composed of 88 progeny from a cross between SES 208 and a diploidized haploid derived from SES 208 by anther culture, ADP 85-0068. This cross allowed direct analysis of meiosis in SES 208 and gametic segregation ratios to be observed. One hundred twenty-seven 10-mer oligonucleotide primers of arbitrary sequence were selected from a pool of 420 primers used to screen the mapping parents. Three hundred thirty-six of the 420 primers amplified 4,540 loci or 13.5 loci per primer. The selected 127 primers revealed 2,160 loci of which 279 were present in SES 208 and absent in ADP 85-0068 and easily scored. Two hundred and eight (74.6%) of these 279 polymorphisms were single-dose polymorphisms (i.e., they displayed 1:1 segregation, chi 2 at 98% confidence level). Linkage analysis (theta = 0.25, LOD = 9.0 for two-point analysis, then theta = 0.25, LOD = 6.0 for multipoint analysis) of single-dose polymorphisms placed them into 42 linkage groups containing at least 2 markers. These single-dose markers span 1,500 contiguous centimorgans (cM) with 32 markers remaining unlinked (15.4%). From this 208-marker map we estimated the genome size of SES 208 to be 2,500 cM. The map has a predicted coverage of 85.1% at 30 cM, meaning that any new marker placed has an 85.1% chance of being within 30 cM of an existing marker. Furthermore, we show that SES 208 behaves like an autopolyploid because (i) the ratio of single-dose markers to higher dose markers fit the assumption of auto-octaploidy and (ii) the absence of repulsion phase linkages. This is the first genetic map constructed directly on a polyploid species for which no diploid relatives are known.

Base Sequence↗

A multivalent pairing model of linkage analysis in autotetraploids.

Polyploidy has been recognized as an important step in the evolutionary diversification of flowering plants and may have a significant impact on plant breeding. Statistical analyses for linkage mapping in polyploid species can be difficult due to considerable complexities in polysomic inheritance. In this article, we develop a novel statistical method for linkage analysis of polymorphic markers in a full-sib family of autotetraploids. This method is established on multivalent pairings of homologous chromosomes at meiosis and can provide a simultaneous maximum-likelihood estimation of the double reduction frequencies of and recombination fraction between two markers. The EM algorithm is implemented to provide a tractable way for estimating relative proportions of different modes of gamete formation that generate identical gamete genotypes due to multivalent pairings. Extensive simulation studies were performed to demonstrate the statistical properties of this method. The implications of the new method for understanding the genome structure and organization of polyploid species are discussed.

Algorithms↗

The chromosomes of turkey embryos during early stages of parthenogenetic development.

In the early stages of parthenogenetic development in turkey eggs, many blastoderms are mosaics of haploid, diploid and polyploid cells. The genome composition of these blastoderms can be identified by C-banding. They may be generally described as either A-Z/2A-ZZ/nA-nZ or A-W/2A-WW/nA-nW and are found in a nearly 1:1 ratio. The blastoderms showing the W body (W+) become lethal within two days of incubation. The haploid cell proportion decreases rapidly during the early stage of development, and, as haploid cells decrease, the proportion of polyploid cells appears to increase. At six days of incubation, various kinds of parthenogenetic development can be observed. Their genome compositions are either diploid (2A-ZZ) or mosaic (A-Z/2A-ZZ). These findings suggest that diploid parthenogenesis occurs by either suppression of meiosis II or chromosome doubling some time after the first cleavage division. The frequent occurrence of mosaic blastoderms indicates that the majority, if not all, of the parthenogenetic embryos initiate their development in haploid ova.

Animals↗

Scanning electron microscopy analysis of the human zona pellucida: influence of maturity and fertilization on morphology and sperm binding pattern.

Human oocytes from the same as well as from different patients have an extremely heterogeneous morphology of the zona pellucida surface as shown by scanning electron microscopy. For years it has been believed that this heterogeneous morphology plays an important part in the sperm-oocyte interaction. It was the aim of this investigation to analyse the morphology and the sperm binding patterns of the human zona pellucida. Oocytes were divided into four categories: mature, immature, fertilized and unfertilized. Four different types of zona morphology were detectable. They ranged from a porous, net-like structure to a nearly smooth and compact surface. No correlation could be established between zona type and oocyte maturity or zona type and achieved fertilization. However, fertilized (polyploid) oocytes had a more compact and smooth zona surface than unfertilized ones. The analysis of the number and distribution patterns of bound spermatozoa on the zona pellucida revealed extremely variable patterns regardless of the zona morphology. Significant differences between mature and immature oocytes did not appear. In both groups there were oocytes with either no or numerous bound spermatozoa on the zona pellucida. Oocytes overloaded with spermatozoa could only be found in the mature group. Unfertilized oocytes had fewer bound spermatozoa on average than polyploid zygotes.

Cell Differentiation↗

Spontaneous blastomere fusion after freezing and thawing of early human embryos leads to polyploidy and chromosomal mosaicism.

The incidence of blastomere fusion after cryopreservation of early human embryos (day 2 and day 3) was investigated using the standard propanediol technique. The process of fusion was observed in all developmental stages (from 2 to 10 cells) and the frequency of this event was 4.6% in day 2 (41/889) and 1.5% in day 3 (10/646) embryos that survived the thawing (embryos with 50-100% intact cells). Fusion of two, and occasionally of several, blastomeres resulted in the formation of multinucleated hybrid cells, which clearly indicated that the ploidy of these newly created cells had been altered. This event, depending on the number of fused cells per embryo, transformed the embryos into either entirely polyploid embryos (complete fusion at 2- or 3-cell stage) or into mosaics being a mixture of polyploid and normal cells. Chromosomal preparations of embryos affected by blastomere fusion indicated the presence of tetraploid mitotic plates. Also, fluorescence in-situ hybridization (FISH) analysis using DNA probes targeting unique sequences on chromosomes 9, 15, 17 and 22 indicated the existence of tetraploid and diploid fluorescence signals in the interphase nuclei within mosaics. Therefore, observations on live and fixed embryos suggested that tetraploid (4n) or hexaploid (6n) and tetraploid-diploid or more complex aberrations of ploidy might be formed as a consequence of blastomere fusion. Furthermore, this demonstrates that freezing and thawing may induce numerical chromosomal changes in human embryos.

Blastomeres↗

Systematic phylogenomic evidence of en bloc duplication of the ancestral 8p11.21-8p21.3-like region.

The genomes of many higher organisms, including plants and bony fish, frequently undergo polyploidization, and it has long been hypothesized that these, and other, large-scale genomic duplications have played an important role in the major evolutionary transitions of our past. Here we build upon an early work to show that the human genomic region 8p11.21-8p21.3 has three paralogous regions on chromosomes 4, 5, and 10 that were produced by two rounds of duplications after the protostomian-deuterostomian split and before the actinopterygian-sarcopterygian split. We base our analysis on the phylogenetic reconstruction of the evolutionary history of 38 gene families located in these regions. Using an alignment centered on protein domains, three different phylogenetic methods, and divergence time estimation, this analysis gives more support in favor of two ancient polyploidization events in the vertebrate ancestral genome.

Animals↗

Evolution of clonality and polyploidy in a weevil system.

The increased interest in asexual organisms calls for in-depth studies of asexual complexes that actively give rise to new clones. We present an extensive molecular study of the Otiorhynchus scaber (Coleoptera, Curculionidae) weevil system. Three forms have traditionally been recognized: diploid sexuals, triploid, and tetraploid parthenogens. All forms coexist in a small central area, but only the polyploid parthenogens have colonized marginal areas. Analyzing the phylogenetic relationship, based on three partial mitochondrial genes, of 95 individuals from 19 populations, we find that parthenogenesis and polyploidy have originated at least three times from different diploid lineages. We observe two major mitochondrial lineages, with over 2.5% sequence divergence between the most basal groups within them, and find that current distribution and phylogenetic relationships are weakly correlated. Quite unexpectedly, we also discover diploid clones that coexist with, and are morphologically indistinguishable from, the diploid sexual females. Our results support that these diploid clones are derived directly from the diploid sexuals. We also find that it is mainly an increase in ploidy level and not the benefits of asexual reproduction that confers to polyploid parthenogens the advantage over their diploid sexual relatives.

Animals↗

Paralogy and orthology in the MALVACEAE rpb2 gene family: investigation of gene duplication in hibiscus.

A sample of the second largest subunit of low-copy nuclear RNA polymerase II (rpb2) sequences from Malvaceae subfamily Malvoideae suggests that rpb2 has been duplicated early in the subfamily's history. Hibiscus and related taxa possess two rpb2 genes, both of which produce congruent phylogenetic patterns that are largely concordant with cpDNA topologies. No evidence of functional divergence or disruption was found among duplicated copies, suggesting that long-term maintenance of duplicated copies of rpb2 is usual in this lineage. Therefore, this gene may be suitable for the potential diagnosis of relatively old polyploid events. One probable pseudogene was found in Radyera farragei and a single chimeric sequence was recovered from Howittia trilocularis, suggesting that the rpb2 locus is not as prone to evolutionary processes that can confound phylogenetic inferences based on nDNA sequences. The pattern of relationships among rpb2 sequences, coupled with chromosome number information and Southern hybridization data, suggests that an early polyploid event was not the cause of the duplication, despite independent evidence of paleopolyploidy in some members of Malvoideae. Rpb2 exons and introns together are suitable for phylogenetic analysis, producing well-resolved and well-supported results that were robust to model permutation and congruent with previous studies of subfamily Malvoideae using cpDNA characters.

Evolution, Molecular↗

Is polyploidy an important genotoxic lesion?

The importance of polyploidy as a genotoxic lesion is uncertain and there have been few publications and no reviews which have included data on spontaneous or induced polyploidy in routine genotoxicity screening. We have attempted to clarify some of the issues by reviewing the published literature and by reference to our historical data base for metaphase analysis of cultured human lymphocytes. In our studies on pharmaceutical compounds polyploidy was the lesion most often found, being induced by approximately 40% of the compounds tested. The mean spontaneous frequency was between 0.1 and 0.3%, and values for polyploidy induction were 5-fold to > 100-fold the spontaneous value. Spontaneous polyploids tended to be near-exact multiples of the haploid chromosome number whereas induced 'polyploids' were, in fact, very heteroploid with a wide range of chromosome numbers. Polyploidy induction often occurred at non-toxic concentrations, usually there were well defined no-effect (threshold) levels and it was unrelated to other genetic effects. Such observations would be expected for inducers of polyploidy because the target molecules are not DNA and for these non-DNA targets there is usually a degree of redundancy. Therefore, inducers of polyploidy are only likely to be a hazard for humans if they are positive at or below therapeutic concentrations. We conclude that polyploidy/near-polyploidy (shown as 'polyploidy' throughout) should be scored as accessory data which becomes important only when induction occurs at therapeutic levels.

Chromosome Aberrations↗

Detection of micronuclei, cell proliferation and hyperdiploidy in bladder epithelial cells of rats treated with o-phenylphenol.

o-Phenylphenol (OPP), a widely used fungicide and antibacterial agent, has been considered to be among the top 10 home and garden pesticides used in the USA. Earlier studies have consistently shown that the sodium salt of OPP (SOPP) causes bladder cancer in male Fischer 344 (F344) rats, whereas OPP has produced variable results. This difference has been attributed to the presence of the sodium salt. To determine cellular and genetic alterations in the rat bladder and the influence of the sodium salt, F344 rats were administered 2% OPP, 2% NaCl and 2% NaCl + 2% OPP in their diet for 14 days. Twenty-four hours before being killed the animals were administered 5-bromo-2'-deoxyuridine (BrdU) by i.p. injection. Bladder cells were isolated, stained with DAPI and scored for the presence of micronuclei and incorporation of BrdU into replicating cells. To determine changes in chromosome number, we used fluorescence in situ hybridization (FISH) with a DNA probe for rat chromosome 4. Significant increases in the frequency of micronuclei and BrdU incorporation were seen in bladder cells of rats from all treatment groups. In contrast, the frequency of hyperdiploidy/polyploidy in treated animals was not increased over that seen in controls. A high control frequency of cells with three or more hybridization signals was seen, probably due to the presence of polyploid cells in the bladder. The presence of polyploid cells combined with cytotoxicity and compensatory cell proliferation makes it difficult to determine whether OPP is capable of inducing aneuploidy in the rat urothelium. In summary, these studies show that OPP can cause cellular and chromosomal alterations in rat bladder cells in the absence of the sodium salt. These results also indicate that at high concentrations the sodium salt can enhance chromosomal damage in the rat urothelium.

Aneuploidy↗

Induction of genetic instability and chromosomal instability by nickel sulfate in V79 Chinese hamster cells.

Nickel compounds are known to be carcinogenic to humans and show genotoxicity, including the ability to induce chromosome aberrations and neoplastic transformation in vitro. The mutagenicity of nickel compounds is, however, equivocal and the mechanisms of carcinogenesis are still not clear. In this study, the possibility that nickel compounds induce genetic or chromosomal instability was examined, because recent studies in cancer research show that these conditions are critically involved in carcinogenesis. V79 Chinese hamster cells were treated with 320 microM nickel sulfate for 24 h at low cell density (100 cells/100 mm diameter dish) and clones derived from single cells surviving Ni treatment were isolated. When cells grew up to 23-25 population doublings post-treatment, mutation frequency at the HPRT locus and the chromosome aberration frequency of each clone were examined. Five out of 37 clones (13.5%) derived from Ni-treated cells showed a remarkably increased frequency of HPRT mutations (>or=1 x 10(-4)), while only one out of 37 control clones (2.7%) showed this high mutation rate. In addition, 17 out of 37 clones (45.9%) from Ni-treated cells showed structural chromosomal aberrations in 10% or more of cells (up to 45.5%), while only three out of 31 control clones (9.7%) showed this high aberration rate. Out of 37 clones derived from Ni-treated cells, eight (21.6%) and 11 (29.7%) clones showed an increased frequency (>or=5%) of aneuploid and polyploid cells, respectively, while only a few control clones showed such an increase in aneuploid and polyploid cells. These results indicate that nickel sulfate can induce genetic and chromosomal instability in V79 cells.

Aneuploidy↗

Post-fertilization polyploidy in human preimplantation embryos fertilized in-vitro.

The development of in-vitro fertilization (IVF) and embryo transfer (ET) as a treatment for certain forms of infertility has emphasized the need for more information on why so many preimplantation embryos fail to develop beyond the very early cleavage stages. Analysis of nuclei from embryos that had ceased to grow or that were morphologically abnormal was carried out using one of two techniques: measurement of the DNA content of their nuclei, or observation of the number of Y chromosomes per nucleus. It was found that there was a correlation between the length of time in culture and abnormal development of polyploidy in a number of the cells. On the other hand, a small proportion of polyploid cells observed in a morphologically normal expanded blastocyst probably represents the normal precursors of polyploid trophoblast cells as observed in other mammalian species.

Blastocyst↗