Cancer origin tracing and timing in two high-risk prostate cancers using multisample whole genome analysis: prospects for personalized medicine.

BACKGROUND: Prostate cancer (PrCa) genomic heterogeneity causes resistance to therapies such as androgen deprivation. Such heterogeneity can be deciphered in the context of evolutionary principles, but current clinical trials do not include evolution as an essential feature. Whether or not analysis of genomic data in an evolutionary context in primary prostate cancer can provide unique added value in the research and clinical domains remains an open question. METHODS: We used novel processing techniques to obtain whole genome data together with 3D anatomic and histomorphologic analysis in two men (GP5 and GP12) with high-risk PrCa undergoing radical prostatectomy. A total of 22 whole genome-sequenced sites (16 primary cancer foci and 6 lymph node metastatic) were analyzed using evolutionary reconstruction tools and spatio-evolutionary models. Probability models were used to trace spatial and chronological origins of the primary tumor and metastases, chart their genetic drivers, and distinguish metastatic and non-metastatic subclones. RESULTS: In patient GP5, CDK12 inactivation was among the first mutations, leading to a PrCa tandem duplicator phenotype and initiating the cancer around age 50, followed by rapid cancer evolution after age 57, and metastasis around age 59, 5 years prior to prostatectomy. In patient GP12, accelerated cancer progression was detected after age 54, and metastasis occurred around age 56, 3 years prior to prostatectomy. Multiple metastasis-originating events were identified in each patient and tracked anatomically. Metastasis from prostate to lymph nodes occurred strictly ipsilaterally in all 12 detected events. In this pilot, metastatic subclone content analysis appears to substantially enhance the identification of key drivers. Evolutionary analysis' potential impact on therapy selection appears positive in these pilot cases. CONCLUSIONS: PrCa evolutionary analysis allows tracking of anatomic site of origin, timing of cancer origin and spread, and distinction of metastatic-capable from non-metastatic subclones. This enables better identification of actionable targets for therapy. If extended to larger cohorts, it appears likely that similar analyses could add substantial biological insight and clinically relevant value.

Expansion of rDNA and pericentromere satellite repeats in the genomes of bank voles Myodes glareolus exposed to environmental radionuclides.

Altered copy number of certain highly repetitive regions of the genome, such as satellite DNA within heterochromatin and ribosomal RNA loci (rDNA), is hypothesized to help safeguard the genome against damage derived from external stressors. We quantified copy number of the 18S rDNA and a pericentromeric satellite DNA (Msat-160) in bank voles (Myodes glareolus) inhabiting the Chernobyl Exclusion Zone (CEZ), an area that is contaminated by radionuclides and where organisms are exposed to elevated levels of ionizing radiation. We found a significant increase in 18S rDNA and Msat-160 content in the genomes of bank voles from contaminated locations within the CEZ compared with animals from uncontaminated locations. Moreover, 18S rDNA and Msat-160 copy number were positively correlated in the genomes of bank voles from uncontaminated, but not in the genomes of animals inhabiting contaminated, areas. These results show the capacity for local-scale geographic variation in genome architecture and are consistent with the genomic safeguard hypothesis. Disruption of cellular processes related to genomic stability appears to be a hallmark effect in bank voles inhabiting areas contaminated by radionuclides.

Applying the Anna Karenina principle for wild animal gut microbiota: Temporal stability of the bank vole gut microbiota in a disturbed environment.

Gut microbiota play an important role in host health. Yet, the drivers and patterns of microbiota imbalance (dysbiosis) in wild animals remain largely unexplored. One hypothesised outcome of stress on animal microbiomes is a destabilised microbial community that is characterised by an increase in inter-individual differences compared with microbiomes of healthy animals, which are expected to be (a) temporally stable and (b) relatively similar among individuals. This set of predictions for response of microbiomes to stressors is known as the Anna Karenina principle (AKP) for animal microbiomes. We examine the AKP in a wild mammal inhabiting disturbed environments by conducting a capture-mark-recapture survey of bank voles Myodes glareolus in areas that contrast in levels of radionuclide contamination (Chernobyl, Ukraine). Counter to key predictions of the AKP, bank voles that are not exposed to radionuclides harbour variable (increased inter-individual differences) and temporally dynamic gut microbiota communities, presumably tracking the natural spatio-temporal variation in resources. Conversely, bank voles exposed to radionuclides host more similar gut microbiota communities that are temporally stable, potentially due to a dysbiosis or selection (on host or bacteria) imposed by chronic radiation exposure. The implication of these data is that environmental stress (radiation exposure) can constrain the natural spatial and temporal variation of wild animal gut microbiota.

The effect of chronic low-dose environmental radiation on organ mass of bank voles in the Chernobyl exclusion zone.

PURPOSE: Animals are exposed to environmental ionizing radiation (IR) externally through proximity to contaminated soil and internally through ingestion and inhalation of radionuclides. Internal organs can respond to radioactive contamination through physiological stress. Chronic stress can compromise the size of physiologically active organs, but studies on wild mammal populations are scarce. The effects of environmental IR contamination on organ masses were studied by using a wild rodent inhabiting the Chernobyl exclusion zone (CEZ). MATERIAL AND METHODS: The masses of brain, heart, kidney, spleen, liver and lung were assessed from bank voles (Myodes glareolus) captured from areas across radioactive contamination gradient within the CEZ. Relative organ masses were used to correct for the body mass of an individual. RESULTS: Results showed a significant negative correlation between IR level in the environment and relative brain and kidney mass. A significant positive correlation between IR and relative heart mass was also found. Principal component analysis (PCA) also suggested positive relationship between IR and relative spleen mass; however, this relationship was not significant when spleen was analyzed separately. There was no apparent relationship between IR and relative liver or lung mass. CONCLUSIONS: Results suggest that in the wild populations even low but chronic doses of IR can lead to changes in relative organ mass. The novelty of these result is showing that exposure to low doses can affect the organ masses in similar fashion as previously shown on high, acute, radiation doses. These data support the hypothesis that wildlife might be more sensitive to IR than animals used in laboratory studies. However, more research is needed to rule out the other indirect effects such as radiosensitivity of the food sources or possible combined stress effects from e.g. infections.

Exposure to environmental radionuclides is associated with altered metabolic and immunity pathways in a wild rodent.

Wildlife inhabiting environments contaminated by radionuclides face putative detrimental effects of exposure to ionizing radiation, with biomarkers such as an increase in DNA damage and/or oxidative stress commonly associated with radiation exposure. To examine the effects of exposure to radiation on gene expression in wildlife, we conducted a de novo RNA sequencing study of liver and spleen tissues from a rodent, the bank vole Myodes glareolus. Bank voles were collected from the Chernobyl Exclusion Zone (CEZ), where animals were exposed to elevated levels of radionuclides, and from uncontaminated areas near Kyiv, Ukraine. Counter to expectations, we did not observe a strong DNA damage response in animals exposed to radionuclides, although some signs of oxidative stress were identified. Rather, exposure to environmental radionuclides was associated with upregulation of genes involved in lipid metabolism and fatty acid oxidation in the livers - an apparent shift in energy metabolism. Moreover, using stable isotope analysis, we identified that fur from bank voles inhabiting the CEZ had enriched isotope values of nitrogen: such an increase is consistent with increased fatty acid metabolism, but also could arise from a difference in diet or habitat between the CEZ and elsewhere. In livers and spleens, voles inhabiting the CEZ were characterized by immunosuppression, such as impaired antigen processing, and activation of leucocytes involved in inflammatory responses. In conclusion, exposure to low dose environmental radiation impacts pathways associated with immunity and lipid metabolism, potentially as a stress-induced coping mechanism.

Corpse management of the invasive Argentine ant inhibits growth of pathogenic fungi.

A dead conspecific poses a potential pathogen risk for social animals. We have discovered that Argentine ants (Linepithema humile) prevent spread of pathogenic fungi from corpses by depositing the dead to combined toilet and refuse areas and applying pygidial gland secretion on them. The presence of a corpse in a nest increases this secretion behaviour. We identified three fungi growing on Argentine ant corpses. Growth of the Argentine ant pathogen Aspergillus nomius and the plant pathogen Fusarium solani on corpses was inhibited as long as the ants were constantly attending them as the ant anal secretion only delayed germination of their spores. In contrast, the effect of the ant anal secretion on the human pathogen Aspergillus fumigatus was much stronger: it prevented spore germination and, accordingly, the fungus no longer grew on the treated corpses. The Argentine ants are one of the world's worst invasive alien species as they cause ecological and economical damage in their new habitats. Our discovery points at a novel method to limit Argentine ant colonies through their natural fungal pathogens.

Exposure to environmental radionuclides associates with tissue-specific impacts on telomerase expression and telomere length.

Telomeres, the protective structures at the ends of chromosomes, can be shortened when individuals are exposed to stress. In some species, the enzyme telomerase is expressed in adult somatic tissues, and potentially protects or lengthens telomeres. Telomeres can be damaged by ionizing radiation and oxidative stress, although the effect of chronic exposure to elevated levels of radiation on telomere maintenance is unknown for natural populations. We quantified telomerase expression and telomere length (TL) in different tissues of the bank vole Myodes glareolus, collected from the Chernobyl Exclusion Zone, an environment heterogeneously contaminated with radionuclides, and from uncontaminated control sites elsewhere in Ukraine. Inhabiting the Chernobyl Exclusion Zone was associated with reduced TL in the liver and testis, and upregulation of telomerase in brain and liver. Thus upregulation of telomerase does not appear to associate with longer telomeres but may reflect protective functions other than telomere maintenance or an attempt to maintain shorter telomeres in a stressful environment. Tissue specific differences in the rate of telomere attrition and apparent radiosensitivity weaken the intra-individual correlation in telomere length among tissues in voles exposed to radionuclides. Our data show that ionizing radiation alters telomere homeostasis in wild animal populations in tissue specific ways.

Infection Load and Prevalence of Novel Viruses Identified from the Bank Vole Do Not Associate with Exposure to Environmental Radioactivity.

Bank voles (Myodes glareolus) are host to many zoonotic viruses. As bank voles inhabiting areas contaminated by radionuclides show signs of immunosuppression, resistance to apoptosis, and elevated DNA repair activity, we predicted an association between virome composition and exposure to radionuclides. To test this hypothesis, we studied the bank vole virome in samples of plasma derived from animals inhabiting areas of Ukraine (contaminated areas surrounding the former nuclear power plant at Chernobyl, and uncontaminated areas close to Kyiv) that differed in level of environmental radiation contamination. We discovered four strains of hepacivirus and four new virus sequences: two adeno-associated viruses, an arterivirus, and a mosavirus. However, viral prevalence and viral load, and the ability to cause a systemic infection, was not dependent on the level of environmental radiation.

Fibroblasts from bank voles inhabiting Chernobyl have increased resistance against oxidative and DNA stresses.

BACKGROUND: Elevated levels of environmental ionizing radiation can be a selective pressure for wildlife by producing reactive oxygen species and DNA damage. However, the underlying molecular mechanisms that are affected are not known. RESULTS: We isolated skin fibroblasts from bank voles (Myodes glareolus) inhabiting the Chernobyl nuclear power plant accident site where background radiation levels are about 100 times greater than in uncontaminated areas. After a 10 Gy dose of gamma radiation fibroblasts from Chernobyl animals recovered faster than fibroblasts isolated from bank voles living in uncontaminated control area. The Chernobyl fibroblasts were able to sustain significantly higher doses of an oxidant and they had, on average, a higher total antioxidant capacity than the control fibroblasts. Furthermore, the Chernobyl fibroblasts were also significantly more resistant than the control fibroblasts to continuous exposure to three DNA damaging drugs. After drug treatment transcription of p53-target gene pro-apoptotic Bax was higher in the control than in the Chernobyl fibroblasts. CONCLUSION: Fibroblasts isolated from bank voles inhabiting Chernobyl nuclear power plant accident site show elevated antioxidant levels, lower sensitivity to apoptosis, and increased resistance against oxidative and DNA stresses. These cellular qualities may help bank voles inhabiting Chernobyl to cope with environmental radioactivity.

De novo transcriptome assembly and developmental mode specific gene expression of Pygospio elegans.

Species with multiple different larval developmental modes are interesting models for the study of mechanisms underlying developmental mode transitions and life history evolution. Pygospio elegans, a small, tube-dwelling polychaete worm commonly found in estuarine and marine habitats around the northern hemisphere, is one species with variable developmental modes. To provide new genomic resources for studying P. elegans and to address the differences in gene expression between individuals producing offspring with different larval developmental modes, we performed whole transcriptome Illumina RNA sequencing of adult worms from two populations and prepared a de novo assembly of the P. elegans transcriptome. The transcriptome comprises 66,233 unigenes, of which 33,807 contain predicted coding sequences, 26,448 have at least one functional annotation, and 3,076 are classified as putative long non-coding RNAs. We found more than 8,000 unigenes significantly differentially expressed between adult worms from populations producing either planktonic or benthic larvae. This comprehensive transcriptome resource for P. elegans adds to the available genomic data for annelids and can be used to uncover mechanisms allowing developmental variation in this and potentially other marine invertebrate species.

Variation in gene expression within clones of the earthworm Dendrobaena octaedra.

Gene expression is highly plastic, which can help organisms to both acclimate and adapt to changing environments. Possible variation in gene expression among individuals with the same genotype (among clones) is not widely considered, even though it could impact the results of studies that focus on gene expression phenotypes, for example studies using clonal lines. We examined the extent of within and between clone variation in gene expression in the earthworm Dendrobaena octaedra, which reproduces through apomictic parthenogenesis. Five microsatellite markers were developed and used to confirm that offspring are genetic clones of their parent. After that, expression of 12 genes was measured from five individuals each from six clonal lines after exposure to copper contaminated soil. Variation in gene expression was higher over all genotypes than within genotypes, as initially assumed. A subset of the genes was also examined in the offspring of exposed individuals in two of the clonal lines. In this case, variation in gene expression within genotypes was as high as that observed over all genotypes. One gene in particular (chymotrypsin inhibitor) also showed significant differences in the expression levels among genetically identical individuals. Gene expression can vary considerably, and the extent of variation may depend on the genotypes and genes studied. Ensuring a large sample, with many different genotypes, is critical in studies comparing gene expression phenotypes. Researchers should be especially cautious inferring gene expression phenotypes when using only a single clonal or inbred line, since the results might be specific to only certain genotypes.

DNA Methylation and Potential for Epigenetic Regulation in Pygospio elegans.

Transitions in developmental mode are common evolutionarily, but how and why they occur is not understood. Developmental mode describes larval phenotypes, including morphology, ecology and behavior of larvae, which typically are generalized across different species. The polychaete worm Pygospio elegans is one of few species polymorphic in developmental mode, with multiple larval phenotypes, providing a possibility to examine the potential mechanisms allowing transitions in developmental mode. We investigated the presence of DNA methylation in P. elegans, and, since maternal provisioning is a key factor determining eventual larval phenotype, we compared patterns of DNA methylation in females during oogenesis in this species. We demonstrate that intragenic CpG site DNA methylation and many relevant genes necessary for DNA methylation occur in P. elegans. Methylation-sensitive AFLP analysis showed that gravid females with offspring differing in larval developmental mode have significantly different methylation profiles and that the females with benthic larvae and non-reproductive females from the same location also differ in their epigenetic profiles. Analysis of CpG sites in transcriptome data supported our findings of DNA methylation in this species and showed that CpG observed/expected ratios differ among females gravid with embryos destined to different developmental modes. The differences in CpG site DNA methylation patterns seen among the samples suggest a potential for epigenetic regulation of gene expression (through DNA methylation) in this species.

Temporal genetic structure in a poecilogonous polychaete: the interplay of developmental mode and environmental stochasticity.

BACKGROUND: Temporal variation in the genetic structure of populations can be caused by multiple factors, including natural selection, stochastic environmental variation, migration, or genetic drift. In benthic marine species, the developmental mode of larvae may indicate a possibility for temporal genetic variation: species with dispersive planktonic larvae are expected to be more likely to show temporal genetic variation than species with benthic or brooded non-dispersive larvae, due to differences in larval mortality and dispersal ability. We examined temporal genetic structure in populations of Pygospio elegans, a poecilogonous polychaete with within-species variation in developmental mode. P. elegans produces either planktonic, benthic, or intermediate larvae, varying both among and within populations, providing a within-species test of the generality of a relationship between temporal genetic variation and larval developmental mode. RESULTS: In contrast to our expectations, our microsatellite analyses of P. elegans revealed temporal genetic stability in the UK population with planktonic larvae, whereas there was variation indicative of drift in temporal samples of the populations from the Baltic Sea, which have predominantly benthic and intermediate larvae. We also detected temporal variation in relatedness within these populations. A large temporal shift in genetic structure was detected in a population from the Netherlands, having multiple developmental modes. This shift could have been caused by local extiction due to extreme environmental conditions and (re)colonization by planktonic larvae from neighboring populations. CONCLUSIONS: In our study of P. elegans, temporal genetic variation appears to be due to not only larval developmental mode, but also the stochastic environment of adults. Large temporal genetic shifts may be more likely in marine intertidal habitats (e.g. North Sea and Wadden Sea) which are more prone to environmental stochasticity than the sub-tidal Baltic habitats. Sub-tidal and/or brackish (less saline) habitats may support smaller P. elegans populations and these may be more susceptible to the effects of random genetic drift. Moreover, higher frequencies of asexual reproduction and the benthic larval developmental mode in these populations leads to higher relatedness and contributes to drift. Our results indicate that a general relationship between larval developmental mode and temporal genetic variation may not exist.

Polymorphism in developmental mode and its effect on population genetic structure of a spionid polychaete, Pygospio elegans.

Population genetic structure of sedentary marine species is expected to be shaped mainly by the dispersal ability of their larvae. Long-lived planktonic larvae can connect populations through migration and gene flow, whereas species with nondispersive benthic or direct-developing larvae are expected to have genetically differentiated populations. Poecilogonous species producing different larval types are ideal when studying the effect of developmental mode on population genetic structure and connectivity. In the spionid polychaete Pygospio elegans, different larval types have been observed between, and sometimes also within, populations. We used microsatellite markers to study population structure of European P. elegans from the Baltic Sea (BS) and North Sea (NS). We found that populations with planktonic larvae had higher genetic diversity than did populations with benthic larvae. However, this pattern may not be related to developmental mode, since in P. elegans, developmental mode may be associated with geography. Benthic larvae were more commonly seen in the brackish BS and planktonic larvae were predominant in the NS, although both larval types also are found from both areas. Significant isolation-by-distance (IBD) was found overall and within regions. Most of the pair-wise F(ST) comparisons among populations were significant, although some geographically close populations with planktonic larvae were found to be genetically similar. However, these results, together with the pattern of IBD, autocorrelation within populations, as well as high estimated local recruitment, suggest that dispersal is limited in populations with planktonic larvae as well as in those with benthic larvae. The decrease in salinity between the NS and BS causes a barrier to gene flow in many marine species. In P. elegans, low, but significant, differentiation was detected between the NS and BS (3.34% in AMOVA), but no clear transition zone was observed, indicating that larvae are not hampered by the change in salinity.

Sexual conflict over the duration of copulation in Drosophila montana: why is longer better?

BACKGROUND: Conflicts of interest between the sexes are increasingly recognized as an engine driving the (co-)evolution of reproductive traits. The reproductive behaviour of Drosophila montana suggests the occurrence of sexual conflict over the duration of copulation. During the last stages of copulation, females vigorously attempt to dislodge the mounting male, while males struggle to maintain genital contact and often successfully extend copulations far beyond the females' preferred duration. RESULTS: By preventing female resistance, we show that females make a substantial contribution towards shortening copulations. We staged matings under different sex ratio conditions, and provide evidence that copulation duration is a form of male reproductive investment that responds to the perceived intensity of sperm competition as predicted by game theoretical models. Further, we investigated potential benefits to persistent males, and costs to females coerced into longer matings. While males did not benefit in terms of increased progeny production by protracting copulation, female remating was delayed after long first copulations. CONCLUSION: Copulation time is a trait subject to sexual conflict. Mating durations exceeding female optima serve males as a form of 'extended mate guarding': by inducing mating refractoriness in the female, a male extends the time over which its sperm is exclusively used to sire progeny and reduces the likelihood of the female being reinseminated by a competitor.