Development of the Myzozoan Aquatic Parasite Perkinsus marinus as A Versatile Experimental Genetic Model Organism.

The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation was previously achieved for Perkinsus spp. but with few tools for transgene expression and limited selection efficacy. We sought to expand the power of experimental genetic tools for Perkinsus using P. marinus as a model. We constructed a modular plasmid assembly system for expression of multiple genes simultaneously. We developed efficient selection systems for three drugs, puromycin, bleomycin and blasticidin, that are effective in as little as three weeks. We developed eleven new promoters of variable expression strength. Furthermore, we identified that genomic integration of transgenes is predominantly via non-homologous recombination but with transgene fragmentation including deletion of some elements. To counter these dynamic processes, we show that bi-cistronic transcripts using the viral 2A peptides can couple selection to the maintenance of the expression of a transgene of interest. Collectively, these new tools and insights provide great new capacity to genetically modify and study Perkinsus as an aquatic parasite and evolutionary model.

Effects of pulse-modulated radiofrequency magnetic field (RF-EMF) exposure on apoptosis, autophagy, oxidative stress and electron chain transport function in human neuroblastoma and murine microglial cells.

The use of body-worn wireless devices with different communication protocols and rapidly changing exposure scenarios is still multiplying and the need to identify possible health effects of radiofrequency electromagnetic field (RF-EMF) exposure with extremely low-frequency (ELF) modulation envelops. In this study, effects of ELF-modulated 935 MHz RF-EMF on apoptosis, autophagy, oxidative stress and electron exchange in N9 microglial and SH-SY5Y neuroblastoma cells were investigated. Cells were exposed at 4 W/kg or sham-exposed for 2 and 24 h. RF-EMF exposure of both cell types did not alter apoptosis, the number of living cells nor the apoptosis-inducing factor (AIF), irrespective of the exposure duration. RF-EMF exposure for 24, but not for 2 h, increased protein levels of the autophagy marker ATG5, whereas LC3B-I and II and pERK were not altered in both cell types and exposure times investigated. A transient increase in glutathione (GSH), but not hydrogen peroxide and cytochrome c oxidase was found only in SH-SY5Y cells, indicating that short-time RF-EMF at SAR levels accepted by today's safety guidelines might cause autophagy and oxidative stress with the effect being dependent on cell type and exposure duration. Further studies are needed to evaluate possible underlying mechanisms involved in pulse-modulated RF-EMF exposure.

Effects of radiofrequency electromagnetic field exposure on neuronal differentiation and mitochondrial function in SH-SY5Y cells.

Exposure to radiofrequency electromagnetic fields (RF-EMF) has dramatically increased in the last decades with expanding use of mobile phones worldwide. The aim of this study was to evaluate effects of RF-EMF on neuronal differentiation and underlying signaling pathways involved in neuronal differentiation, neurodegeneration, and mitochondrial function. Differentiation of SH-SY5Y cells was performed using all-trans retinoic acid or staurosporine to obtain cholinergic and dopaminergic neurons. Exposure of SH-SY5Y cells at 935 MHz, 4 W/kg for 24 h did not alter the neuronal phenotypes quantitatively. Markers of the signaling pathways investigated, namely the mitogen-activated protein kinases (MAPK), extracellular signal-regulated kinases (Erk) 1 and 2 (p-Erk1/2) and protein kinase B (Akt), glycogen synthase kinase 3 ß (GSK3ß) and Wnt/ß-catenin were not significantly affected by RF-EMF compared to sham. RF-EMF-impaired mitochondrial respiration in cells under glucose deprivation, but glutathione levels and mitochondrial fission and fusion markers were not altered. These findings indicate that RF-EMF might lead to an impairment of mitochondrial function that is only manifest at maximal respiration and additional stressors such as glucose deprivation. Further research is needed to investigate the effects of RF-EMF on mitochondrial function in detail because mitochondrial impairment is closely related to the pathogenesis of neurodegenerative diseases.

Activity of mefloquine and mefloquine derivatives against Echinococcus multilocularis.

The cestode E. multilocularis causes the disease alveolar echinococcosis (AE) in humans. The continuously proliferating metacestode (larval stage) of the parasite infects mostly the liver and exhibits tumor-like growth. Current chemotherapeutical treatment options rely on benzimidazoles, which are rarely curative and have to be applied daily and life-long. This can result in considerable hepatotoxicity and thus treatment discontinuation. Therefore, novel drugs against AE are urgently needed. The anti-malarial mefloquine was previously shown to be active against E. multilocularis metacestodes in vitro, and in mice infected by intraperitoneal inoculation of metacestodes when administered at 100 mg/kg by oral gavage twice a week for 12 weeks. In the present study, the same dosage regime was applied in mice infected via oral uptake of eggs representing the natural route of infection. After 12 weeks of treatment, the presence of parasite lesions was assessed in a liver squeeze chamber and by PCR, and a significantly reduced parasite load was found in mefloquine-treated animals. Assessment of mefloquine plasma concentrations by HPLC and modeling using a two-compartment pharmacokinetic model with first-order absorption showed that >90% of the expected steady-state levels (Cmin 1.15 mg/L, Cmax 2.63 mg/L) were reached. These levels are close to concentrations achieved in humans during long-term weekly dosage of 250 mg (dose applied for malaria prophylaxis). In vitro structure-activity relationship analysis of mefloquine and ten derivatives revealed that none of the derivatives exhibited stronger activities than mefloquine. Activity was only observed, when the 2-piperidylmethanol group of mefloquine was replaced by an amino group-containing residue and when the trifluoromethyl residue on position 8 of the quinoline structure was present. This is in line with the anti-malarial activity of mefloquine and it implies that the mode of action in E. multilocularis might be similar to the one against malaria.

Effects of silica nanoparticle exposure on mitochondrial function during neuronal differentiation.

BACKGROUND: Nanomedicine offers a promising tool for therapies of brain diseases, but potential effects on neuronal health and neuronal differentiation need to be investigated to assess potential risks. The aim of this study was to investigate effects of silica-indocyanine green/poly (ε-caprolactone) nanoparticles (PCL-NPs) engineered for laser tissue soldering in the brain before and during differentiation of SH-SY5Y cells. Considering adaptations in mitochondrial homeostasis during neuronal differentiation, metabolic effects of PCL-NP exposure before and during neuronal differentiation were studied. In addition, kinases of the PI3 kinase (PI3-K/Akt) and the MAP kinase (MAP-K/ERK) pathways related to neuronal differentiation and mitochondrial function were investigated. RESULTS: Differentiation resulted in a decrease in the cellular respiration rate and the extracellular acidification rate (ECAR). PCL-NP exposure impaired mitochondrial function depending on the time of exposure. The cellular respiration rate was significantly reduced compared to differentiated controls when PCL-NPs were given before differentiation. The shift in ECAR was less pronounced in PCL-NP exposure during differentiation. Differentiation and PCL-NP exposure had no effect on expression levels and the enzymatic activity of respiratory chain complexes. The activity of the glycolytic enzyme phosphofructokinase was significantly reduced after differentiation with the effect being more pronounced after PCL-NP exposure before differentiation. The increase in mitochondrial membrane potential observed after differentiation was not found in SH-SY5Y cells exposed to PCL-NPs before differentiation. The cellular adenosine triphosphate (ATP) production significantly dropped during differentiation, and this effect was independent of the PCL-NP exposure. Differentiation and nanoparticle exposure had no effect on superoxide levels at the endpoint of the experiments. A slight decrease in the expression of the neuronal differentiation markers was found after PCL-NP exposure, but no morphological variation was observed. CONCLUSIONS: PCL-NP exposure affects mitochondrial function depending on the time of exposure before and during neuronal differentiation. PCL-NP exposure during differentiation was associated with impaired mitochondrial function, which may affect differentiation. Considering the importance of adaptations in cellular respiration for neuronal differentiation and function, further studies are needed to unravel the underlying mechanisms and consequences to assess the possible risks including neurodegeneration.

Evaluation of endocytosis of silica particles used in biodegradable implants in the brain.

Silica nanoparticles embedded in a biodegradable scaffold have been proposed to offer several advantages when used in laser-tissue-soldering of blood vessels in the brain. During degradation, these nanoparticles are likely to be released into the surrounding brain tissue. The aim of this study was to investigate possible cellular uptake mechanism(s) of the two silica nanoparticle types in microglial cells as well as their effect on autophagy and inflammatory cytokines. The nanoparticle uptake was analysed quantitatively using high-content analysis. Nanoparticle incubation did not modulate cytokine secretion and autophagy at any time point investigated. The nanoparticles were taken up by the microglia cells in a time- and particle-dependent manner. The maximal uptake was reached after 4hours and the nanoparticles were found in the endoplasmic reticulum and lysosomes. Macropinocytosis and phagocytosis were predominantly responsible for the uptake, whereas clathrin- and caveolin-independent endocytosis were involved to a minor extent.

Inhibition of in vitro metabolism of testosterone in human, dog and horse liver microsomes to investigate species differences.

Testosterone hydroxylation was investigated in human, canine and equine liver microsomes and in human and canine single CYPs. The contribution of the CYP families 1, 2 and 3 was studied using chemical inhibitors. Testosterone metabolites were analyzed by HPLC. The metabolites androstenedione, 6ß- and 11ß-hydroxytestosterone were found in microsomes of all species, but the pattern of metabolites varied within species. Androstenedione was more prominent in the animal species, and an increase over time was seen in equines. Testosterone hydroxylation was predominantly catalyzed by the CYP3A subfamily in all three species. While CYP2C9 did not metabolise testosterone, the canine ortholog CYP2C21 produced androstenedione. Quercetin significantly inhibited 6ß- and 11ß-hydroxytestosterone in all species investigated, suggesting that CYP2C8 is involved in testosterone metabolism, whereas sulfaphenazole significantly inhibited the formation of 6ß- and 11ß-hydroxytestosterone in human microsomes, at 60 min in equine microsomes, but not in canine microsomes. A contribution of CYP2B6 in testosterone metabolism was only found in human and equine microsomes. Inhibition of 17ß-hydroxysteroid dehydrogenase 2 indicated its involvement in androstenedione formation in humans, increased androstenedione formation was found in equines and no involvement in canines. These findings provide improved understanding of differences in testosterone biotransformation in animal species.

Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94 since the CYP activity was significantly enhanced with co-expressed POR.