Analysis of human invasive cytotrophoblasts demonstrates mosaic aneuploidy.

A total of 24 chromosome-specific fluorescence in situ hybridization probes for interphase nucleus analysis were developed to determine the chromosomal content of individual human invasive cytotrophoblasts derived from in vitro cultured assays. At least 75% of invasive cytotrophoblasts were hyperdiploid and the total number of chromosomes ranged from 47 to 61. The results also demonstrated that these hyperdiploid invasive cytotrophoblasts showed significant heterogeneity. The most copy number gains were observed for chromosomes 13, 14, 15, 19, 21, and 22 with average copy number greater than 2.3. A parallel study using primary invasive cytotrophoblasts also showed a similar trend of copy number changes. Conclusively, 24-chromosome analysis of human non-proliferating cytotrophoblasts (interphase nuclei) was achieved. Hyperdiploidy and chromosomal heterogeneity without endoduplication in invasive cytotrophoblasts may suggest a selective advantage for invasion and short lifespan during normal placental development.

All-male hybrids of a tetrapod Pelophylax esculentus share its origin and genetics of maintenance.

BACKGROUND: Sexual parasites offer unique insights into the reproduction of unisexual and sexual populations. Because unisexuality is almost exclusively linked to the female sex, most studies addressed host-parasite dynamics in populations where sperm-dependent females dominate. Pelophylax water frogs from Central Europe include hybrids of both sexes, collectively named P. esculentus. They live syntopically with their parental species P. lessonae and/or P. ridibundus. Some hybrid lineages consist of all males providing a chance to understand the origin and perpetuation of a host-parasite (egg-dependent) system compared to sperm-dependent parthenogenesis. METHODS: We focused on P. ridibundus-P. esculentus populations where P. ridibundus of both sexes lives together with only diploid P. esculentus males. Based on 17 microsatellite markers and six allozyme loci, we analyzed (i) the variability of individual genomes, (ii) the reproductive mode(s) of all-male hybrids, and (iii) the genealogical relationships between the hybrid and parental genomes. RESULTS: Our microsatellite data revealed that P. esculentus males bear Mendelian-inherited ridibundus genomes while the lessonae genome represents a single clone. Our data indicate that this clone did not recently originate from adjacent P. lessonae populations, suggesting an older in situ or ex situ origin. CONCLUSIONS: Our results confirm that also males can perpetuate over many generations as the unisexual lineage and successfully compete with P. ridibundus males for eggs provided by P. ridibundus females. Natural persistence of such sex-specific hybrid populations allows to studying the similarities and differences between male and female reproductive parasitism in many biological settings.

Full Karyotype Interphase Cell Analysis.

Aneuploidy seems to play not only a decisive role in embryonal development but also in tumorigenesis where chromosomal and genomic instability reflect a universal feature of malignant tumors. The cost of whole genome sequencing has fallen significantly, but it is still prohibitive for many institutions and clinical settings. No applied, cost-effective, and efficient technique has been introduced yet aiming at research to assess the ploidy status of all 24 different human chromosomes in interphases simultaneously, especially in single cells. Here, we present the selection of human probe DNA and a technique using multistep fluorescence in situ hybridization (FISH) employing four sets of six labeled FISH probes able to delineate all 24 human chromosomes in interphase cells. This full karyotype analysis approach will provide additional diagnostic potential for single cell analysis. The use of spectral imaging (SIm) has enabled the use of up to eight different fluorochrome labels simultaneously. Thus, scoring can be easily assessed by visual inspection, because SIm permits computer-assigned and distinguishable pseudo-colors to each probe during image processing. This enables full karyotype analysis by FISH of single-cell interphase nuclei.

Circulating tumor cells: potential markers of minimal residual disease in ovarian cancer? a study of the OVCAD consortium.

PURPOSE: In 75% of ovarian cancer patients the tumor mass is completely eradicated by established surgical and cytotoxic treatment; however, the majority of the tumors recur within 24 months. Here we investigated the role of circulating tumor cells (CTCs) indicating occult tumor load, which remains inaccessible by established diagnostics. EXPERIMENTAL DESIGN: Blood was taken at diagnosis (baseline samples, n = 102) and six months after completion of adjuvant first-line chemotherapy (follow-up samples; n = 78). CTCs were enriched by density gradient centrifugation. A multi-marker immunostaining was established and further complemented by FISH on CTCs and tumor/metastasis tissues using probes for stem-cell like fusion genes MECOM and HHLA1. RESULTS: CTCs were observed in 26.5% baseline and 7.7% follow-up blood samples at a mean number of 12.4 and 2.8 CTCs per ml blood, respectively. Baseline CTCs indicated a higher risk of death in R0 patients with complete gross resection (univariate: HR 2.158, 95% CI 1.111-4.191, p = 0.023; multivariate: HR 2.720, 95% CI 1.340-5.522, p = 0.006). At follow-up, the presence of CTCs was associated with response to primary treatment as assessed using RECIST criteria. Chromosomal gains at MECOM and HHLA1 loci suggest that the observed cells were cancer cells and reflect pathophysiological decisive chromosomal aberrations of the primary and metastatic tumors. CONCLUSIONS: Our data suggest that CTCs detected by the multi-marker protein panel and/or MECOM/HHLA1 FISH represent minimal residual disease in optimally debulked ovarian cancer patients. The role of CTCs cells especially for clinical therapy stratification of the patients has to be validated in consecutive larger studies applying standardized treatment schemes.

Genetic diversity and distribution patterns of diploid and polyploid hybrid water frog populations (Pelophylax esculentus complex) across Europe.

Polyploidization is a rare yet sometimes successful way for animals to rapidly create geno- and phenotypes that may colonize new habitats and quickly adapt to environmental changes. In this study, we use water frogs of the Pelophylax esculentus complex, comprising two species (Pelophylax lessonae, genotype LL; Pelophylax ridibundus, RR) and various diploid (LR) and triploid (LLR, LRR) hybrid forms, summarized as P. esculentus, as a model for studying recent hybridization and polyploidization in the context of speciation. Specifically, we compared the geographic distribution and genetic diversity of diploid and triploid hybrids across Europe to understand their origin, maintenance and potential role in hybrid speciation. We found that different hybrid and parental genotypes are not evenly distributed across Europe. Rather, their genetic diversity is structured by latitude and longitude and the presence/absence of parental species but not of triploids. Highest genetic diversity was observed in central and eastern Europe, the lowest in the northwestern parts of Europe. This gradient can be explained by the decrease in genetic diversity during postglacial expansion from southeastern glacial refuge areas. Genealogical relationships calculated on the basis of microsatellite data clearly indicate that hybrids are of multiple origin and include a huge variety of parental genomes. Water frogs in mixed-ploidy populations without any parental species (i.e. all-hybrid populations) can be viewed as evolutionary units that may be on their way towards hybrid speciation. Maintenance of such all-hybrid populations requires a continuous exchange of genomes between diploids and triploids, but scenarios for alternative evolutionary trajectories are discussed.

Post-zygotic selection against parental genotypes during larval development maintains all-hybrid populations of the frog Pelophylax esculentus.

BACKGROUND: Hybridization between two species usually leads to inviable or infertile offspring, due to endogenous or exogenous selection pressures. Yet, hybrid taxa are found in several plant and animal genera, and some of these hybrid taxa are ecologically and evolutionarily very successful. One example of such a successful hybrid is the water frog, Pelophylax esculentus which originated from matings between the two species P. ridibundus (genotype RR) and P. lessonae (LL). At the northern border of the distribution all-hybrid populations consisting of diploid (LR) and one or two triploid (LLR, LRR) frog types have been established. Here, the hybrid has achieved reproductive independence from its sexual ancestors and forms a self-sustaining evolutionary unit. Based on the gamete production of these hybrids, certain mating combinations should lead to LL and RR offspring, but these parental forms are absent among the adults. RESULTS: In order to investigate the mechanisms that maintain such an all-hybrid system, we performed a field study and a crossing experiment. In the field we sampled several ponds for water frog larvae at different developmental stages. Genotype compositions were then analysed and life-history differences between the genotypes examined. In the experiment we crossed diploid and triploid males and females from different ponds and determined fertilization success as well as development speed and survival rates of the offspring under high, medium and low food availability. In both parts of the study, we found numerous LL and RR offspring during the egg and early larval stages; but the frequency of these parental genotypes decreased drastically during later stages. In natural ponds almost all of them had disappeared already before metamorphosis; under the more benign experimental conditions the last ones died as juveniles during the following year. CONCLUSIONS: From the combined results we conclude that the absence of parental genotypes in all-hybrid populations is due to post-zygotic selection against them, rather than to pre-zygotic mechanisms that might prevent their formation in the first place. For this post-zygotic selection, genetic mechanisms resulting from low genetic diversity and fixation of deleterious mutations seem to be a more likely explanation than ecological factors.

High Performance DNA Probes for Perinatal Detection of Numerical Chromosome Aberrations.

Human reproduction is a tightly controlled process of stepwise evolution with multiple, mostly yet unknown milestones and checkpoints. Healthy halpoid gametes have to be produced by the parents, which will fuse to form the diploid zygote that implants in the female uterus and grows to become first an embryo, then a fetus and finally matures into a newborn. There are several known risk factors that interfere with normal production of gametes, spermatocytes or oocytes, and often cause embryonic mortality and fetal demise at an early stage. Yet some embryos with chomosomal abnormalities can develop beyond the critical first trimester of pregnancy and, while those with supernumary chromosomes in their hyperdiploid cells will be spontaneously aborted, a small fraction of fetuses with an extra chromosome continues to grow to term and will be delivered as a liveborn baby. While minor clinical symptoms displayed by children with trisomies are manageable for many parents, the burden of caring for a child with numerical chromosome abnormalities can be overwhelming to partners or individual families. It also poses a significant financial burden to the society and poses ethical dilemma. In this communication, we will review the progress that has been made in the development of molecular techniques to test individual fetal cells for chromosomal imbalances. We will focus our discussion on the direct visualization of chromosome-specific DNA sequences in live or fixed specimens using fluorescence in situ hybridization (FISH) and, more specifically, talk about the groundbreaking progress that in recent years has been achieved towards an improved diagnosis with novel, chromosome-specific DNA probes.

New exact solution of the relativistic Boltzmann equation and its hydrodynamic limit.

We present an exact solution of the relativistic Boltzmann equation for a system undergoing boost-invariant longitudinal and azimuthally symmetric transverse flow ("Gubser flow"). The resulting exact nonequilibrium dynamics is compared to first and second order relativistic hydrodynamic approximations for various shear viscosity to entropy density ratios. This novel solution can be used to test the validity and accuracy of different hydrodynamic approximations in conditions similar to those generated in relativistic heavy-ion collisions.

Genomic effects on advertisement call structure in diploid and triploid hybrid waterfrogs (Anura, Pelophylax esculentus).

BACKGROUND: In anurans, differences in male mating calls have intensively been studied with respect to taxonomic classification, phylogeographic comparisons among different populations and sexual selection. Although overall successful, there is often much unexplained variation in these studies. Potential causes for such variation include differences among genotypes and breeding systems, as well as differences between populations. We investigated how these three factors affect call properties in male water frogs of Pelophylax lessonae (genotype LL), P. ridibundus (RR) and their interspecific hybrid P. esculentus which comes in diploid (LR) and triploid types (LLR, LRR). RESULTS: We investigated five call parameters that all showed a genomic dosage effect, i.e. they either decreased or increased with the L/R ratio in the order LL-LLR-LR-LRR-RR. Not all parameters differentiated equally well between the five genotypes, but combined they provided a good separation. Two of the five call parameters were also affected by the breeding system. Calls of diploid LR males varied, depending on whether these males mated with one or both of the parental species (diploid systems) or triploid hybrids (mixed ploidy systems). With the exception of the northernmost mixed-ploidy population, call differences were not related to the geographic location of the population and they were not correlated with genetic distances in the R and L genomes. CONCLUSIONS: We found an influence of all three tested factors on call parameters, with the effect size decreasing from genotype through breeding system to geographic location of the population. Overall, results were in line with predictions from a dosage effect in L/R ratios, but in three call parameters all three hybrid types were more similar to one or the other parental species. Also calls of diploid hybrids varied between breeding systems in agreement with the sexual host required for successful reproduction. The lack of hybrid call differences in a mixed-ploidy population at the northern edge of the water frog distribution is likely to be associated with genetic particularities, including a) low genetic variability and/or b) a local loss of genes coding for genotype-dependent call differentiation under conditions where female discrimination between diploid and triploid males is not beneficial.

Gamete production patterns, ploidy, and population genetics reveal evolutionary significant units in hybrid water frogs (Pelophylax esculentus).

The European water frog Pelophylax esculentus is a natural hybrid between P. lessonae (genotype LL) and P. ridibundus (RR). It reproduces through hybridogenesis, eliminating one parental genome from its germline and producing gametes containing the genome of the other parental species. According to previous studies, this elimination and transmission pattern is very diverse. In mixed populations, where only diploid hybrids (LR) live in sympatry and mate with one or both parental species, the excluded genome varies among regions, and the remaining genome is transmitted clonally to haploid gametes. In all-hybrid populations consisting of diploid (LR) and triploid (LLR and/or LRR) frogs, diploid individuals also produce gametes clonally (1n in males, 2n in females), whereas triploids eliminate the genome they have in single copy and produce haploid gametes containing the recombined other genome. However, here, too, regional differences seem to exist, and some triploids have been reported to produce diploid gametes. In order to systematically study such regional and genotype differences in gamete production, their potential origin, and their consequences for the breeding system, we sampled frogs from five populations in three European countries, performed crossing experiments, and investigated the genetic variation through microsatellite analysis. For four populations, one in Poland, two in Germany, and one in Slovakia, our results confirmed the elimination and transmission pattern described above. In one Slovakian population, however, we found a totally different pattern. Here, triploid males (LLR) produce sperm with a clonally transmitted diploid LL genome, rather than a haploid recombined L genome, and LR females clonally produce haploid R eggs, rather than diploid LR eggs. These differences among the populations in gamete production go along with differences in genomotype composition, breeding system (i.e., the way triploids are produced), and genetic variation. These differences are strong evidence for a polyphyletic origin of triploids. Moreover, our findings shed light on the evolutionary potential inherent to the P. esculentus complex, where rare events due to untypical gametogenetic processes can lead to the raise, the perpetuation, and the dispersion of new evolutionary significant lineages which may also deserve special conservation measures.

Analysis of human invasive cytotrophoblasts using multicolor fluorescence in situ hybridization.

Multicolor fluorescence in situ hybridization, or FISH, is a widely used method to assess fixed tissues or isolated cells for numerical and structural chromosome aberrations. Unlike other screening procedures which provide average chromosome numbers for heterogeneous samples, FISH is a sensitive cell-by-cell method to analyze the distribution of abnormal cells in complex tissues. Here, we applied FISH to characterize chromosomal composition of a rare, but very important class of human cells that stabilize the fetal-maternal interface connecting the placenta to the uterine wall during early pregnancy, called invasive cytotrophoblasts (iCTBs). Combining differently-labeled, chromosome-specific DNA probes, we were able to unambiguously determine the number of up to six different autosomes and gonosomes in individual cell nuclei from iCTBs selected on the basis of their invasive behavior. In this manuscript, we describe a method for generation of iCTBs from placental villi, and provide the complete workflow of our FISH experiments including a detailed description of reagents and a trouble-shooting guide. We also include an in-depth discussion of the various types and sources of DNA probes which have evolved considerably in the last two decades. Thus, this communication represents both a complete guide as well as a valuable resource, intended to allow an average laboratory to reproduce the experiments and minimize the amount of specialized, and often costly, equipment.

Long-term study of an infection with ranaviruses in a group of edible frogs (Pelophylax kl. esculentus) and partial characterization of two viruses based on four genomic regions.

Several edible frogs (Pelophylax kl. esculentus) collected into a single group from various ponds in Europe died suddenly with reddening of the skin (legs, abdomen) and haemorrhages in the gastrointestinal tract. Ranavirus was detected in some of the dead frogs using PCR, and virus was also isolated in cell culture. Over the following 3 years, another two outbreaks occurred with low to high mortality in between asymptomatic periods. In the first 2 years, the same ranavirus was detected repeatedly, but a new ranavirus was isolated in association with the second mass-mortality event. The two different ranaviruses were characterized based on nucleotide sequences from four genomic regions, namely, major capsid protein, DNA polymerase, ribonucleoside diphosphate reductase alpha and beta subunit genes. The sequences showed slight variations to each other or GenBank entries and both clustered to the Rana esculenta virus (REV-like) clade in the phylogenetic analysis. Furthermore, a quiescent infection was demonstrated in two individuals. By comparing samples taken before and after transport and caging in groups it was possible to identify the pond of origin and a ranavirus was detected for the first time in wild amphibians in Germany.

Bioinformatics tools allow targeted selection of chromosome enumeration probes and aneuploidy detection.

Accurate determination of cellular chromosome complements is a highly relevant issue beyond prenatal/pre-implantation genetic analyses or stem cell research, because aneusomy may be an important mechanism by which organisms control the rate of fetal cellular proliferation and the fate of regenerating tissues. Typically, small amounts of individual cells or nuclei are assayed by in situ hybridization using chromosome-specific DNA probes. Careful probe selection is fundamental to successful hybridization experiments. Numerous DNA probes for chromosome enumeration studies are commercially available, but their use in multiplexed hybridization assays is hampered due to differing probe-specific hybridization conditions or a lack of a sufficiently large number of different reporter molecules. Progress in the International Human Genome Project has equipped the scientific community with a wealth of unique resources, among them recombinant DNA libraries, physical maps, and data-mining tools. Here, we demonstrate how bioinformatics tools can become an integral part of simple, yet powerful approaches to devise diagnostic strategies for detection of aneuploidy in interphase cells. Our strategy involving initial in silico optimization steps offers remarkable savings in time and costs during probe generation, while at the same time significantly increasing the assay's specificity, sensitivity, and reproducibility.

Multi-Color Spectral Transcript Analysis (SPECTRA) for Phenotypic Characterization of Tumor Cells.

Many human tumors show significant changes in their signal transduction pathways and, thus, the way the cells interact with their environment. Often caused by chromosomal rearrangements, including gene amplifications, translocations or deletions, the altered levels of gene expression may provide a tumor-specific signature that can be exploited for diagnostic or therapeutic purposes. We investigated the utility of multiplexed fluorescence in situ hybridization (FISH) using non-isotopically labeled cDNA probes detected by Spectral Imaging as a sensitive and rapid procedure to measure tumor-specific gene expression signatures. We used a commercially available system to acquire and analyze multicolor FISH images. Initial investigations used panels of fluorescent calibration standards to evaluate the system. These experiments were followed by hybridization of five-to-six differently labeled cDNA probes, which target the transcripts of tyrosine kinase genes known to be differently expressed in normal cells and tumors of the breast or thyroid gland. The relatively simple, yet efficient, molecular cytogenetic method presented here may find many applications in characterization of solid tumors or disseminated tumor cells. Addressing tumor heterogeneity by means of multi-parameter single cell analyses is expected to enable a wide range of investigations in the areas of tumor stem cells, tumor clonality and disease progression.

Chromosome-specific DNA repeats: rapid identification in silico and validation using fluorescence in situ hybridization.

Chromosome enumeration in interphase and metaphase cells using fluorescence in situ hybridization (FISH) is an established procedure for the rapid and accurate cytogenetic analysis of cell nuclei and polar bodies, the unambiguous gender determination, as well as the definition of tumor-specific signatures. Present bottlenecks in the procedure are a limited number of commercial, non-isotopically labeled probes that can be combined in multiplex FISH assays and the relatively high price and effort to develop additional probes. We describe a streamlined approach for rapid probe definition, synthesis and validation, which is based on the analysis of publicly available DNA sequence information, also known as "database mining". Examples of probe preparation for the human gonosomes and chromosome 16 as a selected autosome outline the probe selection strategy, define a timeline for expedited probe production and compare this novel selection strategy to more conventional probe cloning protocols.

Delineating chromosomal breakpoints in radiation-induced papillary thyroid cancer.

Recurrent translocations are well known hallmarks of many human solid tumors and hematological disorders, where patient- and breakpoint-specific information may facilitate prognostication and individualized therapy. In thyroid carcinomas, the proto-oncogenes RET and NTRK1 are often found to be activated through chromosomal rearrangements. However, many sporadic tumors and papillary thyroid carcinomas (PTCs) arising in patients with a history of exposure to elevated levels of ionizing irradiation do not carry these known abnormalities. We developed a rapid scheme to screen tumor cell metaphase spreads and identify candidate genes of tumorigenesis and neoplastic progression for subsequent functional studies. Using a series of overnight fluorescence in situ hybridization (FISH) experiments with pools comprised of bacterial artificial chromosome (BAC) clones, it now becomes possible to rapidly refine breakpoint maps and, within one week, progress from the low resolution Spectral Karyotyping (SKY) maps or Giemsa-banding (G-banding) karyotypes to fully integrated, high resolution physical maps including a list of candiate genes in the critical regions.

Biospecimen Reporting for Improved Study Quality.

Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues, it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The Biospecimen Reporting for Improved Study Quality guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.

200 A GeV Au + Au collisions serve a nearly perfect quark-gluon liquid.

A new robust method to extract the specific shear viscosity (η/s)(QGP) of a quark-gluon plasma (QGP) at temperatures T(c) < T ≲ 2T(c) from the centrality dependence of the eccentricity-scaled elliptic flow v2/ε measured in ultrarelativistic heavy-ion collisions is presented. Coupling viscous fluid dynamics for the QGP with a microscopic transport model for hadronic freeze-out we find for 200 A GeV Au + Au collisions that v2/ε is a universal function of multiplicity density (1/S)(dN(ch)/dy) that depends only on the viscosity but not on the model used for computing the initial fireball eccentricity ε. Comparing with measurements we find 1<4π(η/s)(QGP) < 2.5 where the uncertainty range is dominated by model uncertainties for the values of ε used to normalize the measured v2.

Biospecimen reporting for improved study quality (BRISQ).

Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues, it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality (BRISQ) recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The BRISQ guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.