Live Imaging in Planarians: Immobilization and Real-Time Visualization of Reactive Oxygen Species.

Imaging of living animals allows the study of metabolic processes in relation to cellular structures or larger functional entities. To enable in vivo imaging during long-term time-lapses in planarians, we combined and optimized existing protocols, resulting in an easily reproducible and inexpensive procedure. Immobilization with low-melting-point agarose eliminates the use of anesthetics, avoids interfering with the animal during imaging-functionally or physically-and allows recovering the organisms after the imaging procedure. As an example, we used the immobilization workflow to image the highly dynamic and fast-changing reactive oxygen species (ROS) in living animals. These reactive signaling molecules can only be studied in vivo and mapping their location and dynamics during different physiological conditions is crucial to understand their role in developmental processes and regeneration. In the current protocol, we describe both the immobilization and ROS detection procedure. We used the intensity of the signals together with pharmacological inhibitors to validate the signal specificity and to distinguish it from the autofluorescent nature of the planarian.

Polystyrene Microplastics of Varying Sizes and Shapes Induce Distinct Redox and Mitochondrial Stress Responses in a Caco-2 Monolayer.

Currently, we lack crucial knowledge on how the physicochemical properties of particles affect cellular health, resulting in an important gap in our understanding of the human toxicity of microplastics (MPs). Our aim was to evaluate the impact of the size and the shape of MPs on uptake and the intracellular effects in a human epithelial colorectal adenocarcinoma (Caco-2) cell line. Spherical (200 nm and 2 µm) and fibre-/fragment-shaped (8.9 ± 10.1 µm by 1.14 ± 0.97 µm) polystyrene microplastics (PS-MPs) were used to study their uptake and the potential to induce redox and mitochondrial stress responses after 24 h of exposure. We demonstrated the cellular uptake of both spherical and fibre-/fragment-shaped MPs in a size-dependent manner. In response to 2 µm spheres, we observed differential expressions of redox-related genes, including HMOX1, CAT, and GPX1. All PS-MPs decreased the intracellular H2O2 levels, which can be attributed to mitochondrial stress responses, such as increased mitochondrial DNA content, footprint, and morphology. Altogether, we demonstrated uptakes and changes in redox and mitochondrial parameters for all PS-MPs, with the 200 nm spheres showing the most profound effects. This suggests that the induction of defensive responses in Caco-2 cells mainly correlates with the number of particles taken up.

In Utero Exposure to Air Pollutants and Mitochondrial Heteroplasmy in Neonates.

Mitochondria are sensitive to oxidative stress, which can be caused by traffic-related air pollution. Placental mitochondrial DNA (mtDNA) mutations have been previously linked with air pollution. However, the relationship between prenatal air pollution and cord-blood mtDNA mutations has been poorly understood. Therefore, we hypothesized that prenatal particulate matter (PM2.5) and NO2 exposures are associated with cord-blood mtDNA heteroplasmy. As part of the ENVIRONAGE cohort, 200 mother-newborn pairs were recruited. Cord-blood mitochondrial single-nucleotide polymorphisms were identified by whole mitochondrial genome sequencing, and heteroplasmy levels were evaluated based on the variant allele frequency (VAF). Outdoor PM2.5 and NO2 concentrations were determined by a high-resolution spatial-temporal interpolation method based on the maternal residential address. Distributed lag linear models were used to determine sensitive time windows for the association between NO2 exposure and cord-blood mtDNA heteroplasmy. A 5 µg/m3 increment in NO2 was linked with MT-D-Loop16311T>C heteroplasmy from gestational weeks 17-25. MT-CYTB14766C>T was negatively associated with NO2 exposure in mid pregnancy, from weeks 14-17, and positively associated in late pregnancy, from weeks 31-36. No significant associations were observed with prenatal PM2.5 exposure. This is the first study to show that prenatal NO2 exposure is associated with cord-blood mitochondrial mutations and suggests two critical windows of exposure in mid-to-late pregnancy.

In utero particulate matter exposure in association with newborn mitochondrial ND4L10550A>G heteroplasmy and its role in overweight during early childhood.

BACKGROUND: Mitochondria play an important role in the energy metabolism and are susceptible to environmental pollution. Prenatal air pollution exposure has been linked with childhood obesity. Placental mtDNA mutations have been associated with prenatal particulate matter exposure and MT-ND4L10550A>G heteroplasmy has been associated with BMI in adults. Therefore, we hypothesized that in utero PM2.5 exposure is associated with cord blood MT-ND4L10550A>G heteroplasmy and early life growth. In addition, the role of cord blood MT-ND4L10550A>G heteroplasmy in overweight during early childhood is investigated. METHODS: This study included 386 mother-newborn pairs. Outdoor PM2.5 concentrations were determined at the maternal residential address. Cord blood MT-ND4L10550A>G heteroplasmy was determined using Droplet Digital PCR. Associations were explored using logistic regression models and distributed lag linear models. Mediation analysis was performed to quantify the effects of prenatal PM2.5 exposure on childhood overweight mediated by cord blood MT-ND4L10550A>G heteroplasmy. RESULTS: Prenatal PM2.5 exposure was positively associated with childhood overweight during the whole pregnancy (OR = 2.33; 95% CI: 1.20 to 4.51; p = 0.01), which was mainly driven by the second trimester. In addition, prenatal PM2.5 exposure was associated with cord blood MT-ND4L10550A>G heteroplasmy from gestational week 9 - 13. The largest effect was observed in week 10, where a 5 µg/m3 increment in PM2.5 was linked with cord blood MT-ND4L10550A>G heteroplasmy (OR = 0.93; 95% CI: 0.87 to 0.99). Cord blood MT-ND4L10550A>G heteroplasmy was also linked with childhood overweight (OR = 3.04; 95% CI: 1.15 to 7.50; p = 0.02). The effect of prenatal PM2.5 exposure on childhood overweight was mainly direct (total effect OR = 1.18; 95% CI: 0.99 to 1.36; natural direct effect OR = 1.20; 95% CI: 1.01 to 1.36)) and was not mediated by cord blood MT-ND4L10550A>G heteroplasmy. CONCLUSIONS: Cord blood MT-ND4L10550A>G heteroplasmy was linked with childhood overweight. In addition, in utero exposure to PM2.5 during the first trimester of pregnancy was associated with cord blood MT-ND4L10550A>G heteroplasmy in newborns. Our analysis did not reveal any mediation of cord blood MT-ND4L10550A>G heteroplasmy in the association between PM2.5 exposure and childhood overweight.

Explosive networking: The role of adaptive host radiations and ecological opportunity in a species-rich host-parasite assembly.

Many species-rich ecological communities emerge from adaptive radiation events. Yet the effects of adaptive radiation on community assembly remain poorly understood. Here, we explore the well-documented radiations of African cichlid fishes and their interactions with the flatworm gill parasites Cichlidogyrus spp., including 10,529 reported infections and 477 different host-parasite combinations collected through a survey of peer-reviewed literature. We assess how evolutionary, ecological, and morphological parameters determine host-parasite meta-communities affected by adaptive radiation events through network metrics, host repertoire measures, and network link prediction. The hosts' evolutionary history mostly determined host repertoires of the parasites. Ecological and evolutionary parameters predicted host-parasite interactions. Generally, ecological opportunity and fitting have shaped cichlid-Cichlidogyrus meta-communities suggesting an invasive potential for hosts used in aquaculture. Meta-communities affected by adaptive radiations are increasingly specialised with higher environmental stability. These trends should be verified across other systems to infer generalities in the evolution of species-rich host-parasite networks.

Somewhere I belong: phylogeny and morphological evolution in a species-rich lineage of ectoparasitic flatworms infecting cichlid fishes.

A substantial portion of biodiversity has evolved through adaptive radiation. However, the effects of explosive speciation on species interactions remain poorly understood. Metazoan parasites infecting radiating host lineages could improve our knowledge because of their intimate host relationships. Yet limited molecular, phenotypic and ecological data discourage multivariate analyses of evolutionary patterns and encourage the use of discrete characters. Here, we assemble new molecular, morphological and host range data widely inferred from a species-rich lineage of parasites (Cichlidogyrus, Platyhelminthes: Monogenea) infecting cichlid fishes to address data scarcity. We infer a multimarker (28S/18S rDNA, ITS1, COI mtDNA) phylogeny of 58 of 137 species and characterize major lineages through synapomorphies inferred from mapping morphological characters. We predict the phylogenetic position of species without DNA data through shared character states, a morphological phylogenetic analysis, and a classification analysis with support vector machines. Based on these predictions and a cluster analysis, we assess the systematic informativeness of continuous characters, search for continuous equivalents for discrete characters, and suggest new characters for morphological traits not analysed to date. We also model the attachment/reproductive organ and host range evolution using the data for 136 of 137 described species and multivariate phylogenetic comparative methods (PCMs). We show that discrete characters not only can mask phylogenetic signals, but also are key for characterizing species groups. Regarding the attachment organ morphology, a divergent evolutionary regime for at least one lineage was detected and a limited morphological variation indicates host and environmental parameters affecting its evolution. However, moderate success in predicting phylogenetic positions, and a low systematic informativeness and high multicollinearity of morphological characters call for a revaluation of characters included in species characterizations.

Population genomics of introduced Nile tilapia Oreochromis niloticus (Linnaeus, 1758) in the Democratic Republic of the Congo: Repeated introductions since colonial times with multiple sources.

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.

Dactylogyridae 2022: a meta-analysis of phylogenetic studies and generic diagnoses of parasitic flatworms using published genetic and morphological data.

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.

Interactive toxicity of copper and cadmium in regenerating and adult planarians.

In a polluted environment, metals are present as complex mixtures. As a result, organisms are exposed to different metals at the same time, which affects both metal-specific as well as overall toxicity. Detailed information about the molecular mechanisms underlying the adverse effects of combined exposures remains limited in terms of different life stages. In this study, the freshwater planarian Schmidtea mediterranea was used to investigate developmental and physiological responses associated with a combined exposure to Cu and Cd. In addition, the cellular and molecular mechanisms underlying the provoked adverse effects were studied in different exposure scenarios. Mixed exposure resulted in a decline in survival, diverse non-lethal morphological changes, neuroregenerative impairments, altered behaviour and a limited repair capacity. Underlying to these effects, the cellular redox state was altered in all exposure conditions. In adult animals, this led to DNA damage and corresponding transcriptional changes in cell cycle and DNA repair genes. In regenerating animals, changes in hydrogen peroxide and glutathione contents led to regenerative defects. Overall, our results demonstrate that (1) developing organisms are more susceptible to metal exposures, and (2) the toxicity of an individual metal increases significantly in a mixed exposure scenario. These aspects have to be included in current risk assessment strategies.

Glyphosate and AMPA exposure in relation to markers of biological aging in an adult population-based study.

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. OBJECTIVE: To determine the association between glyphosate and AMPA exposure and biomarkers of biological aging. 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. 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. CONCLUSIONS: This study showed that AMPA exposure may be associated with telomere biology in adults.

A Spatiotemporal Characterisation of Redox Molecules in Planarians, with a Focus on the Role of Glutathione during Regeneration.

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.

Taxonomy and phylogeny of Koinocystididae (Platyhelminthes, Kalyptorhynchia), with the description of three new genera and twelve new species.

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.

Differential effect of silver nanoparticles on the microbiome of adult and developing planaria.

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.

DNA diet profiles with high-resolution animal tracking data reveal levels of prey selection relative to habitat choice in a crepuscular insectivorous bird.

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.

Physico-chemical characterisation of the fraction of silver (nano)particles in pristine food additive E174 and in E174-containing confectionery.

Silver (E174) is authorised as a food additive in the EU. The unknown particle size distribution of E174 is a specific concern for the E174 risk assessment. This study characterised the fraction of silver (nano)particles in 10 commercially available pristine E174 food additives and 10 E174-containing products by transmission electron microscopy (TEM) and single-particle inductively coupled plasma-mass spectrometry (spICP-MS). TEM analysis showed that all samples contained micrometre-sized flakes and also a fraction of (nano)particles. Energy-dispersive X-ray spectroscopy (EDX) and electron diffraction confirmed that the (nano)particles and micrometre-sized flakes consisted of silver. A higher amount of (nano)particles was observed in the products than in the food additives. In addition, the surface of the micrometre-sized flakes was rougher in products. The median of the minimum external dimension, assessed as minimal Feret diameter, of the fraction of (nano)particles determined by quantitative TEM analysis was 11 ± 4 nm and 18 ± 7 nm (overall mean ± standard deviation), for food additives and products, respectively. Similar size distributions were obtained by spICP-MS and TEM, considering the limit of detection of spICP-MS. The median of the equivalent spherical diameter of the fraction of (nano)particles determined by spICP-MS was 19 ± 4 nm and 21 ± 2 nm (overall mean ± standard deviation), for food additives and products, respectively. In all samples, independent of the choice of technique, the nano-sized particles represented more than 97% (by number) of the silver particles, even though the largest mass of silver was present as flakes.

The first mitochondrial genomes of endosymbiotic rhabdocoels illustrate evolutionary relaxation of atp8 and genome plasticity in flatworms.

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.

Regenerative responses following DNA damage - ß-catenin mediates head regrowth in the planarian Schmidtea mediterranea.

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 (MMS) revealed that despite a similar DNA-damaging effect along the anteroposterior axis of intact animals, 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 ß-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.

Breastfeeding predicts blood mitochondrial DNA content in adolescents.

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.

In vivo Toxicity Assessment of Silver Nanoparticles in Homeostatic versus Regenerating Planarians.

Silver nanoparticles (AgNPs) 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.

A carcinogenic trigger to study the function of tumor suppressor genes in Schmidtea mediterranea.

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.