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The comet assay is a versatile method to detect nuclear DNA damage in individual eukaryotic cells, from yeast to human. The types of damage detected encompass DNA strand breaks and alkali-labile sites (e.g., apurinic/apyrimidinic sites), alkylated and oxidized nucleobases, DNA-DNA crosslinks, UV-induced cyclobutane pyrimidine dimers and some chemically induced DNA adducts. Depending on the specimen type, there are important modifications to the comet assay protocol to avoid the formation of additional DNA damage during the processing of samples and to ensure sufficient sensitivity to detect differences in damage levels between sample groups. Various applications of the comet assay have been validated by research groups in academia, industry and regulatory agencies, and its strengths are highlighted by the adoption of the comet assay as an in vivo test for genotoxicity in animal organs by the Organisation for Economic Co-operation and Development. The present document includes a series of consensus protocols that describe the application of the comet assay to a wide variety of cell types, species and types of DNA damage, thereby demonstrating its versatility.The comet assay is commonly used to assess DNA damage. This collection of consensus protocols includes adaptations for a wide range of species and sample types, assay formats and detection of different types of DNA lesions.

Dactylogyridae is one of the most studied families of parasitic flatworms with more than 1000 species and 166 genera described to date including ecto- and endoparasites. Dactylogyrid monogeneans were suggested as model organisms for host-parasite macroevolutionary and biogeographical studies due to the scientific and economic importance of some of their host lineages. Consequently, an array of phylogenetic research into different dactylogyrid lineages has been produced over the past years but the last family-wide study was published 16 years ago. Here, we provide a meta-analysis of the phylogenetic relationships of Dactylogyridae including representatives of all genera with available molecular data (n = 67). First, we investigate the systematic informativeness of morphological characters widely used to diagnose dactylogyrid genera through a parsimony analysis of the characters, character mapping, and phylogenetic comparative methods. Second, we provide an overview of the current state of the systematics of the family and its subfamilies, and summarise potentially poly- and paraphyletic genera. Third, we elaborate on the implications of taxonomic, citation, and confirmation bias in past studies. Fourth, we discuss host range, biogeographical, and freshwater-marine patterns. We found two well-supported macroclades which we assigned to the subfamilies Dactylogyrinae and Ancyrocephalinae. These subfamilies further include 16 well-supported clades with only a few synapomorphies that could be deduced from generic diagnoses in the literature. Furthermore, few morphological characters considered systematically informative at the genus level display a strong phylogenetic signal. However, the parsimony analysis suggests that these characters provide little information on the relationships between genera. We conclude that a strong taxonomic bias and low coverage of DNA sequences and regions limit knowledge on morphological and biogeographical evolutionary patterns that can be inferred from these results. We propose addressing potential citation and confirmation biases through a 'level playing field' multiple sequence alignment as provided by this study. This research was funded by Czech Science Foundation (GACˇ R) standard project GA19-13573S. N.K. (BOF21PD01), A.J.C.L. (BOF19OWB02) and M.P.M.V. (BOF20TT06) are financed by the Special Research Fund of Hasselt University, Belgium. Walter A. Boeger (Universidade Federal do Paraná, Brazil) is thanked for fruitful discussions on this matter

During colonial times, Nile tilapia Oreochromis niloticus (Linnaeus, 1758) was introduced into non-native parts of the Congo Basin (Democratic Republic of the Congo, DRC) for the first time. Currently, it is the most farmed cichlid in the DRC, and is present throughout the Congo Basin. Although Nile tilapia has been reported as an invasive species, documentation of historical introductions into this basin and its consequences are scant. Here, we study the genetic consequences of these introductions by genotyping 213 Nile tilapia from native and introduced regions, focusing on the Congo Basin. Additionally, 48 specimens from 16 other tilapia species were included to test for hybridization. Using RAD sequencing (27,611 single nucleotide polymorphisms), we discovered genetic admixture with other tilapia species in several morphologically identified Nile tilapia from the Congo Basin, reflecting their ability to interbreed and the potential threat they pose to the genetic integrity of native tilapias. Nile tilapia populations from the Upper Congo and those from the Middle-Lower Congo are strongly differentiated. The former show genetic similarity to Nile tilapia from the White Nile, while specimens from the Benue Basin and Lake Kariba are similar to Nile tilapia from the Middle-Lower Congo, suggesting independent introductions using different sources. We conclude that the presence of Nile tilapia in the Congo Basin results from independent introductions, reflecting the dynamic aquaculture history, and that their introduction probably leads to genetic interactions with native tilapias, which could lower their fitness. We therefore urge avoiding further introductions of Nile tilapia in non-native regions and to use native tilapias in future aquaculture efforts.

Species interactions are a key aspect of evolutionary biology. Parasites, specifically, are drivers of the evolution of species communities and impact biosecurity and public health. However, when using interaction networks for evolutionary studies, interdependencies between distantly related species in these networks are shaped by ancient and complex processes. We propose using recent interacting host–parasite radiations, e.g. African cichlid fishes and cichlid gill parasites belonging to Cichlidogyrus (Dactylogyridae, Monogenea), as macroevolutionary model of species interactions. The cichlid–Cichlidogyrus network encompasses 138 parasite species and 416 interactions identified through morphological characteristics and genetic markers in 160 publications. We discuss the steps required to develop this model system based on data resolution, sampling bias, and reporting quality. In addition, we propose the following steps to guide efforts for a macroevolutionary model system for species interactions: first, evaluating and expanding model system outcome measures to increase data resolution; second, closing knowledge gaps to address underreporting and sampling bias arising from limited human and financial resources. Identifying phylogenetic and geographic targets, creating systematic overviews, enhancing scientific collaborations, and avoiding data loss through awareness of predatory journal publications can accelerate this process; and third, standardising data reporting to increase reporting quality and to facilitate data accessibility.

Metazoan parasites encompass a significant portion of the global biodiversity. Their relevance for environmental and human health calls for a better understanding as parasite macroevolution remains mostly understudied. Yet limited molecular, phenotypic, and ecological data have so far discouraged complex analyses of evolutionary mechanisms and encouraged the use of data discretisation and body-size correction. In this case study, we aim to highlight the limitations of these methods and propose new methods optimised for small datasets. We apply multivariate phylogenetic comparative methods (PCMs) and statistical classification using support vector machines (SVMs) to a data-deficient host-parasite system. We use continuous morphometric and host range data currently widely inferred from a species-rich lineage of parasites (Cichlidogyrus incl. Scutogyrus - Platyhelminthes: Monogenea, Dactylogyridae) infecting cichlid fishes. For PCMs, we modelled the attachment organ and host range evolution using the data of 135 species and an updated multi-marker (28S and 18S rDNA, ITS1, COI mtDNA) phylogenetic reconstruction of 58/137 described species. Through a cluster analysis, SVM-based classification, and taxonomic literature survey, we infered the systematic informativeness of discretised and continuous characters. We demonstrate that an update to character coding and size-correction techniques is required as some techniques mask phylogenetic signals but remain useful for characterising species groups of Cichlidogyrus. Regarding the attachment organ evolution, PCMs suggest a pattern associated with genetic drift. Yet host and environmental parameters might put this structure under stabilising selection as indicated by a limited morphological variation. This contradiction, the absence of a phylogenetic signal and multicollinearity in most measurements, a moderate 73% accordance rate of taxonomic approach and SVMs, and a low phylogenetic informativeness of reproductive organ data suggest an overall limited systematic value of the measurements included in most species characterisations. We conclude that PCMs and SVM-based approaches are suitable tools to investigate the character evolution of data-deficient taxa.

Many species-rich ecological communities result from adaptive radiation events. The effects of these explosive speciation events on community assembly remain poorly understood. Here, we explore the well-documented radiations of African cichlid fishes and interactions with their flatworm gill parasites (Cichlidogyrus spp.) including 10529 reported infections and 477 different host-parasite combinations collected through a survey of peer-reviewed literature. We assess the evolutionary, ecological, and morphological parameters on meta-communities partially affected by adaptive radiation evens using network metrics, host repertoire measures, and network link prediction (NLP). The hosts’ evolutionary history mostly determined host repertoires. Ecological and evolutionary parameters predicted host-parasite associations, but many interactions remain undetected according to NLP. Parasite meta-communities under host adaptive radiation are more specialised and stable while ecological opportunity and ecological fitting have shaped interactions elsewhere. The cichlid-Cichlidogyrus network is a suitable eco-evolutionary study system but future studies should validate our findings in other radiating host-parasite systems.

Hosts and parasites have often intimate associations. Therefore, the evolution of their interactions is crucial for understanding species-rich host-parasite communities. Yet relatively few studies investigate eco-evolutionary feedbacks in these systems as large datasets remain scarce. Here, we explore African cichlid fishes and their flatworm gill parasites (Cichlidogyrus spp.) including 9901 reported infections and 473 different host-parasite combinations collected through a survey of peer-reviewed literature. We apply network metrics, estimate host repertoires, and use network link prediction (NLP) algorithms to investigate meta-community structures and their predictors including evolutionary, ecological, and morphological parameters. Host repertoire was mostly determined by the hosts’ evolutionary history. Both ecological and evolutionary parameters predicted host parasite associations but many interactions remain undetected according to NLP. We conclude that ecological opportunity paired with ecological fitting has shaped interactions. The cichlid-Cichlidogyrus network is a suitable study system for eco-evolutionary feedbacks but taxonomic research remains key to finding undetected interactions.

A strict coordination between pro- and antioxidative molecules is needed for normal animal physiology, although their exact function and dynamics during regeneration and development remains largely unknown. Via in vivo imaging, we were able to locate and discriminate between reactive oxygen species (ROS) in real-time during different physiological stages of the highly regenerative planarian Schmidtea mediterranea. All ROS signals were strong enough to overcome the detected autofluorescence. Combined with an in situ characterisation and quantification of the transcription of several antioxidant genes, our data showed that the planarian gut and epidermis have a well-equipped redox system. Pharmacological inhibition or RNA interference of either side of the redox balance resulted in alterations in the regeneration process, characterised by decreased blastema sizes and delayed neurodevelopment, thereby affecting tails more than heads. Focusing on glutathione, a central component in the redox balance, we found that it is highly present in planarians and that a significant reduction in glutathione content led to regenerative failure with tissue lesions, characterised by underlying stem cell alterations. This exploratory study indicates that ROS and antioxidants are tightly intertwined and should be studied as a whole to fully comprehend the function of the redox balance in animal physiology. His work was supported by the Research Foundation Flanders (FWO) (G.0B83.17N, N1522719 and 1522015N and the Bijzonder OnderzoeksFonds of Hasselt University (BOF16NI03). The research leading to results presented in this publication was carried out with infrastructure funded by The European Marine Biological Resource Centre (EMBRC) Belgium—FWO project GOH3817N and I001219N. The authors thank Natascha Steffanie and Ria Vanderspikken for their skilful technical assistance.

The taxon Koinocystididae is the third most species-rich family within Eukalyptorhynchia. However, its diversity and phylogeny have been largely neglected in former studies. We introduce three new genera and twelve new species of Koinocystididae including Simplexcystis asymmetrica gen. n. sp. n., Galapagetula cubensis sp. n., eight species of Reinhardorhynchus gen. n. and two species of Itaipusa. This raises the total number of species within Koinocystididae from 51 to 63. We also report on new distribution records for six known species: I. divae (Cuba, Panama and New Caledonia), I. karlingi (Sardinia and Lanzarote), Reinhardorhynchus riegeri comb. n. (Cuba), R. ruffinjonesi comb. n. (Cuba and Panama), Utelga heinckei (Cuba and Lanzarote), and U. pseudoheinckei (Sardinia). Simplexcystis asymmetrica gen. n. sp. n. is characterised by a male duct running eccentrically through the copulatory bulb, lack of any hard structures in the male system, lack of a bursa, and the fact that the epithelia of the female, the male, and part of the common atrium are covered by a brush border. Galapagetula cubensis sp. n. has a caudal gonopore, a divisa-type copulatory bulb with an unarmed penis papilla, and a female duct without a sphincter. The new species of Itaipusa and Reinhardorhynchus gen. n. differ from their congeners in the detailed structure of the copulatory bulb and especially the hard structures associated with it. In a molecular phylogenetic analysis based on all available 18S and 28S rDNA sequences of koinocystidids, we found support for the monophyly of the family and the genus Utelga Marcus, 1949. The genus Itaipusa is not monophyletic in that I. sinensis forms a clade with Rhinolasius dillonicus, while other species of Itaipusa that have a copulatory bulb armed with hooks form a clade together with Sekerana stolzi. As the type species of Itaipusa (I. divae) is in neither of these clades, we erected a new genus for I. sinensis (Koinogladius gen. n.) and one for species of Itaipusa having a hook-bearing copulatory bulb (Reinhardorhynchus gen. n.), respectively. Whether the remaining species of Itaipusa form a monophylum remains uncertain. The taxon Koinocystididae is the third most species-rich family within Eukalyptorhynchia. However, its diversity and phylogeny have been largely neglected in former studies. We introduce three new genera and twelve new species of Koinocystididae including Simplexcystis asymmetrica gen. n. sp. n., Galapagetula cubensis sp. n., eight species of Reinhardorhynchus gen. n. and two species of Itaipusa. This raises the total number of species within Koinocystididae from 51 to 63. We also report on new distribution records for six known species: I. divae (Cuba, Panama and New Caledonia), I. karlingi (Sardinia and Lanzarote), Reinhardorhynchus riegeri comb. n. (Cuba), R. ruffinjonesi comb. n. (Cuba and Panama), Utelga heinckei (Cuba and Lanzarote), and U. pseudoheinckei (Sardinia). Simplexcystis asymmetrica gen. n. sp. n. is characterised by a male duct running eccentrically through the copulatory bulb, lack of any hard structures in the male system, lack of a bursa, and the fact that the epithelia of the female, the male, and part of the common atrium are covered by a brush border. Galapagetula cubensis sp. n. has a caudal gonopore, a divisa-type copulatory bulb with an unarmed penis papilla, and a female duct without a sphincter. The new species of Itaipusa and Reinhardorhynchus gen. n. differ from their congeners in the detailed structure of the copulatory bulb and especially the hard structures associated with it. In a molecular phylogenetic analysis based on all available 18S and 28S rDNA sequences of koinocystidids, we found support for the monophyly of the family and the genus Utelga Marcus, 1949. The genus Itaipusa is not monophyletic in that I. sinensis forms a clade with Rhinolasius dillonicus, while other species of Itaipusa that have a copulatory bulb armed with hooks form a clade together with Sekerana stolzi. As the type species of Itaipusa (I. divae) is in neither of these clades, we erected a new genus for I. sinensis (Koinogladius gen. n.) and one for species of Itaipusa having a hook bearing copulatory bulb (Reinhardorhynchus gen. n.), respectively. Whether the remaining species of Itaipusa form a monophylum remains uncertain.

Many nominal species of microscopic animals traditionally fitting the 'everything is everywhere' paradigm have been revealed to be complexes of cryptic species. Here, we explore species diversity within the micrometazoan flatworm Gyratrix hermaphroditus-unique among meiofauna because of its global occurrence in a wide variety of brackish, freshwater and marine environments. With maximum likelihood and Bayesian approaches, we analysed 18S, 28S, 5.8S and ITS2 rDNA sequences from 401 specimens across the global distribution of G. hermaphroditus. Generalized Mixed Yule Coalescent (GMYC) and Automatic Barcode Gap Discovery (ABGD) methods delineated 78 and 62 putative species, respectively. This renders G. hermaphroditus one of the most species-rich complexes known to date. Based on shape variations of the male copulatory organ, 14 morphotypes corresponding with molecular clades were identified within the species complex. Within morphotypes, morphometric measurements were able to further discriminate between GMYC species using discriminant analyses. While most putative species occur on local or regional scales, over 10% are distributed over vast distances (>500 km apart) and two GMYC and six ABGD species have colonised multiple continents. This suggests that the cosmopolitanism of the G. hermaphroditus species complex is not just caused by mixing cryptic species with a more limited geographic distribution, but is due to the presence of previously unrecognised cosmopolitan taxa. The wide variation in distribution patterns between putative species indicates that meiofaunal biogeography should not be simplified into 'everything is everywhere', but rather entails every ecological state, extending from local endemism to true cosmopolitanism. Fonds Wetenschappelijk Onderzoek, Grant/Award Number: G.08.208.08 and W0.009.11N (BeBol); Belgian Federal Science Policy Office; European Marine Biological Resource Centre Belgium, Grant/Award Number: FWO project GOH3817. This work was performed in the context of the FWO project G.08.208.08, the FWO research network W0.009.11N (BeBol), the BELSPO–IAP project SPEEDY and personal FWO grants to Bart Tessens and Marlies Monnens. The research leading to results presented in this publication was carried out with infrastructure funded by EMBRC Belgium - FWO project GOH3817N. Financial support was provided by the Belgian Science Policy Office (BELSPO) through the ‘Joint Experimental Molecular Unit' (JEMU: http://jemu. myspecies.info/) at RBINS and RMCA. Additional collection campaigns were funded by FWO travel grants to Niels Van Steenkiste. Mrs. Ria Vanderspikken and Mrs. Natascha Steffanie are gratefully acknowledged for their invaluable practical assistance in the lab at Hasselt University. Finally, we would like to thank everyone involved in the sampling campaigns that made this research possible: Prof. Ernest Schockaert is thanked for providing numerous specimens from all around the globe and Dr Wim Willems and Prof. Patrick Reygel for their help during the sampling campaigns to Australia and South Africa. Special acknowledgement also goes out to Dr Ricky Taylor, S'Bu Mfeka and the entire team at KZN wildlife for their support in St Lucia. We are grateful to Prof. Ana Maria Leal-Zanchet and João Braccini for their help during the expedition to Brazil. Prof. Marco CuriniGalletti is cordially thanked for his invitation to Sardinia and for sharing his extensive knowledge of Gyratrix hermaphroditus. The authors are grateful to Dr Chris Glasby for access to his lab at the Museum and Art Gallery of the Northern Territory in Darwin and to Prof. Sabine Dittmann for her valuable assistance at the Lincoln Marine Science Centre in Port Lincoln.

Given the global decline of many invertebrate food resources, it is fundamental to understand the dietary requirements of insectivores. We give new insights into the functional relationship between the spatial habitat use, food availability, and diet of a crepuscular aerial insectivore, the European Nightjar (Caprimulgus europaeus) by relating spatial use data with high‐throughput sequencing (HTS) combined with DNA metabarcoding. Our study supports the predictions that nightjars collect a substantial part of their daily nourishment from foraging locations, sometimes at considerable distance from nesting sites. Lepidopterans comprise 65% of nightjars' food source. Nightjars tend to select larger species of Lepidoptera (>19 mm) which suggests that nightjars optimize the efficiency of foraging trips by selecting the most energetically favorable—larger—prey items. We anticipate that our findings may shed additional light on the interactions between invertebrate communities and higher trophic levels, which is required to understand the repercussions of changing food resources on individual‐ and population‐level processes., We give new insights into the functional relationship between the spatial habitat use, food availability, and diet of a crepuscular aerial insectivore. Nightjars collect a substantial part of their daily nourishment from foraging locations which were presumed to be unsuitable for the species. These foraging habitats are found at considerable distances from breeding habitats and comprise grasslands and meadows.

Background/Aim: Glyphosate, a broad-spectrum herbicide, and its main metabolite aminomethylphosphonic acid (AMPA) are persistent in the environment. Studies showed associations between glyphosate or AMPA exposure and several adverse cellular processes, including metabolic alterations and oxidative stress.& nbsp;Objective: To determine the association between glyphosate and AMPA exposure and biomarkers of biological aging.& nbsp;Methods: We examined glyphosate and AMPA exposure, mtDNA content and leukocyte telomere length in 181 adults, included in the third cycle of the Flemish Environment and Health Study (FLEHSIII). DNA was isolated from leukocytes and the relative mtDNA content and telomere length were determined using qPCR. Urinary glyphosate and AMPA concentrations were measured by Gas Chromatography-Tandem Mass Spectrometry (GC-MS-MS). We used multiple linear regression models to associate mtDNA content and leukocyte telomere length with glyphosate or AMPA exposure while adjusting for confounding variables.& nbsp;Results: A doubling in urinary AMPA concentration was associated with 5.19% (95% CI: 0.49 to 10.11; p = 0.03) longer leukocyte telomere length, while no association was observed with urinary glyphosate concentration. No association between mtDNA content and urinary glyphosate nor AMPA levels was observed.& nbsp;Conclusions: This study showed that AMPA exposure may be associated with telomere biology in adults.

Despite extensive research on molecular pathways controlling the process of regeneration in model organisms, little is known about the actual initiation signals necessary to induce regeneration. Recently, the activation of ERK signaling has been shown to be required to initiate regeneration in planarians. However, how ERK signaling is activated remains unknown. Reactive Oxygen Species (ROS) are well-known early signals necessary for regeneration in several models, including planarians. Still, the probable interplay between ROS and MAPK/ERK has not yet been described. Here, by interfering with major mediators (ROS, EGFR and MAPK/ERK), we were able to identify wound-induced ROS, and specifically H2O2, as upstream cues in the activation of regeneration. Our data demonstrate new relationships between regeneration-related ROS production and MAPK/ERK activation at the earliest regeneration stages, as well as the involvement of the EGFR-signaling pathway. Our results suggest that (1) ROS and/or H2O2 have the potential to rescue regeneration after MEK-inhibition, either by H2O2-treatment or light therapy, (2) ROS and/or H2O2 are required for the activation of MAPK/ERK signaling pathway, (3) the EGFR pathway can mediate ROS production and the activation of MAPK/ERK during planarian regeneration. Despite extensive research on molecular pathways controlling the process of regeneration in model organisms, little is known about the actual initiation signals necessary to induce regeneration. Recently, the activation of ERK signaling has been shown to be required to initiate regeneration in planarians. However, how ERK signaling is activated remains unknown. Reactive Oxygen Species (ROS) are well-known early signals necessary for regeneration in several models, including planarians. Still, the probable interplay between ROS and MAPK/ERK has not yet been described. Here, by interfering with major mediators (ROS, EGFR and MAPK/ERK), we were able to identify woundinduced ROS, and specifically H2O2, as upstream cues in the activation of regeneration. Our data demonstrate new relationships between regeneration-related ROS production and MAPK/ERK activation at the earliest regeneration stages, as well as the involvement of the EGFR-signaling pathway. Our results suggest that (1) ROS and/or H2O2 have the potential to rescue regeneration after MEK-inhibition, either by H2O2-treatment or light therapy, (2) ROS and/or H2O2 are required for the activation of MAPK/ERK signaling pathway, (3) the EGFR pathway can mediate ROS production and the activation of MAPK/ERK during planarian regeneration.

Silver nanoparticles (AgNPs) are widely incorporated in household, consumer and medical products. Their unintentional release via wastewaters raises concerns on their environmental impact, particularly for aquatic organisms and their associated bacterial communities. It is known that the microbiome plays an important role in its host's health and physiology, e.g. by producing essential nutrients and providing protection against pathogens. A thorough understanding of the effects of AgNPs on bacterial communities and on their interactions with the host is crucial to fully assess AgNP toxicity on aquatic organisms. Our results indicate that the microbiome of the invertebrate Schmidtea mediterranea, a freshwater planarian, is affected by AgNP exposure at the tested 10 μg/ml concentration. Using targeted amplification of the bacterial 16S rRNA gene V3-V4 region, two independent experiments on the microbiomes of adult worms revealed a consistent decrease in Betaproteobacteriales after AgNP exposure, mainly attributed to a decrease in Curvibacter and Undibacterium. Although developing tissues and organisms are known to be more sensitive to toxic compounds, three independent experiments in regenerating worms showed a less pronounced effect of AgNP exposure on the microbiome, possibly because underlying bacterial community changes during development mask the AgNP induced effect. The presence of a polyvinyl-pyrrolidone (PVP) coating did not significantly alter the outcome of the experiments compared to those with uncoated particles. The observed variation between the different experiments underlines the highly variable nature of microbiomes and emphasises the need to repeat microbiome experiments, within and between physiological states of the animal.

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian Schmidtea mediterranea. Exposure to the genotoxic compound methyl methanesulfonate revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, stem cell responses differed between anterior and posterior fragments after amputation. Stem cell proliferation and differentiation proceeded successfully in the amputated heads, leading to regeneration of missing tissues. Stem cells in the amputated tails showed decreased proliferation and differentiation capacity. As a result, tails could not regenerate. Interference with the body-axis-associated component β-catenin-1 increased regenerative success in tail fragments by stimulating proliferation at an early time point. Our results suggest that differences in the Wnt-signalling gradient along the body axis modulate stem cell responses to MMS.

Nutrition during early childhood is linked to metabolic programming. We hypothesized that breastfeeding has long-term consequences on the energy metabolism exemplified by mitochondrial DNA (mtDNA). As part of the third cycle of the Flemish Environment and Health Study (FLEHSIII) cohort, 303 adolescents aged 14-15 years were included. We associated breastfeeding and blood mtDNA content 14-15 years later while adjusting for confounding variables. Compared with non-breastfed adolescents, mtDNA content was 23.1% (95%CI: 4.4-45.2; p = 0.013) higher in breastfed adolescents. Being breastfed for 1-10 weeks, 11-20 weeks, and >20 weeks, was associated with a higher mtDNA content of respectively 16.0% (95%CI: -7.1-44.9; p = 0.191), 23.5% (95%CI: 0.8-51.3; p = 0.042), and 31.5% (95%CI: 4.3-65.7; p = 0.021). Our study showed a positive association between breastfeeding and mtDNA content in adolescents which gradually increased with longer periods of breastfeeding. Higher mtDNA content may be an underlying mechanism of the beneficial effects of breastfeeding on children's metabolism. ispartof: SCIENTIFIC REPORTS vol:10 issue:1 ispartof: location:England status: published

The first three mitochondrial (mt) genomes of endosymbiotic turbellarian flatworms are characterised for the rhabdocoels Graffilla buccinicola, Syndesmis echinorum and S. kurakaikina. Interspecific comparison of the three newly obtained sequences and the only previously characterised rhabdocoel, the free-living species Bothromesostoma personatum, reveals high mt genomic variability, including numerous rearrangements. The first intrageneric comparison within rhabdocoels shows that gene order is not fully conserved even between congeneric species. Atp8, until recently assumed absent in flatworms, was putatively annotated in two sequences. Selection pressure was tested in a phylogenetic framework and is shown to be significantly relaxed in this and another protein-coding gene: cox1. If present, atp8 appears highly derived in platyhelminths and its functionality needs to be addressed in future research. Our findings for the first time allude to a large degree of undiscovered (mt) genomic plasticity in rhabdocoels. It merits further attention whether this variation is correlated with a symbiotic lifestyle. Our results illustrate that this phenomenon is widespread in flatworms as a whole and not exclusive to the better-studied neodermatans. ispartof: International Journal Of Biological Macromolecules vol:162 pages:454-469 ispartof: location:Netherlands status: published

Pluripotent stem cells hold great potential for regenerative medicine. Increased replication and division, such is the case during regeneration, concomitantly increases the risk of adverse outcomes through the acquisition of mutations. Seeking for driving mechanisms of such outcomes, we challenged a pluripotent stem cell system during the tightly controlled regeneration process in the planarian

Despite the extensive research on molecular pathways controlling the process of regeneration in planarians and other regeneration models, little is known about the actual initiation signals necessary to induce regeneration. Previously the involvement of ROS, EGFR and MAPK/ERK has been demonstrated during planarian regeneration, however the exact interplay has not been yet described. By selectively interfering with major mediators in all three key parts (ROS, EGFR & MAPK/ERK), we were able to identify amputation/wound-induced ROS, and H2O2specifically, as upstream cue in activating regeneration-initiation. In addition, our results demonstrate new relationships between regeneration related ROS production and MAPK/ERK activation at early regeneration stages, as well as the involvement of the EGFR-signaling pathway. In summary, our results suggest a new and more extensive signaling model with ROS, and H2O2, highlighted as upstream initiation-factor and its important functions in the downstream EGFR-MAPK/ERK pathway during planarian regeneration.

Silver nanoparticles (AgNPs) currently belong to the most commercialized nanomaterials, used in both consumer products and medical applications. Despite its omnipresence, in-depth knowledge on the potential toxicity of nanosilver is still lacking, especially for developing organisms. Research on vertebrates is limited due to ethical concerns, and planarians are an ideal invertebrate model to study the effects of AgNPs on stem cells and developing tissues in vivo, as regeneration mimics development by triggering massive stem cell proliferation. Our results revealed a strong interference of AgNPs with tissue- and neuroregeneration which was related to an altered stem cell cycle. The presence of a PVP-coating significantly influenced toxicity outcomes, leading to elevated DNA-damage and decreased stem cell proliferation. Non-coated AgNPs had an inhibiting effect on stem cell and early progeny numbers. Overall, regenerating tissues were more sensitive to AgNP toxicity, and careful handling and appropriate decision making is needed in AgNP applications for healing and developing tissues. We emphasize on the importance of AgNP characterization, as we showed that changes in physicochemical properties influence toxicity. This study was financially supported by Bijzonder Onderzoeksfonds Hasselt University; Fonds Wetenschappelijk Onderzoek Onderzoek Vlaanderen; Province of Limburg and Vlaamse Instelling voor Technologisch Onderzoek.

Background: Telomere length and mitochondrial DNA (mtDNA) content are markers of aging and aging-related diseases. There is inconclusive evidence concerning the mechanistic effects of airborne particulate matter (PM) exposure on biomolecular markers of aging. Objective: The present study examines the association between short- and long-term PM exposure with telomere length and mtDNA content in the elderly and investigates to what extent this association is mediated by expression of genes playing a role in the telomere–mitochondrial axis of aging. Methods: Among 166 nonsmoking elderly participants, we used qPCR to measure telomere length and mtDNA content in leukocytes and RNA from whole blood to measure expression of SIRT1, TP53, PPARGC1A, PPARGC1B, NRF1, and NFE2L2. Associations between PM exposure and markers of aging were estimated using multivariable linear regression models adjusted for sex, age, BMI, socioeconomic status, statin use, past smoking status, white blood cell count, and percentage of neutrophils. Mediation analysis was performed to explore the role of age-related markers between the association of PM exposure and outcome. Annual PM2.5 exposure was calculated for each participant’s home address using a high-resolution spatial–temporal interpolation model. Results: Annual PM2.5 concentrations ranged from 15 to 23 μg/m3. A 5-μg/m3 increment in annual PM2.5 concentration was associated with a relative decrease of 16.8% (95% CI: –26.0%, –7.4%, p = 0.0005) in telomere length and a relative decrease of 25.7% (95% CI: –35.2%, –16.2%, p < 0.0001) in mtDNA content. Assuming causality, results of the mediation analysis indicated that SIRT1 mediated 19.5% and 22.5% of the estimated effect of PM2.5 exposure on telomere length and mtDNA content, respectively. Conclusions: Our findings suggest that the estimated effects of PM2.5 exposure on the telomere–mitochondrial axis of aging may play an important role in chronic health effects of PM2.5. Citation: Pieters N, Janssen BG, Dewitte H, Cox B, Cuypers A, Lefebvre W, Smeets K, Vanpoucke C, Plusquin M, Nawrot TS. 2016. Biomolecular markers within the core axis of aging and particulate air pollution exposure in the elderly: a cross-sectional study. Environ Health Perspect 124:943–950; http://dx.doi.org/10.1289/ehp.1509728

The importance of nerve-derived signalling for correct regeneration has been the topic of research for more than a hundred years, but we are just beginning to identify the underlying molecular pathways of this process. Within the current review, we attempt to provide an extensive overview of the neural influences during early and late phases of both vertebrate and invertebrate regeneration. In general, denervation impairs limb regeneration, but the presence of nerves is not essential for the regeneration of aneurogenic extremities. This observation led to the “neurotrophic factor(s) hypothesis”, which states that certain trophic factors produced by the nerves are necessary for proper regeneration. Possible neuron-derived factors which regulate regeneration as well as the denervation-affected processes are discussed.

Planarians have been long known for their regenerative ability, which hinges on pluripotency. Recently, however, the planarian model has been successfully established for routine toxicological screens aimed to assess overproliferation, mutagenicity and tumorigenesis. In this study, we focused on planarian tumor suppressor genes (TSGs) and their role during chemically induced carcinogenic stress in Schmidtea mediterranea. Combining in silico and proteomic screens with exposure to human carcinogen type 1A agent cadmium (Cd), we showed that many TSGs have a function in stem cells and that, in general, exposure to Cd accelerated the onset and increased the severity of the observed phenotype. This suggested that the interaction between environmental and genetic factors plays an important role in tumor development in S. mediterranea. Therefore, we further focused on the synergistic effects of Cd exposure and p53 knockdown (KD) at the cellular and molecular levels. Cd also produced a specific proteomic landscape in homeostatic animals, with 172 proteins differentially expressed, 43 of which were downregulated. Several of these proteins have tumor suppressor function in human and other animals, namely Wilms Tumor 1 Associated Protein (WT1), Heat Shock Protein 90 (HSP90), Glioma Pathogenesis-Related Protein 1 (GLIPR1) and Matrix Metalloproteinase B (Smed-MMPB). Both Glipr1 and MmpB KD produced large outgrowths, epidermal lesions and epidermal blisters. The epidermal blisters that formed as a consequence of Smed-MmpB KD were populated by smedwi1+ cells, many of which were actively proliferating, while large outgrowths contained ectopically differentiated structures, such as photoreceptors, nervous tissue and a small pharynx. In conclusion, Smed-MmpB is a planarian TSG that prevents stem cell proliferation and differentiation outside the proper milieu. This work was supported by Fonds Wetenschappelijk Onderzoek (R-6411 to A.W.; 1522015N and G0B8317N to A.W., A.V.R., K.S.), Universität des Saarlandes (T.A.T.), Universiteit Hasselt (A.W., A.V.R., K.S.), Bijzonder Onderzoeksfonds (G0B81317 to A.W., A.V.R., K.S.), Deutscher Akademischer Austauschdienst (50753940 to L.G.), Hercules Foundation (R-3986), Egyptian Cultural Affair Mission Sector of the Ministry of Higher Education/National Research Centre (A.Z.A.-Z.B.), Max Planck Institute for Molecular Biomedicine (L.G.) and Fraunhofer Institute for Biomedical Engineering (L.G.).

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Aiming to in vivo characterize the responses of pluripotent stem cells and regenerative tissues to carcinogenic stress, we employed the highly regenerative organism Schmidtea mediterranea. Its broad regenerative capacities are attributable to a large pool of pluripotent stem cells, which are considered key players in the lower vulnerability toward chemically induced carcinogenesis observed in regenerative organisms. Schmidtea mediterranea is, therefore, an ideal model to study pluripotent stem cell responses with stem cells residing in their natural environment. Including microenvironmental alterations is important, as the surrounding niche influences the onset of oncogenic events. Both short-(3 days) and long-term (17 days) exposures to the genotoxic carcinogen methyl methanesulfonate (50 mu M) were evaluated during homeostasis and animal regeneration, two situations that render altered cellular niches. In both cases, MMS-induced DNA damage was observed, which provoked a decrease in proliferation on the short term. The outcome of DNA damage responses following long-term exposure differed between homeostatic and regenerating animals. During regeneration, DNA repair systems were more easily activated than in animals in homeostasis, where apoptosis was an important outcome. Knockdown experiments confirmed the importance of DNA repair systems during carcinogenic exposure in regenerating animals as knockdown of rad51 induced a stem cell-depleted phenotype, after regeneration was completed. Bijzonder OnderzoeksFonds of Hasselt University (grant number BOF08G01); Hasselt University tUL-impulsfinanciering (project toxicology) Fonds voor Wetenschappelijk Onderzoek (grant number 1522015N and PhD grant number R6411 to A.W.); Agentschap voor Innovatie door Wetenschap en Technologie (grant number 101442 to A.-S.S. and OZM grant number 100631 to M.W.).

Planarians have been long known for their regenerative ability, which hinges on pluripotency. Recently, however, the planarian model has been successfully established for routine toxicological screens aimed to assess overproliferation, mutagenicity and tumorigenesis. In this study, we focused on planarian tumor suppressor genes (TSGs) and their role during chemically induced carcinogenic stress in Schmidtea mediterranea. Combining in silico and proteomic screens with exposure to human carcinogen type 1A agent cadmium (Cd), we showed that many TSGs have a function in stem cells and that, in general, exposure to Cd accelerated the onset and increased the severity of the observed phenotype. This suggested that the interaction between environmental and genetic factors plays an important role in tumor development in S. mediterranea. Therefore, we further focused on the synergistic effects of Cd exposure and p53 knockdown (KD) at the cellular and molecular levels. Cd also produced a specific proteomic landscape in homeostatic animals, with 172 proteins differentially expressed, 43 of which were downregulated. Several of these proteins have tumor suppressor function in human and other animals, namely Wilms Tumor 1 Associated Protein (WT1), Heat Shock Protein 90 (HSP90), Glioma Pathogenesis-Related Protein 1 (GLIPR1) and Matrix Metalloproteinase B (Smed-MMPB). Both Glipr1 and MmpB KD produced large outgrowths, epidermal lesions and epidermal blisters. The epidermal blisters that formed as a consequence of Smed-MmpB KD were populated by smedwi1+ cells, many of which were actively proliferating, while large outgrowths contained ectopically differentiated structures, such as photoreceptors, nervous tissue and a small pharynx. In conclusion, Smed-MmpB is a planarian TSG that prevents stem cell proliferation and differentiation outside the proper milieu., Summary: Combining exposure to the human carcinogen cadmium with in silico and proteomic top-down approaches, we disclosed the conserved function of two novel tumor suppressor genes in planarian.

Reverse transcription-quantitative PCR (RT-qPCR) has been widely adopted to measure differences in mRNA levels; however, biological and technical variation strongly affects the accuracy of the reported differences. RT-qPCR specialists have warned that, unless researchers minimize this variability, they may report inaccurate differences and draw incorrect biological conclusions. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines describe procedures for conducting and reporting RT-qPCR experiments. The MIQE guidelines enable others to judge the reliability of reported results; however, a recent literature survey found low adherence to these guidelines. Additionally, even experiments that use appropriate procedures remain subject to individual variation that statistical methods cannot correct. For example, since ideal reference genes do not exist, the widely used method of normalizing RT-qPCR data to reference genes generates background noise that affects the accuracy of measured changes in mRNA levels. However, current RT-qPCR data reporting styles ignore this source of variation. In this commentary, we direct researchers to appropriate procedures, outline a method to present the remaining uncertainty in data accuracy, and propose an intuitive way to select reference genes to minimize uncertainty. Reporting the uncertainty in data accuracy also serves for quality assessment, enabling researchers and peer reviewers to confidently evaluate the reliability of gene expression data.

Over the years, anthropogenic factors have led to cadmium (Cd) accumulation in the environment causing various health problems in humans. Although Cd is not a Fenton-like metal, it induces oxidative stress in various animal models via indirect mechanisms. The degree of Cd-induced oxidative stress depends on the dose, duration and frequency of Cd exposure. Also the presence or absence of serum in experimental conditions, type of cells and their antioxidant capacity, as well as the speciation of Cd are important determinants. At the cellular level, the Cd-induced oxidative stress either leads to oxidative damage or activates signal transduction pathways to initiate defence responses. This balance is important on how different organ systems respond to Cd stress and ultimately define the pathological outcome. In this review, we highlight the Cd-induced oxidant/antioxidant status as well as the damage versus signalling scenario in relation to Cd toxicity. Emphasis is addressed to Cd-induced pathologies of major target organs, including a section on cell proliferation and carcinogenesis. Furthermore, attention is paid to Cd-induced oxidative stress in undifferentiated stem cells, which can provide information for future therapies in preventing Cd-induced pathologies.

The hypothesis that mitogen-activated protein kinase (MAPK) signalling is important in plant defences against metal stress has become accepted in recent years. To test the role of oxidative signal-inducible kinase (OXI1) in metal-induced oxidative signalling, the responses of oxi1 knockout lines to environmentally realistic cadmium (Cd) and copper (Cu) concentrations were compared with those of wild-type plants. A relationship between OXI1 and the activation of lipoxygenases and other initiators of oxylipin production was observed under these stress conditions, suggesting that lipoxygenase-1 may be a downstream component of OXI1 signalling. Metal-specific differences in OXI1 action were observed. For example, OXI1 was required for the up-regulation of antioxidative defences such as catalase in leaves and Fe-superoxide dismutase in roots, following exposure to Cu, processes that may involve the MEKK1-MKK2-WRKY25 cascade. Moreover, the induction of Cu/Zn superoxide dismutases in Cu-exposed leaves was regulated by OXI1 in a manner that involves fluctuations in the expression of miRNA398. These observations contrast markedly with the responses to Cd exposure, which also involves OXI1-independent pathways but rather involves changes in components mediating intracellular communication.

A delicate balance exists between the process of carcinogenesis and tissue regeneration. A number of malignant tumours are considered the outcome of an impaired or incomplete regeneration process, resulting in persistently dividing cells. Regeneration-competent tissues and animals are able to prevent and counteract growth abnormalities and seem to have a low vulnerability to chemical carcinogenesis. Cancer cell survival depends, among other things, on various redox-related mechanisms, which are targets of currently developed therapies. Disadvantages of these therapies are a lack of specificity and drug resistance. As the majority of these redox-related mechanisms also play an important role in successful and coordinated cell functioning and reproduction, the regeneration process offers a unique parallel context for modern cancer research. This review focuses on the interconnections between regeneration and carcinogenesis and how an understanding of regenerative forces and redox-controlled mechanisms could contribute to the identification of new therapeutic targets to block the growth and survival of cancer cells.

One of the major challenges in the development of alternative carcinogenicity assays is the prediction of non-genotoxic carcinogens. The variety of non-genotoxic cancer pathways complicates the search for reliable parameters expressing their carcinogenicity. As non-genotoxic and genotoxic carcinogens have different cancer risks, the objective of this study was to develop a concept for an in vivo test, based on flatworm stem cell dynamics, to detect and classify carcinogenic compounds. Our methodology entails an exposure to carcinogenic compounds during the animal's regeneration process, which revealed differences in proliferative responses between non-genotoxic and genotoxic carcinogens during the initial stages of the regeneration process. A proof of concept was obtained after an extensive study of proliferation dynamics of a genotoxic and a non-genotoxic compound. A pilot validation with a limited set of compounds showed that the proposed concept not only enabled a simple prediction of genotoxic and non-genotoxic carcinogens, but also had the power to discriminate between both. We further optimized this discrimination by combining stem cell proliferation responses with a phenotypic screening and by using specific knockdowns. In the future, more compounds will be tested to further validate and prove this concept. This work was supported by Hasselt University tUL-impulsfinanciering (project toxicology) and FWO (1522015N). The work of An-Sofie Stevens and Maxime Willems was financed by the agency for Innovation by Science and Technology (IWT).

This study aims to investigate the colonization of poplar by the endophyte Pseudomonas putida W619 and its capacity to promote plant growth. Poplar cuttings were inoculated with P. putida W619 (wild-type or gfp-labelled). The colonization of both strains was investigated and morphological, physiological and biochemical parameters were analyzed to evaluate plant growth promotion. Inoculation with P. putida W619 (wild-type) resulted in remarkable growth promotion, decreased activities of antioxidative defence related enzymes, and reduced stomatal resistance, all indicative of improved plant health and growth in comparison with the non-inoculated cuttings. In contrast, inoculation with gfp-labelled P. putida W619 did not promote growth; it even had a negative effect on plant health and growth. Furthermore, compared to the wildtype strain, colonization by the gfp-labelled P. putida W619:gfp1 was much lower; it only colonized the rhizosphere and root cortex while the wild-type strain also colonized the root xylem vessels. Despite the strong plant growth promoting capacity of P. putida W619 (wild-type), after gfp labelling its growth promoting characteristics disappeared and its colonization capacity was strongly influenced; for these reasons gfp labelling should be applied with sufficient caution.

The cellular redox balance seems an important modulator under heavy metal stress. While for other heavy metals these processes are well studied, oxidative stress related responses are also known to be triggered under uranium stress but information remains limited. This study aimed to further unravel the mechanisms by which plants respond to uranium stress. Seventeen-day-old Arabidopsis thaliana seedlings, grown on a modified Hoagland solution under controlled conditions, were exposed to 0, 0.1, 1, 10 and 100 μM uranium for 1, 3 and 7 days. While in Part I of this study oxidative stress related responses in the roots were discussed, this second Part II discusses oxidative stress related responses in the leaves and general conclusions drawn from the results of the roots and the leaves will be presented. As several responses were already visible following 1 day exposure, when uranium concentrations in the leaves were negligible, a root-to-shoot signaling system was suggested in which plastids could be important sensing sites. While lipid peroxidation, based on the amount of thiobarbituric acid reactive compounds, was observed after exposure to 100 μM uranium, affecting membrane structure and function, a transient concentration dependent response pattern was visible for lipoxygenase initiated lipid peroxidation. This transient character of uranium stress responses in leaves was emphasized by results of lipoxygenase (LOX2) and antioxidative enzyme transcript levels, enzyme capacities and glutathione concentrations both in time as with concentration. The ascorbate redox balance seemed an important modulator of uranium stress responses in the leaves as in addition to the previous transient responses, the total ascorbate concentration and ascorbate/dehydroascorbate redox balance increased in a concentration and time dependent manner. This could represent either a slow transient response or a stable increase with regard to plant acclimation to uranium stress.

When aiming to evaluate the environmental impact of uranium contamination, it is important to unravel the mechanisms by which plants respond to uranium stress. As oxidative stress seems an important modulator under other heavy metal stress, this study aimed to investigate oxidative stress related responses in Arabidopsis thaliana exposed to uranium concentrations ranging from 0.1 to 100 μM for 1, 3 and 7 days. Besides analyzing relevant reactive oxygen species-producing and -scavenging enzymes at protein and transcriptional level, the importance of the ascorbate–glutathione cycle under uranium stress was investigated. These results are reported separately for roots and leaves in two papers: Part I dealing with responses in the roots and Part II unraveling responses in the leaves and presenting general conclusions. Results of Part I indicate that oxidative stress related responses in the roots were only triggered following exposure to the highest uranium concentration of 100 μM. A fast oxidative burst was suggested based on the observed enhancement of lipoxygenase (LOX1) and respiratory burst oxydase homolog (RBOHD) transcript levels already after 1 day. The first line of defense was attributed to superoxide dismutase (SOD), also triggered from the first day. The enhanced SOD-capacity observed at protein level corresponded with an enhanced expression of iron SOD (FSD1) located in the plastids. For the detoxification of H2O2, an early increase in catalase (CAT1) transcript levels was observed while peroxidase capacities were enhanced at the later stage of 3 days. Although the ascorbate peroxidase capacity and gene expression (APX1) increased, the ascorbate/dehydroascorbate redox balance was completely disrupted and shifted toward the oxidized form. This disrupted balance could not be inverted by the glutathione part of the cycle although the glutathione redox balance could be maintained.

We focus on the recent evidence that elucidates our understanding about the effects of cadmium (Cd) on human health and their prevention. Recently, there has been substantial progress in the exploration of the shape of the Cd concentration-response function on osteoporosis and mortality. Environmental exposure to Cd increases total mortality in a continuous fashion without evidence of a threshold, independently of kidney function and other classical factors associated with mortality including age, gender, smoking and social economic status. Pooled hazard rates of two recent environmental population based cohort studies revealed that for each doubling of urinary Cd concentration, the relative risk for mortality increases with 17% (95% CI 4.2-33.1%; P < 0.0001). Tubular kidney damage starts at urinary Cd concentrations ranging between 0.5 and 2 mug urinary Cd/g creatinine, and recent studies focusing on bone effects show increased risk of osteoporosis even at urinary Cd below 1 mug Cd/g creatinine. The non-smoking adult population has urinary Cd concentrations close to or higher than 0.5 mug Cd/g creatinine. To diminish the transfer of Cd from soil to plants for human consumption, the bioavailability of soil Cd for the plants should be reduced (external bioavailability) by maintaining agricultural and garden soils pH close to neutral (pH-H(2)O of 7.5; pH-KCL of 6.5). Reducing the systemic bioavailability of intestinal Cd can be best achieved by preserving a balanced iron status. The latter might especially be relevant in groups with a lower intake of iron, such as vegetarians, and women in reproductive phase of life. In exposed populations, house dust loaded with Cd is an additional relevant exposure route. In view of the insidious etiology of health effects associated with low dose exposure to Cd and the current European Cd intake which is close to the tolerable weekly intake, one should not underestimate the importance of the recent epidemiological evidence on Cd toxicity as to its medical and public health implications. The Environmental Health research at Hasselt University is supported by grants from the Flemish Fund for Scientific Research (FWO, krediet aan navorsers to T.N.) and internal Hasselt University grants (Bijzonder Onderzoekdsfonds, BOF).

Quantitative real time polymerase chain reaction (qPCR) has become a widely used tool to examine gene expression levels. Reliable quantification, however, depends on a proper normalization strategy. Normalization with multiple reference genes is becoming the standard, although the most suitable reference genes depend on the applied treatment as well as the tissue or cell type studied. In this study the stability of various reference genes was investigated in cultures of oligodendrocytes derived from either mature or neonatal rats, the latter also in the presence of the liver X receptor (LXR) agonist. The expression stability of ten commonly used reference genes (HPRT, GAPDH, 18S, ActB, CycA, Tbp, Rpl13A, YWHAZ, HMBS, Pgk1) was analyzed using geNorm and NormFinder. When comparing the different types of cell cultures, Rpl13A, CycA, Pgk1 and YWHAZ were identified as most stable genes. After LXR agonist treatment, CycA, Pgk1 and Rpl13A were found to be the most stable by both geNorm and NormFinder. HMBS and the commonly used housekeeping genes GAPDH and 18S turned out to be the most variable according to geNorm and NormFinder. In conclusion, the use of multiple reference genes, instead of only one, in qPCR experiments with rat oligodendrocytes is strongly advised and standard housekeeping genes such as GAPDH and 18S are not recommended as they appear to be relatively unstable under the experimental conditions used. Reference gene selection should always be performed for each individual experiment, since useful reference genes are very specific for every situation. (C) 2010 Elsevier B.V. All rights reserved.

The reactive oxygen species (ROS)-signaling pathway is very important in heavy metal toxicity. Induction of the antioxidative defense mechanism, comprising ROS-scavenging enzymes and metabolites, in plants after environmental uranium contamination has been insufficiently studied in the past. This study aimed to analyze oxidative stress related responses in Arabidopsis thaliana after uranium exposure. Seventeen-day-old seedlings were exposed to 0, 0.1, 1, 10 and 100 μM uranium for 3 days. After exposure to 100 μM uranium, a decrease in fresh weight for leaves and roots was observed, leaves colored anthocyanous and roots were stunted and yellow. To reveal the importance of oxidative stress in uranium toxicity, alterations in ROS-scavenging enzymes were studied at protein and transcriptional level. Superoxide dismutase (SOD) capacities increased in leaves and roots after exposure to 100 μM uranium but no differences were observed for catalase (CAT) capacities. Transcript levels of different SODs located at various cellular compartments were affected depending on the place of action. Gene expression of CAT in leaves and roots was also affected after uranium exposure. Results indicate that oxidative stress plays an important role in uranium toxicity but suggest that plant responses differ for leaves and roots.

Recent research highlighted the impact of ROS as upstream regulators of tissue regeneration. We investigated their role and targeted processes during the regeneration of different body structures using the planarian Schmidtea mediterranea, an organism capable of regenerating its entire body, including its brain. The amputation of head and tail compartments induces a ROS burst at the wound site independently of the orientation. Inhibition of ROS production by diphenyleneiodonium (DPI) or apocynin (APO) causes regeneration defaults at both the anterior and posterior wound sites, resulting in reduced regeneration sites (blastemas) and improper tissue homeostasis. ROS signaling is necessary for early differentiation and inhibition of the ROS burst results in defects on the regeneration of the nervous system and on the patterning process. Stem cell proliferation was not affected, as indicated by histone H3-P immunostaining, fluorescence-activated cell sorting (FACS), in situ hybridization of smedwi-1, and transcript levels of proliferation-related genes. We showed for the first time that ROS modulate both anterior and posterior regeneration in a context where regeneration is not limited to certain body structures. Our results indicate that ROS are key players in neuroregeneration through interference with the differentiation and patterning processes. Bijzonder Onderzoeks- Fonds of Hasselt University (BOF08G01. Hasselt University tUL-impulsfinanciering (project toxicology). Grant BFU2012-31701 (Spain). Agency for Innovation by Science and Technology (IWT)

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants that are formed in combustion processes. At the cellular level, exposure to PAHs causes oxidative stress and/or some of it congeners bind to DNA, which may interact with mitochondrial function. However, the influence of these pollutants on mitochondrial DNA (mtDNA) content remains largely unknown. We determined whether indoor exposure to PAHs is associated with mitochondrial damage as represented by blood mtDNA content. Blood mtDNA content (ratio mitochondrial/nuclear DNA copy number) was determined by real-time qPCR in 46 persons, both in winter and summer. Indoor PAH exposure was estimated by measuring PAHs in sedimented house dust, including 6 volatile PAHs and 8 non-volatile PAHs. Biomarkers of oxidative stress at the level of DNA and lipid peroxidation were measured. In addition to the epidemiologic enquiry, we exposed human TK6 cells during 24 h at various concentrations (range: 0 to 500 mu M) of benzo(a) pyrene and determined mtDNA content. Mean blood mtDNA content averaged (+/- SD) 0.95 +/- 0.185. The median PAH content amounted 554.1 ng/g dust (25th-75th percentile: 390.7-767.3) and 1385 ng/g dust (25th-75th percentile: 1000-1980) in winter for volatile and non-volatile PAHs respectively. Independent for gender, age, BMI and the consumption of grilled meat or fish, blood mtDNA content decreased by 9.85% (95% CI: -15.16 to -4.2; p = 0.002) for each doubling of non-volatile PAH content in the house dust in winter. The corresponding estimate for volatile PAHs was -7.3% (95% CI: -13.71 to -0.42; p = 0.04). Measurements of oxidative stress were not correlated with PAH exposure. During summer months no association was found between mtDNA content and PAH concentration. The ability of benzo(a)pyrene (range 0 mu M to 500 mu M) to lower mtDNA content was confirmed in vitro in human TK6 cells. Based on these findings, mtDNA content can be a target of PAH toxicity in humans. Study supported by the Flemish Administration for Environment (Leefmilieu, Natuur & Energie) and the Flemish Scientific Fund (G.0.873.11.N.10/G029212N) and Bijzonder Onderzoeksfonds (BOF) UHasselt. Tim Nawrot is holder of a grant from the European Research Council, ERC starting grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

BACKGROUND: Studies emphasize the importance of particulate matter (PM) in the formation of reactive oxygen species and inflammation. We hypothesized that these processes can influence mitochondrial function of the placenta and fetus. OBJECTIVES: We investigated the influence of PM10 exposure during pregnancy on the mitochondrial DNA6content (mtDNA content) of the placenta and umbilical cord blood. METHODS: DNA was extracted from placental tissue (n = 174) and umbilical cord leukocytes (n = 176). Relative mtDNA copy numbers (i.e. mtDNA content) were determined by real6time PCR. Multiple regression models were used to link mtDNA content and in utero exposure to PM10 over various time windows during pregnancy. RESULTS: In multivariate6adjusted analysis, a 106Cg/m³ increase in PM10 exposure during the last month of pregnancy was associated with a 16.1% decrease (95% CI: 625.2, 66.0%, p = 0.003) in placental mtDNA content. The corresponding effect6size for average PM10 exposure during the third trimester was 17.4% (95% CI: 631.8, 60.1%, p = 0.05). Furthermore, we found that each doubling in residential distance to major roads was associated with an increase in placental mtDNA content of 4.0% (95% CI: 0.4, 7.8%, p = 0.03). No association was found between cord blood mtDNA content and PM10 exposure. CONCLUSIONS: Prenatal PM10 exposure was associated with placental mitochondrial alterations, which may both reflect and intensify oxidative stress production. The potential health consequences of decreased placental mtDNA content in early life must be further elucidated. This work was supported by grants from the Flemish Scientific Fund (FWO, G.0.873.11.N.10/1516112N), BOF and tUL6impulse financing (Transnational University Limburg, Hasselt6Maastricht Impuls Financing)

The planarian Schmidtea mediterranea is a well-studied model organism for developmental research, because of its stem cell system. This characteristic also provides a unique opportunity to study stress management and the effect of stress on stem cells. In this study, we characterised the stress signature at different levels of biological organization. The carcinogenic metal cadmium was used as a model chemical stressor. We focused on stem cell activity and its interaction with other known stress parameters. Here, we have found that S. mediterranea is able to cope with high internal levels of cadmium. At endpoints such as size and mobility, cadmium-related stress effects were detected but all of these responses were transient. Correspondingly, cadmium exposure led to an elevated mitotic activity of the neob-lasts, at the same time points when the other responses disappeared. At the molecular level, we observed redox-related responses that can be linked with both repair as well as proliferation mechanisms. Together, our results suggest that these animals have a high plasticity. The induc-tion of stem cell activity may underlie this restoring effect, although a carcinogenic outcome after longer exposure times cannot be excluded.

Accurate quantification by real-time RT-PCR relies on normalisation of the measured gene expression data. Normalisation with multiple reference genes is becoming the standard, but the best reference genes for gene expression studies within one organism may depend on the applied treatments or the organs and tissues studied. Ideally, reference genes should be evaluated in all experimental systems. A number of candidate reference genes for Arabidopsis have been proposed, which can be used as a starting point to evaluate their expression stability in individual experimental systems by available computer algorithms like geNorm and NormFinder. Using this approach, we identified the best three reference genes from a set of ten candidates, which included three traditional "housekeeping" genes, for normalisation of gene expression when roots and leaves of Arabidopsis thaliana are exposed to cadmium (Cd) and copper (Cu). The expression stabilities of AT5G15710 (F-box protein), AT2G28390 (SAND family protein) and AT5G08290 (mitosis protein YLS8) were the highest when considering the effect to the roots and shoots of Cd and Cu treatments. Even though the effect of Cd and excess Cu on the plants is very different, the same best reference genes were identified when considering Cd or Cu treatments separately. This suggests that these three genes may also be suitable when studying the gene expression after exposure of Arabidopsis thaliana to increased concentrations of other metals.

We examined the subcellular cadmium (Cd) localization in roots and leaves of wild-type Arabidopsis thaliana (ecotype Columbia) exposed to environmentally relevant Cd concentrations. Energy-dispersive X-ray microanalysis (EDXMA) was performed on high-pressure frozen and freeze-substituted tissues. In the root cortex, Cd was associated with phosphorus (Cd/P) in the apoplast and sulfur (Cd/S) in the symplast, suggesting phosphate and phytochelatin sequestration, respectively. In the endodermis, sequestration of Cd/S was present as fine granular deposits in the vacuole and as large granular deposits in the cytoplasm. In the central cylinder, symplastic accumulation followed a distinct pattern illustrating the importance of passage cells for the uptake of Cd. In the apoplast, a shift of Cd/S granular deposits from the middle lamella towards the plasmalemma was observed. Large amounts of precipitated Cd in the phloem suggest retranslocation from the shoot. In leaves, Cd was detected in tracheids but not in the mesophyll tissue. Extensive symplastic and apoplastic sequestration in the root parenchyma combined with retranslocation via the phloem confirms the excluder strategy of Arabidopsis thaliana.