Bacterial communities in ballast tanks of cargo vessels - Shaped by salinity, treatment and the point of origin of the water but "hatch" its typical microbiome.

Ballast water is a main vector of introduction of potentially harmful or pathogenic aquatic organisms. The development of genetic tools for ballast water monitoring has been underway and highlighted as a source for accurate and reliable data for decision making. We used 16S rRNA gene amplicon sequencing to analyze the microbial communities found in the ballast water of fifteen commercial ships routed through two Estonian ports. In parallel, samples from the port area were collected at the same time each ship visited. Fluorescence microscopy was utilized to assess the effectiveness of the treatment applied to ballast water. In addition, supplemental samples were collected from Hamburg Port (Germany) and a ballast tank decontamination system used at this port. The composition and diversity of bacterial communities varied greatly between obtained samples. The application of UV treatment did not demonstrate significant reduction in species richness estimates. The composition of microbial communities was significantly influenced by salinity, treatment (mainly untreated or UV treated) and the point of origin of the ballast water. Over a hundred potentially pathogenic bacterial taxa were found in relatively high abundance, including in ballast water that had received UV treatment. These shortcomings of stand-alone UV treatment of ballast water, especially when weak treatment is applied insufficiently, highlight the danger of possible harmful effects arising over time and the need for genetic tools for ballast water monitoring and management.

Amyloid ß25-35 induced ROS-burst through NADPH oxidase is sensitive to iron chelation in microglial Bv2 cells.

Iron chelation therapy and inhibition of glial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can both represent possible routes for Alzheimer's disease modifying therapies. The metal hypothesis is largely focused on direct binding of metals to the N-terminal hydrophilic 1-16 domain peptides of Amyloid beta (Aß) and how they jointly give rise to reactive oxygen species (ROS) production. The cytotoxic effects of Aß through ROS and metals are mainly studied in neuronal cells using full-length Aß1-40/42 peptides. Here we study cellularly-derived ROS during 2-60min in response to non-metal associated mid domain Aß25-35 in microglial Bv2 cells by fluorescence based spectroscopy. We analyze if Aß25-35 induce ROS production through NADPH oxidase and if the production is sensitive to iron chelation. NADPH oxidase inhibitor diphenyliodonium (DPI) is used to confirm the production of ROS through NADPH oxidase. We modulate cellular iron homeostasis by applying cell permeable iron chelators desferrioxamine (DFO) and deferiprone (DFP). NADPH oxidase subunit gp91-phox level was analyzed by Western blotting. Our results show that Aß25-35 induces strong ROS production through NADPH oxidase in Bv2 microglial cells. Intracellular iron depletion resulted in restrained Aß25-35 induced ROS.

A novel method for comparison of biocidal properties of nanomaterials to bacteria, yeasts and algae.

Toxicity testing of nanomaterials (NMs) is experimentally challenging because NMs may interfere with test environment and assay components. In this work we propose a simple and reliable method--a 'spot test' to compare biocidal potency of NMs to unicellular microorganisms such as bacteria, yeasts and algae. The assay is straightforward: cells are incubated in deionized water suspensions of NMs for up to 24h and then pipetted as a 'spot' on agarized medium. Altogether seven bacterial strains, yeast and a microalga were tested. CuO, TiO2 and two different Ag NPs, multi-wall C-nanotubes (MWCNTs), AgNO3, CuSO4, 3,5-dichlorophenol, triclosan and H2O2 were analyzed. The biocidal potency of tested substances ranged from 0.1mg/L to >1000 mg/L; whereas, the least potent NMs toward all test species were TiO2 NPs and MWCNTs and most potent Ag and CuO NPs. Based on the similar toxicity pattern of the tested chemicals on the nine unicellular organisms in deionized water we conclude that toxicity mechanism of biocidal chemicals seems to be similar, whatever the organism (bacteria, yeast, alga). Therefore, when the organisms are not 'protected' by their environment that usually includes various organic and inorganic supplements their tolerance to toxicants is chemical- rather than organism-dependent.

Environmental effects of soil contamination by shale fuel oils.

Estonia is currently one of the leading producers of shale oils in the world. Increased production, transportation and use of shale oils entail risks of environmental contamination. This paper studies the behaviour of two shale fuel oils (SFOs)--'VKG D' and 'VKG sweet'--in different soil matrices under natural climatic conditions. Dynamics of SFOs' hydrocarbons (C10-C40), 16 PAHs, and a number of soil heterotrophic bacteria in oil-spiked soils was investigated during the long-term (1 year) outdoor experiment. In parallel, toxicity of aqueous leachates of oil-spiked soils to aquatic organisms (crustaceans Daphnia magna and Thamnocephalus platyurus and marine bacteria Vibrio fischeri) and terrestrial plants (Sinapis alba and Hordeum vulgare) was evaluated. Our data showed that in temperate climate conditions, the degradation of SFOs in the oil-contaminated soils was very slow: after 1 year of treatment, the decrease of total hydrocarbons' content in the soil did not exceed 25 %. In spite of the comparable chemical composition of the two studied SFOs, the VKG sweet posed higher hazard to the environment than the heavier fraction (VKG D) due to its higher mobility in the soil as well as higher toxicity to aquatic and terrestrial species. Our study demonstrated that the correlation between chemical parameters (such as total hydrocarbons or total PAHs) widely used for the evaluation of the soil pollution levels and corresponding toxicity to aquatic and terrestrial organisms was weak.

Dissolution of silver nanowires and nanospheres dictates their toxicity to Escherichia coli.

Silver nanoparticles are extensively used in antibacterial applications. However, the mechanisms of their antibacterial action are not yet fully explored. We studied the solubility-driven toxicity of 100 × 6100 nm (mean primary diameter × length) silver nanowires (NWs) to recombinant bioluminescent Escherichia coli as a target representative of enteric pathogens. The bacteria were exposed to silver nanostructures in water to exclude the speciation-driven alterations. Spherical silver nanoparticles (83 nm mean primary size) were used as a control for the effect of NPs shape. Toxicity of both Ag NWs and spheres to E. coli was observed at similar nominal concentrations: the 4h EC50 values, calculated on the basis of inhibition of bacterial bioluminescence, were 0.42 ± 0.06 and 0.68 ± 0.01 mg Ag/L, respectively. Dissolution and bioavailability of Ag from NWs and nanospheres, analyzed with AAS or Ag-sensor bacteria, respectively, suggested that the toxic effects were caused by solubilized Ag(+) ions. Moreover, the antibacterial activities of Ag NWs suspension and its ultracentrifuged particle-free supernatant were equal. The latter indicated that the toxic effects of ~80-100 nm Ag nanostructures to Escherichia coli were solely dependent on their dissolution and no shape-induced/related effects were observed. Yet, additional nanospecific effects could come into play in case of smaller nanosilver particles.

Toxicity of CuO nanoparticles to yeast Saccharomyces cerevisiae BY4741 wild-type and its nine isogenic single-gene deletion mutants.

A suite of eight tentatively oxidative stress response-deficient Saccharomyces cerevisiae BY4741 single-gene mutants (sod1Δ, sod2Δ, yap1Δ, cta1Δ, ctt1Δ, gsh1Δ, glr1Δ, and ccs1Δ) and one copper-vulnerable mutant (cup2Δ) was used to elucidate weather the toxicity of CuO nanoparticles to S. cerevisiae is mediated by oxidative stress (OS). Specifically, sensitivity profiles of mutants' phenotypes and wild-type (wt) upon exposure to nano-CuO were compared. As controls, CuSO4 (solubility), bulk-CuO (size), H2O2, and menadione (OS) were used. Growth inhibition of wt and mutant strains was studied in rich YPD medium and cell viability in deionized water (DI). Dissolved Cu-ions were quantified by recombinant metal-sensing bacteria and chemical analysis. To wt strain nano-CuO was 32-fold more toxic than bulk-CuO: 24-h IC50 4.8 and 155 mg/L in DI and 643 and >20000 mg/L in YPD, respectively. In toxicant-free YPD medium, all mutants had practically similar growth patterns as wt. However, the mutant strains sod1Δ, sod2Δ, ccs1Δ, and yap1Δ showed up to 12-fold elevated sensitivity toward OS standard chemicals menadione and H2O2 but not to nano-CuO, indicating that CuO nanoparticles exerted toxicity to yeast cells via different mechanisms. The most vulnerable strain to all studied Cu compounds was the copper stress response-deficient strain cup2Δ (∼16-fold difference with wt), indicating that the toxic effect of CuO (nano)particles proceeds via dissolved Cu-ions. The dissolved copper solely explained the toxicity of nano-CuO in DI but not in YPD. Assumingly, in YPD nano-CuO acquired a coating of peptides/proteins and sorbed onto the yeast's outer surface, resulting in their increased solubility in the close vicinity of yeast cells and increased uptake of Cu-ions that was not registered by the assays used for the analysis of dissolved Cu-ions in the test medium. Lastly, as yeast retained its viability in DI even by 24th hour of incubation, the profiling of the acute basal toxicity of chemicals toward yeasts may be conducted in DI.

Interactions of PLA2-s from Vipera lebetina, Vipera berus berus and Naja naja oxiana venom with platelets, bacterial and cancer cells.

Secretory phospholipasesA(2) (sPLA(2)s) form a large family of structurally related enzymes widespread in nature. Herein, we studied the inhibitory effects of sPLA(2)s from Vipera lebetina (VLPLA(2)), Vipera berus berus (VBBPLA(2)), and Naja naja oxiana (NNOPLA(2)) venoms on (i) human platelets, (ii) four different bacterial strains (gram-negative Escherichia coli and Vibrio fischeri; gram-positive Staphylococcus aureus and Bacillus subtilis) and (iii) five types of cancer cells (PC-3, LNCaP, MCF-7, K-562 and B16-F10) in vitro. sPLA(2)s inhibited collagen-induced platelet aggregation: VBBPLA(2) IC(50) = 0.054, VLPLA(2) IC(50) = 0.072, NNOPLA(2) IC(50) = 0.814 µM. p-Bromophenacylbromide-inhibited sPLA(2) had no inhibitory action on platelets. 36.17 µM VBBPLA(2 )completely inhibited the growth of gram-positive Bacillus subtilis whereas no growth inhibition was observed towards gram-negative Escherichia coli. The inhibitory action of sPLA(2)s (~0.7 µM and ~7 µM) towards cancer cells depended on both venom and cell type. VBBPLA(2 )(7.2 µM) inhibited significantly the viability of K-562 cells and the cell death appeared apoptotic. The sPLA(2)s exhibited no inhibitory effect towards LNCaP cells and some effect (8%-20%) towards other cells. Thus, already sub-µM concentrations of sPLA(2)s inhibited collagen-induced platelet aggregation and from the current suite of studied svPLA(2)s and test cells, VBBPLA(2) was the most growth inhibitory towards Bacillus subtilis and K-562 cells.

Pro-survival effects of JNK and p38 MAPK pathways in LPS-induced activation of BV-2 cells.

Identifying MAPK pathways and understanding their role in microglial cells may be crucial for understanding the pathogenesis of neurodegenerative diseases since activated microglia could contribute to the progressive nature of neurodegeneration. In this study we show that the JNK pathway plays an important role in the survival of resting microglia BV-2 cells, as evidenced by Annexin-V positive staining and caspase-3 activation in cells treated with the specific JNK inhibitor SP600125. During LPS-induced activation of BV-2 cells inhibition of the p38 and JNK pathways with SB203580 and SP600125, respectively, results in apoptosis as detected by apoptotic markers. In the presence SP600125 the phosphorylation of p38 was significantly increased both in control and LPS-activated BV-2 cells. This suggests that the pro-survival role of JNK is possible due to its abrogation of a potentially apoptotic signal mediated by p38 MAPK pathway. Furthermore, inhibition of the p38 MAPK pathway during LPS-induced activation of BV-2 cells resulted in an increased phosphorylation of c-Jun, suggesting that the pro-survival effect of p38 MAPK during inflammatory conditions involves the JNK pathway. In conclusion, the results of this study demonstrate that both the JNK and p38 MAPK pathways possess anti-apoptotic functions in the microglial cell line BV-2 during LPS-induced activation.