ABSTRACT
There is an ongoing process in which mitochondrial sequences are being integrated into the nuclear genome. The importance of these sequences has already been revealed in cancer biology, forensic, phylogenetic studies and in the evolution of the eukaryotic genetic information. Human and numerous model organisms' genomes were described from those sequences point of view. Furthermore, recent studies were published on the patterns of these nuclear localised mitochondrial sequences in different taxa.However, the results of the previously released studies are difficult to compare due to the lack of standardised methods and/or using few numbers of genomes. Therefore, in this paper our primary goal is to establish a uniform mining pipeline to explore these nuclear localised mitochondrial sequences.Our results show that the frequency of several repetitive elements is higher in the flanking regions of these sequences than expected. A machine learning model reveals that the flanking regions' repetitive elements and different structural characteristics are highly influential during the integration process.In this paper, we introduce a general mining pipeline for all mammalian genomes. The workflow is publicly available and is believed to serve as a validated baseline for future research in this field. We confirm the widespread opinion, on - as to our current knowledge - the largest dataset, that structural circumstances and events corresponding to repetitive elements are highly significant. An accurate model has also been trained to predict these sequences and their corresponding flanking regions.
Subject(s)
Genome, Mitochondrial , Animals , Humans , Phylogeny , DNA, Mitochondrial/genetics , Mammals/genetics , Repetitive Sequences, Nucleic AcidABSTRACT
The conjunctival bacterial resident and opportunistic flora of dogs may represent a major source of dissemination of pathogens throughout the environment or to other animals and humans. Nevertheless, contamination with bacteria from external sources is common. In this context, the study of the antimicrobial resistance (AMR) pattern may represent an indicator of multidrug resistant (MDR) strains exchange. The present study was focused on a single predisposed breed-Saint Bernard. The evaluated animals were healthy, but about half had a history of ocular disease/treatment. The swabs collected from conjunctival sacs were evaluated by conventional microbiological cultivation and antimicrobial susceptibility testing (AST). The most prevalent Gram-positive was Staphylococcus spp.; regardless of the history, while Gram-negative was Pseudomonas spp.; exclusively from dogs with a history of ocular disease/treatment. Other identified genera were represented by Bacillus, Streptococcus, Trueperella, Aeromonas and Neisseria. The obtained results suggest a possible association between the presence of mixed flora and a history of ocular disease/treatment. A high AMR was generally observed (90%) in all isolates, especially for kanamycin, doxycycline, chloramphenicol and penicillin. MDR was recorded in Staphylococcus spp. and Pseudomonas spp. This result together with a well-known zoonotic potential may suggest an exchange of these strains within animal human populations and the environment.
Subject(s)
Bacteria/isolation & purification , Conjunctiva/microbiology , Drug Resistance, Bacterial , Eye Infections/microbiology , Eye Infections/veterinary , Animals , Dogs , Female , Male , PrevalenceABSTRACT
Tumor-initiating cells are a subpopulation of cells that have self-renewal capacity to regenerate a tumor. Here, we identify stem cell-like chromatin features in human glioblastoma initiating cells (GICs) and link them to a loss of the repressive histone H3 lysine 9 trimethylation (H3K9me3) mark. Increasing H3K9me3 levels by histone demethylase inhibition led to cell death in GICs but not in their differentiated counterparts. The induction of apoptosis was accompanied by a loss of the activating H3 lysine 9 acetylation (H3K9ac) modification and accumulation of DNA damage and downregulation of DNA damage response genes. Upon knockdown of histone demethylases, KDM4C and KDM7A both differentiation and DNA damage were induced. Thus, the H3K9me3-H3K9ac equilibrium is crucial for GIC viability and represents a chromatin feature that can be exploited to specifically target this tumor subpopulation.
Subject(s)
Epigenesis, Genetic , Gene Expression Regulation, Neoplastic , Glioblastoma/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Neoplastic Stem Cells/metabolism , Acetylation , Animals , Apoptosis/genetics , Cell Line, Tumor , Cell Self Renewal/genetics , Chromatin/metabolism , DNA Methylation , DNA Repair/genetics , Gene Knockdown Techniques , Glioblastoma/pathology , HEK293 Cells , Histones , Humans , Jumonji Domain-Containing Histone Demethylases/genetics , Lysine/metabolism , Mice , Promoter Regions, Genetic/genetics , RNA, Small Interfering/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Populations of ectothermic vertebrates are vulnerable to environmental pollution and climate change because certain chemicals and extreme temperatures can cause sex reversal during early ontogeny (i.e. genetically female individuals develop male phenotype or vice versa), which may distort population sex ratios. However, we have troublingly little information on sex reversals in natural populations, due to unavailability of genetic sex markers. Here, we developed a genetic sexing method based on sex-linked single nucleotide polymorphism loci to study the prevalence and fitness consequences of sex reversal in agile frogs (Rana dalmatina). Out of 125 juveniles raised in laboratory without exposure to sex-reversing stimuli, 6 showed male phenotype but female genotype according to our markers. These individuals exhibited several signs of poor physiological condition, suggesting stress-induced sex reversal and inferior fitness prospects. Among 162 adults from 11 wild populations in North-Central Hungary, 20% of phenotypic males had female genotype according to our markers. These individuals occurred more frequently in areas of anthropogenic land use; this association was attributable to agriculture and less strongly to urban land use. Female-to-male sex-reversed adults had similar body mass as normal males. We recorded no events of male-to-female sex reversal either in the laboratory or in the wild. These results support recent suspicions that sex reversal is widespread in nature, and suggest that human-induced environmental changes may contribute to its pervasiveness. Furthermore, our findings indicate that sex reversal is associated with stress and poor health in early life, but sex-reversed individuals surviving to adulthood may participate in breeding.
Subject(s)
Ranidae , Sex Ratio , Adult , Animals , Breeding , Female , Genetic Markers , Genotype , Humans , Male , Ranidae/geneticsABSTRACT
Glioblastoma (GBM) is a highly aggressive brain tumor and still remains incurable. Among others, an immature subpopulation of self-renewing and therapy-resistant tumor cells-often referred to as glioblastoma stem-like cells (GSCs)-has been shown to contribute to disease recurrence. To target these cells personalized immunotherapy has gained a lot of interest, e.g. by reactivating pre-existing anti-tumor immune responses against GSC antigens. To identify T cell targets commonly presented by GSCs and their differentiated counterpart, we used a proteomics-based separation of GSC proteins in combination with a T cell activation assay. Altogether, 713 proteins were identified by LC-ESI-MS/MS mass spectrometry. After a thorough filtering process, 32 proteins were chosen for further analyses. Immunogenicity of corresponding peptides was tested ex vivo. A considerable number of these antigens induced T cell responses in GBM patients but not in healthy donors. Moreover, most of them were overexpressed in primary GBM and also highly expressed in recurrent GBM tissues. Interestingly, expression of the most frequent T cell target antigens could also be confirmed in quiescent, slow-cycling GSCs isolated in high purity by the DEPArray technology. Finally, for a subset of these T cell target antigens, an association between expression levels and higher T cell infiltration as well as an increased expression of positive immune modulators was observed. In summary, we identified novel immunogenic proteins, which frequently induce tumor-specific T cell responses in GBM patients and were also detected in vitro in therapy-resistant quiescent, slow-cycling GSCs. Stable expression of these T cell targets in primary and recurrent GBM support their suitability for future clinical use.
Subject(s)
Antigens, Neoplasm/metabolism , Brain Neoplasms/pathology , Glioblastoma/pathology , Proteomics , T-Lymphocyte Subsets/pathology , Animals , Annexin A1/metabolism , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Carcinogenicity Tests , Carrier Proteins/metabolism , Cells, Cultured , Chaperonin 60/metabolism , Cystatin A/metabolism , Disease Models, Animal , Epitope Mapping , Female , Humans , Interferon-gamma/metabolism , Ki-67 Antigen/metabolism , Male , Mice , Microfilament Proteins/metabolism , Mitochondrial Proteins/metabolism , Neoplastic Stem Cells/pathologyABSTRACT
The widespread application of pesticides emphasises the importance of understanding the impacts of these chemicals on natural communities. The most commonly applied broad-spectrum herbicides in the world are glyphosate-based herbicides, which have been suggested to induce significant behavioural changes in non-target organisms even at low environmental concentrations. To scrutinize the behavioural effects of herbicide-exposure we exposed agile frog (Rana dalmatina) tadpoles in an outdoor mesocosm experiment to three concentrations of a glyphosate-based herbicide (0, 2 and 6.5mg acid equivalent (a.e.) / L). To assess whether anti-predator behaviour is affected by the pesticide, we combined all levels of herbicide-exposure with three predator treatments (no predator, caged Aeshna cyanea dragonfly larvae or Lissotriton vulgaris newt adults) in a full factorial design. We observed hiding, activity, proximity to the predator cage and vertical position of tadpoles. We found that at the higher herbicide concentration tadpoles decreased their activity and more tadpoles were hiding, and at least at the lower concentration their vertical position was closer to the water surface than in tadpoles of the control treatment. Tadpoles also decreased their activity in the presence of dragonfly larvae, but did not hide more in response to either predator, nor did tadpoles avoid predators spatially. Further, exposure to the herbicide did not significantly influence behavioural responses to predation threat. Our study documents a definite influence of glyphosate-based herbicides on the behaviour of agile frog tadpoles and indicates that some of these changes are similar to those induced by dangerous predators. This may suggest that the underlying physiological mechanisms or the adaptive value of behavioural changes may similar.
Subject(s)
Escape Reaction/drug effects , Glycine/analogs & derivatives , Herbicides/toxicity , Larva/drug effects , Predatory Behavior/drug effects , Water Pollutants, Chemical/toxicity , Animals , Dose-Response Relationship, Drug , Environmental Monitoring/methods , Glycine/toxicity , Odonata/physiology , Ranidae , Spatial Behavior/drug effects , GlyphosateABSTRACT
Despite a steeply increasing number of ecotoxicological studies on the effects of pesticides on nontarget organisms, studies assessing the adequacy and reliability of different experimental approaches have remained scarce. We scrutinized effects of a glyphosate-based herbicide on larvae of two European anuran amphibians by estimating species-specific LC50 values, assessing how an additional stress factor may influence outcomes, and investigating whether replicate experiments yielded qualitatively the same results. We exposed Rana dalmatina and Bufo bufo tadpoles to two predator treatments (no predator vs. predator chemical cues) combined with varying herbicide concentrations, repeated the experiment with a subset of the experimental treatments and partly with slight modifications 1 week later and assessed survival. Our results indicated that the herbicide was moderately toxic to tadpoles. The presence of predator chemical cues did not affect the lethality of the herbicide in either species. The estimated sensitivity of R. dalmatina tadpoles varied considerably across experiments, whereas in case of B. bufo LC50 values remained very similar. Our results suggest that differences in the experimental setup may often have no influence on the measured effects of pesticides, whereas replicated experiments can deliver widely differing results in other cases, perhaps depending on the studied species, the population origin of the tested individuals, or the test conditions. This draws attention to the suggestion that strict standardization may not deliver widely applicable insights into the toxicity of contaminants and, instead, intentionally introducing variation into the design of ecotoxicological experiments and replicating entire experiments may prove highly beneficial.
Subject(s)
Herbicides/toxicity , Ranidae/physiology , Water Pollutants, Chemical/toxicity , Animals , Ecotoxicology , Larva/drug effects , Reproducibility of Results , Toxicity TestsABSTRACT
Defensive toxins are widespread in nature, yet we know little about how various environmental factors shape the evolution of chemical defense, especially in vertebrates. In this study we investigated the natural variation in the amount and composition of bufadienolide toxins, and the relative importance of ecological factors in predicting that variation, in larvae of the common toad, Bufo bufo, an amphibian that produces toxins de novo. We found that tadpoles' toxin content varied markedly among populations, and the number of compounds per tadpole also differed between two geographical regions. The most consistent predictor of toxicity was the strength of competition, indicating that tadpoles produced more compounds and larger amounts of toxins when coexisting with more competitors. Additionally, tadpoles tended to contain larger concentrations of bufadienolides in ponds that were less prone to desiccation, suggesting that the costs of toxin production can only be afforded by tadpoles that do not need to drastically speed up their development. Interestingly, this trade-off was not alleviated by higher food abundance, as periphyton biomass had negligible effect on chemical defense. Even more surprisingly, we found no evidence that higher predation risk enhances chemical defenses, suggesting that low predictability of predation risk and high mortality cost of low toxicity might select for constitutive expression of chemical defense irrespective of the actual level of predation risk. Our findings highlight that the variation in chemical defense may be influenced by environmental heterogeneity in both the need for, and constraints on, toxicity as predicted by optimal defense theory.
Subject(s)
Bufo bufo/physiology , Environment , Larva/chemistry , Larva/physiology , Animals , Biomass , Bufanolides/analysis , Bufanolides/chemistry , Linear ModelsABSTRACT
Cancer cells with enhanced self-renewal capacity influence tumour growth in glioblastoma. So far, a variety of surrogate markers have been proposed to enrich these cells, emphasizing the need to devise new characterization methods. Here, we screen a large panel of glioblastoma cultures (n = 21) cultivated under stem cell-permissive conditions and identify several cell lines with enhanced self-renewal capacity. These cell lines are capable of matrix-independent growth and form fast-growing, orthotopic tumours in mice. Employing isolation, re-plating, and label-retention techniques, we show that self-renewal potential of individual cells is partitioned asymmetrically between daughter cells in a robust and cell line-specific fashion. This yields populations of fast- and slow-cycling cells, which differ in the expression of cell cycle-associated transcripts. Intriguingly, fast-growing cells keep their slow-cycling counterparts in a reversible state of quiescence associated with high chemoresistance. Our results suggest that two different subpopulations of tumour cells contribute to aberrant growth and tumour recurrence after therapy in glioblastoma.
Subject(s)
Brain Neoplasms/pathology , Glioblastoma/pathology , Neoplasm Recurrence, Local/pathology , Neoplastic Stem Cells/pathology , Animals , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation , Comparative Genomic Hybridization , Disease Models, Animal , Gene Dosage/genetics , Gene Expression Profiling , Glioblastoma/metabolism , Humans , Mice , Neoplastic Stem Cells/metabolism , Oligonucleotide Array Sequence AnalysisABSTRACT
The enzyme dUTPase has an essential role in maintaining genomic integrity. In mouse, nuclear and mitochondrial isoforms of the enzyme have been described. Here we present the isoform-specific mRNA expression levels in different murine organs during development using RT-qPCR. In this study, we analyzed organs of 14.5-day embryos and of postnatal 2-, 4-, 10-week- and 13-month-old mice. We demonstrate organ-, sex- and developmental stage-specific differences in the mRNA expression levels of both isoforms. We found high mRNA expression level of the nuclear isoform in the embryo brain, and the expression level remained relatively high in the adult brain as well. This was surprising, since dUTPase is known to play an important role in proliferating cells, and mass production of neural cells is completed by adulthood. Thus, we investigated the pattern of the dUTPase protein expression specifically in the adult brain with immunostaining and found that dUTPase is present in the germinative zones, the subventricular and the subgranular zones, where neurogenesis occurs and in the rostral migratory stream where neuroblasts migrate to the olfactory bulb. These novel findings suggest that dUTPase may have a role in cell differentiation and indicate that accurate dTTP biosynthesis can be vital, especially in neurogenesis.
Subject(s)
Brain , Neurogenesis , Pyrophosphatases , Animals , Pyrophosphatases/metabolism , Pyrophosphatases/genetics , Mice , Female , Male , Brain/metabolism , Brain/growth & development , Gene Expression Regulation, Developmental , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Environmentally sensitive sex determination may help organisms adapt to environmental change but also makes them vulnerable to anthropogenic stressors, with diverse consequences for population dynamics and evolution. The mechanisms translating environmental stimuli to sex are controversial: although several fish experiments supported the mediator role of glucocorticoid hormones, results on some reptiles challenged it. We tested this hypothesis in amphibians by investigating the effect of corticosterone on sex determination in agile frogs (Rana dalmatina). This species is liable to environmental sex reversal whereby genetic females develop into phenotypic males. After exposing tadpoles during sex determination to waterborne corticosterone, the proportion of genetic females with testes or ovotestes increased from 11% to up to 32% at 3 out of 4 concentrations. These differences were not statistically significant except for the group treated with 10 nM corticosterone, and there was no monotonous dose-effect relationship. These findings suggest that corticosterone is unlikely to mediate sex reversal in frogs. Unexpectedly, animals originating from urban habitats had higher sex-reversal and corticosterone-release rates, reduced body mass and development speed, and lower survival compared to individuals collected from woodland habitats. Thus, anthropogenic environments may affect both sex and fitness, and the underlying mechanisms may vary across ectothermic vertebrates.
Subject(s)
Corticosterone , Glucocorticoids , Male , Female , Animals , Glucocorticoids/pharmacology , Corticosterone/pharmacology , Anura , Ranidae , TestisABSTRACT
Chytridiomycosis, an emerging infectious disease caused by Batrachochytrium dendrobatidis (Bd), poses a serious threat to amphibians. The thermal optimum of Bd is lower than that of most amphibians, providing an opportunity to cure infected individuals with elevated temperature. However, this approach presupposes detailed knowledge about the thermal tolerance of the fungus. To determine the temperature that may effectively reduce infection burdens in vivo, detailed in vitro studies are needed to characterize thermal tolerance of the fungus without complexities introduced by the species-specific characteristics of hosts' immune systems. The aim of our study was to evaluate the thermal tolerance of a hypervirulent isolate of Bd, considering the limits of its thermal tolerance and its capacity to rebound following heat treatment. We incubated Bd cell cultures at five different temperatures (21, 25.5, 27, 29, or 30.5 C) for one of six exposure durations (3, 4, 5, 6, 7, or 8 days) and subsequently counted the number of zoospores to assess the temperature dependence of Bd growth. We observed intensive Bd growth at 21 C. At 25.5 C, the number of zoospores also increased over time, but the curve plateaued at about half of the maximum values observed in the lower temperature treatment. At temperatures of 27 C and above, the fungus showed no measurable growth. However, when we moved the cultures back to 21 C after the elevated temperature treatments, we observed recovery of Bd growth in all cultures previously treated at 27 C. At 29 C, a treatment duration of 8 days was necessary to prevent recovery of Bd growth, and at 30.5 C a treatment duration of 5 days was needed to achieve the same result, revealing that these moderately elevated temperatures applied for only a few days have merely a fungistatic rather than a fungicidal effect under in vitro conditions.
Subject(s)
Chytridiomycota , Mycoses , Batrachochytrium , Humans , Mycoses/microbiology , TemperatureABSTRACT
Numtogenesis is observable in the mammalian genomes resulting in the integration of mitochondrial segments into the nuclear genomes (numts). To identify numts in rabbit, we aligned mitochondrial and nuclear genomes. Alignment significance threshold was calculated and individual characteristics of numts were analysed. We found 153 numts in the nuclear genome. The GC content of numts were significantly lower than the GC content of their genomic flanking regions or the genome itself. The frequency of three mammalian-wide interspersed repeats were increased in the proximity of numts. The decreased GC content around numts strengthen the theory which supposes a link between DNA structural instability and numt integration.
Subject(s)
DNA, Mitochondrial , Genome, Mitochondrial , Animals , Cell Nucleus/genetics , DNA, Mitochondrial/chemistry , DNA, Mitochondrial/genetics , Genome , Mammals/genetics , Mitochondria/genetics , Phylogeny , Rabbits , Sequence Analysis, DNAABSTRACT
Extreme temperatures during heat waves can induce mass-mortality events, but can also exert sublethal negative effects by compromising life-history traits and derailing sexual development. Ectothermic animals may, however, also benefit from increased temperatures via enhanced physiological performance and the suppression of cold-adapted pathogens. Therefore, it is crucial to address how the intensity and timing of naturally occurring or human-induced heat waves affect life-history traits and sexual development in amphibians, to predict future effects of climate change and to minimize risks arising from the application of elevated temperature in disease mitigation. We raised agile frog (Rana dalmatina) and common toad (Bufo bufo) tadpoles at 19 °C and exposed them to a simulated heat wave of 28 or 30 °C for six days during one of three ontogenetic periods (early, mid or late larval development). In agile frogs, exposure to 30 °C during early larval development increased mortality. Regardless of timing, all heat-treatments delayed metamorphosis, and exposure to 30 °C decreased body mass at metamorphosis. Furthermore, exposure to 30 °C during any period and to 28 °C late in development caused female-to-male sex reversal, skewing sex ratios strongly towards males. In common toads, high temperature only slightly decreased survival and did not influence phenotypic sex ratio, while it reduced metamorph mass and length of larval development. Juvenile body mass measured 2 months after metamorphosis was not adversely affected by temperature treatments in either species. Our results indicate that heat waves may have devastating effects on amphibian populations, and the severity of these negative consequences, and sensitivity can vary greatly between species and with the timing and intensity of heat. Finally, thermal treatments against cold-adapted pathogens have to be executed with caution, taking into account the thermo-sensitivity of the species and the life stage of animals to be treated.
Subject(s)
Anura , Hot Temperature , Animals , Bufo bufo , Female , Larva , Male , Ranidae , Sexual DevelopmentABSTRACT
The antiferromagnetic structures of the layered oxychalcogenides (Sr(1-x)Ba(x))(2)CoO(2)Cu(2)S(2) (0 ≤ x ≤ 1) have been determined by powder neutron diffraction. In these compounds Co(2+) is coordinated by four oxide ions in a square plane and two sulfide ions at the apexes of an extremely tetragonally elongated octahedron; the polyhedra share oxide vertexes. The magnetic reflections present in the diffraction patterns can in all cases be indexed using a â2a × â2a × c expansion of the nuclear cell, and nearest-neighbor Co(2+) moments couple antiferromagnetically within the CoO(2) planes. The ordered magnetic moment of Co(2+) in Sr(2)CoO(2)Cu(2)S(2) (x = 0) is 3.8(1) µ(B) at 5 K, consistent with high-spin Co(2+) ions carrying three unpaired electrons and with an additional significant unquenched orbital component. Exposure of this compound to moist air is shown to result in copper deficiency and a decrease in the size of the ordered moment to about 2.5 µ(B); there is a strong correlation between the size of the long-range ordered moment and the occupancy of the Cu site. Both the tetragonal elongation of the CoO(4)S(2) polyhedron and the ordered moment in (Sr(1-x)Ba(x))(2)CoO(2)Cu(2)S(2) increase with increasing Ba content, and in Ba(2)CoO(2)Cu(2)S(2), which has Co(2+) in an environment that is close to purely square planar, the ordered moment of 4.5(1) µ(B) at 5 K is over 0.7 µ(B) larger than that in Sr(2)CoO(2)Cu(2)S(2), so the unquenched orbital component in this case is even larger than that observed in octahedral Co(2+) systems such as CoO. The experimental observations of antiferromagnetic ground states and the changes in properties resulting from replacement of Sr by Ba are supported by ab initio calculations on Sr(2)CoO(2)Cu(2)S(2) and Ba(2)CoO(2)Cu(2)S(2). The large orbital moments in these systems apparently result from spin-orbit mixing of the unequally populated d(xz), d(yz), and d(z(2)) orbitals, which are reckoned to be almost degenerate when the CoO(4)S(2) polyhedron reaches its maximum elongation. The magnitudes of the ordered moments in high-spin Co(2+) oxide, oxychalcogenide, and oxyhalide systems are shown to correlate well with the tetragonal elongation of the coordination environment. The large orbital moments lead to an apparently magnetostrictive distortion of the crystal structures below the Neél temperature, with the symmetry lowered from tetragonal I4/mmm to orthorhombic Immm and the size of the distortion correlating well with the size of the long-range ordered moment for all compositions and for temperature-dependent data gathered on Ba(2)CoO(2)Cu(2)S(2).
Subject(s)
Cobalt/chemistry , Magnetics , Metals, Alkaline Earth/chemistry , Oxygen/chemistry , Sulfides/chemistry , Ions/chemistry , Molecular Structure , Solutions , Sulfides/chemical synthesisABSTRACT
Anthropogenic environmental change poses a special threat to species in which genetic sex determination can be overwritten by the thermal and chemical environment. Endocrine disrupting chemicals as well as extreme temperatures can induce sex reversal in such species, with potentially wide-ranging consequences for fitness, demography, population viability and evolution. Despite accumulating evidence suggesting that chemical and thermal effects may interact in ecological contexts, little is known about their combined effects on sex reversal. Here we assessed the simultaneous effects of high temperature (female-to-male sex-reversing agent) and 17α-ethinylestradiol (EE2), a widespread xenoestrogen (male-to-female sex-reversing agent), on sexual development and fitness-related traits in agile frogs (Rana dalmatina). We exposed tadpoles to a six-days heat wave (30 °C) and/or an ecologically relevant concentration of EE2 (30 ng/L) in one of three consecutive larval periods, and diagnosed sex reversals two months after metamorphosis using species-specific markers for genetic sexing. We found that high temperature induced female-to-male sex reversal, decreased survival, delayed metamorphosis, decreased body mass at metamorphosis, and increased the proportion of animals that had no fat bodies, while EE2 had no effect on these traits. Simultaneous exposure to heat and EE2 had non-additive effects on juvenile body mass, which were dependent on treatment timing and further complicated by a negative effect of sex reversal on body mass. These results show that environmentally relevant exposure to EE2 does not diminish the female-to-male sex-reversing effects of high temperature. Instead, our findings on growth suggest that climate change and chemical pollution may have complex consequences for individual fitness and population persistence in species with environment-sensitive sex determination.
Subject(s)
Endocrine Disruptors , Water Pollutants, Chemical , Animals , Anura , Climate Change , Endocrine Disruptors/toxicity , Ethinyl Estradiol , Female , Male , Temperature , Water Pollutants, Chemical/toxicityABSTRACT
The coupling of nucleotide biosynthesis and genome integrity plays an important role in ensuring faithful maintenance and transmission of genetic information. The enzyme dUTPase is a prime example of such coupling, as it generates dUMP for thymidylate biosynthesis and removes dUTP for synthesis of uracil-free DNA. Despite its significant role, the expression patterns of dUTPase isoforms in animals have not yet been described. Here, we developed a detailed optimization procedure for RT-qPCR-based isoform-specific analysis of dUTPase expression levels in various organs of adult mice. Primer design, optimal annealing temperature, and primer concentrations were specified for both nuclear and mitochondrial dUTPase isoforms, as well as two commonly used reference genes, GAPDH and PPIA. The linear range of the RNA concentration for the reverse transcription reaction was determined. The PCR efficiencies were calculated using serial dilutions of cDNA. Our data indicate that organs involved in lymphocyte production, as well as reproductive organs, are characterized by high levels of expression of the nuclear dUTPase isoform. On the other hand, we observed that expression of the mitochondrial dUTPase isoform is considerably increased in heart, kidney, and ovary. Despite the differences in expression levels among the various organs, we also found that the mitochondrial dUTPase isoform shows a much more uniform expression pattern as compared to the reference genes GAPDH and PPIA.
Subject(s)
Isoenzymes/genetics , Pyrophosphatases/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Transcriptome , Animals , Cell Nucleus/enzymology , DNA Primers/genetics , DNA, Complementary/metabolism , Deoxyuracil Nucleotides/metabolism , Female , Kidney/enzymology , Male , Mice , Mitochondria/enzymology , Myocardium/enzymology , Osmolar Concentration , Ovary/enzymology , Sensitivity and Specificity , Transition TemperatureABSTRACT
Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2'-deoxyuridine 5'-triphosphate (dUTP) into 2'-deoxyuridine 5'-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous dut +/- animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early dut -/- embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice.
Subject(s)
Embryonic Development/genetics , Gene Deletion , Pyrophosphatases/genetics , Animals , Blastocyst/metabolism , Blastocyst/pathology , CRISPR-Cas Systems , Cells, Cultured , Heterozygote , Homozygote , Mice , Mice, Knockout , Pyrophosphatases/metabolismABSTRACT
The absolute and relative configurations of 1-epialexine are established by X-ray crystallographic analysis, giving (1S,2R,3R,7S,7aS)-1,2,7-trihydroxy-3-(hydroxymethyl)pyrrolizidine. The compound crystallizes as the hemihydrate C(8)H(15)NO(4) x 0.5H(2)O, with hydrogen bonds holding the water molecule in a hydrophilic pocket between epialexine bilayers. In addition, a comparison was made between results obtained from examination of the Bijvoet pairs from data sets collected using molybdenum and copper radiation.
Subject(s)
Pyrrolizidine Alkaloids/chemistry , Crystallography, X-Ray , Molecular ConformationABSTRACT
The quality of structural models for 1,2,4,5-tetra-bromo-benzene (TBB), C6H2Br4, based on data collected from a single crystal in a diamond anvil cell at 0.4â GPa in situ using two different diffractometers belonging to different generations have been compared, together with the effects of applying different data-processing strategies.