Synergistic antibacterial effect of copper and silver nanoparticles and their mechanism of action.

Bacterial infections are one of the leading causes of death worldwide. In the case of topical bacterial infections such as wound infections, silver (Ag) has historically been one of the most widely used antibacterials. However, scientific publications have demonstrated the adverse effects of silver on human cells, ecotoxicity and insufficient antibacterial effect for the complete elimination of bacterial infections. The use of Ag in the form of nanoparticles (NPs, 1-100 nm) allows to control the release of antibacterial Ag ions but is still not sufficient to eliminate infection and avoid cytotoxicity. In this study, we tested the potency of differently functionalized copper oxide (CuO) NPs to enhance the antibacterial properties of Ag NPs. The antibacterial effect of the mixture of CuO NPs (CuO, CuO-NH2 and CuO-COOH NPs) with Ag NPs (uncoated and coated) was studied. CuO and Ag NP combinations were more efficient than Cu or Ag (NPs) alone against a wide range of bacteria, including antibiotic-resistant strains such as gram-negative Escherichia coli and Pseudomonas aeruginosa as well as gram-positive Staphylococcus aureus, Enterococcus faecalis and Streptococcus dysgalactiae. We showed that positively charged CuO NPs enhanced the antibacterial effect of Ag NPs up to 6 times. Notably, compared to the synergy of CuO and Ag NPs, the synergy of respective metal ions was low, suggesting that NP surface is required for the enhanced antibacterial effect. We also studied the mechanisms of synergy and showed that the production of Cu+ ions, faster dissolution of Ag+ from Ag NPs and lower binding of Ag+ by proteins of the incubation media in the presence of Cu2+ were the main mechanisms of the synergy. In summary, CuO and Ag NP combinations allowed increasing the antibacterial effect up to 6 times. Thus, using CuO and Ag NP combinations enables to retain excellent antibacterial effects due to Ag and synergy and enhances beneficial effects, since Cu is a vital microelement for human cells. Thus, we suggest using combinations of Ag and CuO NPs in antibacterial materials, such as wound care products, to increase the antibacterial effect of Ag, improve safety and prevent and cure topical bacterial infections.

Multi-generation exposure to polystyrene nanoplastics showed no major adverse effects in Daphnia magna.

Long-term impacts of plastics exposure to organisms, especially to the smallest plastics fraction, nanoplastics (NPs; ≤1 µm), are yet to be fully understood. The data concerning multiple generations are especially rare - an exposure scenario that is the most relevant from the standpoint of environmental reality aspect. Using Pd-doped 200 nm polystyrene NPs, which allowed for quantification of NPs in trace concentrations, the aim of the study was to evaluate the multigenerational impact of NPs for the freshwater crustacean Daphnia magna. Four consecutive 21-day exposures involving F0-F3 generations of D. magna were conducted according to OECD211. NPs impact (at 0.1 mg/L and 1 mg/L) was assessed in parallel to a comparative particle mesoporous SiO2 of similar size and shape (at 1 mg/L) to deconvolute impacts of variable particle chemistry. D. magna mortality, reproductive endpoints, body length (adults and offspring) and lipid content (offspring) were assessed upon NPs and SiO2 exposures. NPs association with adults and offspring was quantified by ICP-MS through the NPs Pd-dopant. The results showed no NPs impact on D. magna at 0.1 mg/L. At 1 mg NPs/L, the only statistically significant effect on adult organisms was increased fertility in the F3 generation. Conversely, SiO2 induced 80% mortality in F3 adult D. magna and the survived adults were significantly smaller and less fertile than those of other treatments. Both particles induced decreased size and lipid content in F3 offspring. The average NPs body burdens (ng NPs/mg D. magna dwt) for the adult and offspring D. magna were 105 ± 12 and 823 ± 440, respectively at 0.1 mg/L exposure and 503 ± 176 and 621 ± 235, respectively at 1 mg/L exposure. Finally, the results of this study add to the previous findings showing that multi-generation exposure to synthetic nano-sized particles of different chemistries may disturb the energy balance of D. magna.

Visible-Light Active Flexible and Durable Photocatalytic Antibacterial Ethylene-co-vinyl Acetate-Ag/AgCl/α-Fe2O3 Composite Coating.

When particles are mixed in polymer, particle surfaces become passivated by polymer matrix, leading to significantly reduced photocatalytic and, thus, also reduced antibacterial activity, as the catalytic particles become isolated from the outer environment and microorganisms reaching the surface. Herein, we demonstrate a facile and rapid approach for coating preparation at room temperature, yielding good adhesion of particles in combination with the particles' interface location. Flexible ethylene-co-vinyl acetate Ag/AgCl/α-Fe2O3 composite coatings were prepared by the spin-coating method. The synthesized photocatalytically active coating surface exhibited a distinct and rapid inhibition of bacterial growth, with at least a 7-log reduction of gram-positive bacteria Staphylococcus aureus viability after 30 min of visible-light illumination. We also analyzed the shedding of the Ag-ions and reactive oxygen species production from the composite coating and showed that reactive oxygen species played the main role in the photocatalytic bacterial inactivation, destroying the bacteria cell as proven by the Confocal Laser Scanning Microscopy.

Stable Isotope Tracing Uncovers Reduced γ/ß-ATP Turnover and Metabolic Flux Through Mitochondrial-Linked Phosphotransfer Circuits in Aggressive Breast Cancer Cells.

Changes in dynamics of ATP γ- and ß-phosphoryl turnover and metabolic flux through phosphotransfer pathways in cancer cells are still unknown. Using 18O phosphometabolite tagging technology, we have discovered phosphotransfer dynamics in three breast cancer cell lines: MCF7 (non-aggressive), MDA-MB-231 (aggressive), and MCF10A (control). Contrary to high intracellular ATP levels, the 18O labeling method revealed a decreased γ- and ß-ATP turnover in both breast cancer cells, compared to control. Lower ß-ATP[18O] turnover indicates decreased adenylate kinase (AK) flux. Aggressive cancer cells had also reduced fluxes through hexokinase (HK) G-6-P[18O], creatine kinase (CK) [CrP[18O], and mitochondrial G-3-P[18O] substrate shuttle. Decreased CK metabolic flux was linked to the downregulation of mitochondrial MTCK1A in breast cancer cells. Despite the decreased overall phosphoryl flux, overexpression of HK2, AK2, and AK6 isoforms within cell compartments could promote aggressive breast cancer growth.

Cubic Iron Core-Shell Nanoparticles Functionalized to Obtain High-Performance MRI Contrast Agents.

Nanoparticles with SiO2 coating were synthesized to have a cubic iron core. These were found to have saturation magnetization very close to the highest possible value of any iron-containing nanoparticles and the bulk iron saturation magnetization. The in vitro toxicology studies show that they are highly biocompatible and possess better MRI contrast agent potential than iron oxide NPs.

Long-Term Toxicity of Gadolinium to the Freshwater Crustacean Daphnia magna.

The lanthanides are considered emerging contaminants but information on their long-term toxicity to aquatic species under environmentally relevant conditions is scarce. We aimed to fill this gap by evaluating the long-term adverse effects of gadolinium on the freshwater model-crustacean Daphnia magna. The exposure of D. magna for up to 39 days to 0.1 mg Gd/L (a 21-days chronic toxicity NOEC value derived by us formerly) in the lake water had no negative effect (p > 0.05) on vitality, size and reproduction of parent animals as well as their offspring. Thus, assumingly the current Gd contamination levels of surface waters pose no hazard to aquatic crustaceans that in general are very sensitive to various pollutants. Moreover, presence of 0.1 mg Gd/L in the lake water even mitigated the long-term toxic effect of 0.2 mg Ni/L (studied as a model co-contaminant) to D. magna's vitality and productivity.

Long Term Exposure to Virgin and Recycled LDPE Microplastics Induced Minor Effects in the Freshwater and Terrestrial Crustaceans Daphnia magna and Porcellio scaber.

The effects of microplastics (MP) are extensively studied, yet hazard data from long-term exposure studies are scarce. Moreover, for sustainable circular use in the future, knowledge on the biological impact of recycled plastics is essential. The aim of this study was to provide long-term toxicity data of virgin vs recycled (mechanical recycling) low density polyethylene (LDPE) for two commonly used ecotoxicity models, the freshwater crustacean Daphnia magna and the terrestrial crustacean Porcellio scaber. LDPE MP was tested as fragments of 39.8 ± 8.82 µm (virgin) and 205 ± 144 µm (recycled) at chronic exposure levels of 1-100 mg LDPE/L (D. magna) and 0.2-15 g LDPE/kg soil (P. scaber). Mortality, reproduction, body length, total lipid content, feeding and immune response were evaluated. With the exception of very low inconsistent offspring mortality at 10 mg/L and 100 mg/L of recycled LDPE, no MP exposure-related adverse effects were recorded for D. magna. For P. scaber, increased feeding on non-contaminated leaves was observed for virgin LDPE at 5 g/kg and 15 g/kg. In addition, both LDPE induced a slight immune response at 5 g/kg and 15 g/kg with more parameters altered for virgin LDPE. Our results indicated different sublethal responses upon exposure to recycled compared to virgin LDPE MP.

Colon cancer cell differentiation by sodium butyrate modulates metabolic plasticity of Caco-2 cells via alteration of phosphotransfer network.

The ability of butyrate to promote differentiation of cancer cells has important implication for colorectal cancer (CRC) prevention and therapy. In this study, we examined the effect of sodium butyrate (NaBT) on the energy metabolism of colon adenocarcinoma Caco-2 cells coupled with their differentiation. NaBT increased the activity of alkaline phosphatase indicating differentiation of Caco-2 cells. Changes in the expression of pluripotency-associated markers OCT4, NANOG and SOX2 were characterized during the induced differentiation at mRNA level along with the measures that allowed distinguishing the expression of different transcript variants. The functional activity of mitochondria was studied by high-resolution respirometry. Glycolytic pathway and phosphotransfer network were analyzed using enzymatical assays. The treatment of Caco-2 cells with NaBT increased production of ATP by oxidative phosphorylation, enhanced mitochondrial spare respiratory capacity and caused rearrangement of the cellular phosphotransfer networks. The flexibility of phosphotransfer networks depended on the availability of glutamine, but not glucose in the cell growth medium. These changes were accompanied by suppressed cell proliferation and altered gene expression of the main pluripotency-associated transcription factors. This study supports the view that modulating cell metabolism through NaBT can be an effective strategy for treating CRC. Our data indicate a close relationship between the phosphotransfer performance and metabolic plasticity of CRC, which is associated with the cell differentiation state.

Surface carboxylation or PEGylation decreases CuO nanoparticles' cytotoxicity to human cells in vitro without compromising their antibacterial properties.

Clinical use of CuO nanoparticles (NPs) as antibacterials can be hampered by their toxicity to human cells. We hypothesized that certain surface functionalizations of CuO NPs may render NPs toxic to bacteria, but still be relatively harmless to human cells. To control this hypothesis, the toxicity of differently functionalized CuO NPs to bacteria Escherichia coli vs human cells (THP-1 macrophages and HACAT keratinocytes) was compared using similar conditions and end points. CuO NPs functionalized with polyethylene glycol (CuO-PEG), carboxyl (CuO-COOH, anionic), ammonium (CuO-NH4+, cationic) and unfunctionalized CuO NPs and CuSO4 (controls) were tested. In general, the toxicity of Cu compounds decreased in the following order: CuO-NH4+ > unfunctionalized CuO > CuSO4 > CuO-COOH > CuO-PEG. Positively charged unfunctionalized CuO and especially CuO-NH4+ proved most toxic (24-h EC50 = 21.7-47 mg/l) and had comparable toxicity to bacterial and mammalian cells. The multivariate analysis revealed that toxicity of these NPs was mostly attributed to their positive zeta potential, small hydrodynamic size, high Cu dissolution, and induction of reactive oxygen species (ROS) and TNF-α. In contrast, CuO-COOH and CuO-PEG NPs had lower toxicity to human cells compared to bacteria despite efficient uptake of these NPs by human cells. In addition, these NPs did not induce TNF-α and ROS. Thus, by varying the NP functionalization and Cu form (soluble salt vs NPs), it was possible to "target" the toxicity of Cu compounds, whereas carboxylation and PEGylation rendered CuO NPs that were more toxic to bacteria than to human cells envisaging their use in medical antibacterial products.

Hazard evaluation of polystyrene nanoplastic with nine bioassays did not show particle-specific acute toxicity.

Plastic is a wide-spread pollutant and must be evaluated for potential adverse effects of its breakdown product, microplastic (≤5 mm) along with its subfraction, nanoplastic (1-100 nm). Risk assessment of pollutants cannot be conducted without their toxicity (dose-response) data. In this study, toxicity of polystyrene nanoplastics (PS-NPL) was evaluated using 8 acute and 1 subchronic toxicity assays with 10 organisms of different biological complexity (bacteria, yeast, algae, protozoans, mammalian cells in vitro, crustaceans, midge larvae). Commercial 26 and 100 nm carboxylated PS-NPL spheres were chosen as model and tested in nominal concentrations up to 100 mg/L (1.025·1016 26 nm and 1.83·1014 100 nm particles/L). In most of the assays, both PS-NPL proved non-toxic (L(E)C50 > 100 mg/L) but three tests (V. fischeri, R. subcapitata, D. magna) flagged toxicity in 'as received' 26 nm PS-NPL and D. magna also in 100 nm PS-NPL (EC50 ranging from 13 to 71 mg/L). As, according to manufacturers, both PS-NPL suspensions contained additives (surfactants and biocidal NaN3), the three toxicity tests were repeated also on dialysed PS-NPL and on NaN3. Non-toxicity of dialysed PS-NPL indicated that the toxicity of 'as-received' PS-NPL was not particle-specific but false positive due to water-soluble additives in the PS-NPL preparations. NaN3 was very toxic to D. magna (48 h EC50 = 0.05 ± 0.03 mg NaN3/L), toxic to R. subcapitata (72 h EC50 = 4.97 ± 3.7 mg NaN3/L) and non-toxic to V. fischeri. Toxicity of 'as-received' PS-NPL was not fully explainable by NaN3 but also attributable to other additives in the suspensions. Toxicity research of microplastic using commercial model particles must always consider the potential influence of additives, e.g. test the toxicity of dialysed NPL for comparison. In our study, D. magna, R. subcapitata and V. fischeri were the most sensitive to PS-NPL water-soluble additives and flagged their presence in NPL preparations.

Toxicity of differently sized and charged silver nanoparticles to yeast Saccharomyces cerevisiae BY4741: a nano-biointeraction perspective.

In the current study, we evaluated the modulatory effects of size and surface coating/charge of AgNPs on their toxicity to a unicellular yeast Saccharomyces cerevisiae BY4741 - a fungal model. For that, the toxicity of a set of 10 and 80 nm citrate-coated (negatively charged) and branched polyethylenimine (bPEI) coated (positively charged) AgNPs was evaluated in parallel with AgNO3 as ionic control. Yeast cells were exposed to different concentrations of studied compounds in deionized water for 24 h at 30 °C and evaluated for the viability by the post-exposure colony-forming ability. Particle-cell interactions were assessed by SEM, TEM and confocal laser scanning microscopy (CLSM) in the reflection mode. AgNPs toxicity to yeast was size and charge-dependent: 24-h IC50 values ranged from 0.04 (10nAg-bPEI) up to 8.3 mg Ag/L (80nAg-Cit). 10 nm AgNPs were 5-27 times more toxic than 80 nm AgNPs and bPEI-AgNPs 8-44 times more toxic than citrate-AgNPs. SEM and TEM visualization showed that bPEI-AgNPs but not citrate-AgNPs adsorbed onto the yeast cell's surface. However, according to CLSM all the studied AgNPs, whatever the size and coating, ended up within the yeast cell. Toxicity of citrate-AgNPs was largely explained by the dissolved Ag ions but the bPEI-AgNPs showed mainly particle-driven effects leading to the cellular internalization and/or to more pronounced dissolution of AgNPs in the close vicinity of the cell wall. Therefore, the size, and especially the coating/charge of AgNPs can be efficiently used for the design of new more efficient antifungals.

Antimicrobial potency of differently coated 10 and 50 nm silver nanoparticles against clinically relevant bacteria Escherichia coli and Staphylococcus aureus.

Silver nanoparticles (nanoAg) are effective antimicrobials and promising alternatives to traditional antibiotics. This study aimed at evaluating potency of different nanoAg against healthcare infections associated bacteria: Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. A library of differently coated nanoAg of two different sizes (10 and 50 nm) were prepared using coating agents poly-L-Lysine (PLL), cetyltrimethyl-ammonium bromide (CTAB), citrate (CIT), polyvinyl-pyrrolidone (PVP), polysorbate 80 (Tween 80), and dioctyl-sodium sulfosuccinate (AOT). Stability evaluation by means of agglomeration and dissolution behaviour was performed for all nanoAg under conditions relevant for this study. Antibacterial properties of nanoAg were addressed by determining their minimal bactericidal concentrations (MBC) in deionised (DI) water to minimise the influence of silver speciation on its bioavailability. In parallel, AgNO3 was analysed as an ionic control. Studied nanoAg were efficient antimicrobials being remarkably more potent towards E. coli than to S. aureus (4 h MBC values for different nanoAg ranged from 0.08 to 5.0 mg Ag/L and 1.0-10 mg Ag/L, respectively). The toxicity of all nanoAg to S. aureus (but not to E. coli) increased with exposure time (4 h vs 24 h). 10 nm sized nanoAg released more Ag-ions and were more toxic than 50 nm nanoAg. Coating-dependent toxicity was more prominent for 50 nm nanoAg coated with Tween 80 or CTAB rendering the least toxic nanoAg. Obtained results showed that the antimicrobial effects of nanoAg were driven by shed Ag-ions, depended on target bacteria, exposure time and were the interplay of NP size, solubility and surface coating.

Rapid in situ assessment of Cu-ion mediated effects and antibacterial efficacy of copper surfaces.

Release of metal ions from metal-based surfaces has been considered one of the main drivers of their antimicrobial activity. Here we describe a method that enables parallel assessment of metal ion release from solid metallic surfaces and antimicrobial efficacy of these surfaces in a short time period. The protocol involves placement of a small volume of bioluminescent bacteria onto the tested surface and direct measurement of bioluminescence at various time points. In this study, two recombinant Escherichia coli strains, one expressing bioluminescence constitutively and applicable for general antimicrobial testing, and the other induced by Cu ions, were selected. Decrease in bioluminescence of constitutive E. coli on the surfaces showed a good correlation with the decrease in bacterial viability. Response of Cu-inducible E. coli showed a correlation with Cu content in the tested surfaces but not with Cu dissolution suggesting the role of direct bacteria-surface contact in Cu ion-driven antibacterial effects. In summary, the presented protocol enables the analysis of microbial toxicity and bioavailability of surface-released metal ions directly on solid surfaces within 30-60 min. Although optimized for copper and copper alloy surfaces and E. coli, the method can be extended to other types of metallic surfaces and bacterial strains.

UVA-induced antimicrobial activity of ZnO/Ag nanocomposite covered surfaces.

Application of efficient antimicrobial surfaces has been estimated to decrease both, the healthcare-associated infections and the spread of antibiotic-resistant bacteria. In this paper, we prepared ZnO and ZnO/Ag nanoparticle covered surfaces and evaluated their antimicrobial efficacy towards a Gram-negative bacterial model (Escherichia coli), a Gram-positive bacterial model (Staphylococcus aureus) and a fungal model (Candida albicans) in the dark and under UVA illumination. The surfaces were prepared by spin coating aliquots of ZnO and ZnO/Ag nanoparticle suspensions onto glass substrates. Surfaces contained 2 or 20 µg Zn/cm2 and 0-0.02 µg Ag/cm2. No significant antimicrobial activity of the surfaces, except of those with the highest Ag or Zn content was observed in the dark. On the other hand, UVA illuminated surfaces containing 20 µg Zn/cm2 and 2 µg Zn plus 0.02 µg Ag/cm2 caused >3 log decrease in the viable counts of E. coli and S. aureus in 30 min. As proven by brilliant blue FCF dye degradation and elemental analysis of the surfaces, this remarkable antimicrobial activity was a combined result of photocatalytic effect and release of Zn and Ag ions from surfaces. Surfaces retained significant antibacterial and photocatalytic properties after several usage cycles. Compared to bacteria, yeast C. albicans was significantly less sensitive to the prepared surfaces and only about 1 log reduction of viable count was observed after 60 min UVA illumination. In conclusion, the developed ZnO/Ag surfaces exhibit not only high antibacterial activity but also some antifungal activity.

Assessment of the hazard of nine (doped) lanthanides-based ceramic oxides to four aquatic species.

The risk of environmental pollution with rare earth oxides rises in line with increasing application of these compounds in different sectors. However, data on potential environmental hazard of lanthanides is scarce and concerns mostly Ce and Gd. In this work, the aquatic toxicity of eight doped lanthanide-based ceramic oxides (Ce0.9Gd0.1O2, LaFeO3, Gd0.97CoO3, LaCoO3, (La0.5Sr0.5)0.99MnO3, Ce0.8Pr0.2O2, (La0.6Sr0.4)0.95CoO3, LaNiO4) and one non-doped oxide (CeO2) with primary size from 23 to 590nm were evaluated in four short-term laboratory assays with freshwater crustaceans and duckweeds. Results showed no acute toxicity (EC50>100mg/L) or very low acute toxicity for most studied oxides. Observed toxicity was probably due to bioavailable fraction of dopant metals (Ni and Co) but in the case of aquatic plants, decrease of nutrient availability (complexing of phosphorus by lanthanides) was also presumed. Studied oxides/metals accumulated in the aquatic plant tissue and in the gut of crustaceans and thus may be further transferred via the aquatic food chain. Accumulation of metals in the duckweed Lemna minor may be recommended as a cost-effective screening bioassay for assessment of potential hazard of poorly soluble oxides to aquatic ecosystems.

Uptake and transcytosis of functionalized superparamagnetic iron oxide nanoparticles in an in vitro blood brain barrier model.

Two major hurdles in nanomedicine are the limited strategies for synthesizing stealth nanoparticles and the poor efficacy of the nanoparticles in translocating across the blood brain barrier (BBB). Here we examined the uptake and transcytosis of iron oxide nanoparticles (IONPs) grafted with biomimetic phosphorylcholine (PC) brushes in an in vitro BBB model system, and compared them with bare, PEG or PC-PEG mixture grafted IONPs. Hyperspectral imaging indicated IONP co-localization with cells. Quantitative analysis with total reflection X-ray fluorescence spectrometry showed that after 24 h, 78% of PC grafted, 68-69% of PEG or PC-PEG grafted, and 30% of bare IONPs were taken up by the BBB. Transcytosis of IONPs was time-dependent and after 24 h, 16-17% of PC or PC-PEG mixture grafted IONPs had passed the BBB model, significantly more than PEG grafted or bare IONPs. These findings point out that grafting of IONPs with PC is a viable strategy for improving the uptake and transcytosis of nanoparticles.

Evaluation of the effect of test medium on total Cu body burden of nano CuO-exposed Daphnia magna: A TXRF spectroscopy study.

Toxicity of Cu and Cu-based nanoparticles (NPs) to aquatic biota is usually mitigated in natural freshwater compared to organics-free artificial freshwater. The main aim of this study was to evaluate whether mitigated toxicity is accompanied by lower total copper body burden in the freshwater crustacean Daphnia magna and whether CuO NPs are more hazardous in this aspect than soluble Cu salts. Total copper body burden in different media (OECD202 artificial freshwater and two natural freshwaters) was measured by a relatively novel technique - total reflection X-ray fluorescence (TXRF) spectroscopy - which proved suitable for the analysis of individual juvenile daphnids. Mean copper body burden was 2.8-42 times higher in daphnids exposed to CuO NPs (0.05 mg Cu/L and 1 mg Cu/L) than in daphnids exposed to equal or equitoxic concentrations (0.025 mg Cu/L and 0.05 mg Cu/L) of CuSO4. Using natural freshwater instead of artificial one resulted in increased copper burden after exposure to CuO NPs but not after exposure to Cu salt. After 24 h post-exposure depuration in the presence of algae Raphidocelis subcapitata, total copper body burden in daphnids exposed to CuO NPs sharply decreased while in daphnids exposed to Cu salt it did not. Despite the CuO NP toxicity mitigating effect of natural freshwater, total copper body burden of aquatic crustaceans in natural waterbodies may be greater than could be predicted based on the results obtained using artificial freshwater as the test medium.

Toxicity of Nine (Doped) Rare Earth Metal Oxides and Respective Individual Metals to Aquatic Microorganisms Vibrio fischeri and Tetrahymena thermophila.

Despite the increasing use of rare earth elements (REEs) and oxides (REOs) in various technologies, the information on their ecotoxicological hazard is scarce. Here, the effects of La3+, Ce3+, Pr3+, Nd3+, Gd3+, CeO2, and eight doped REOs to marine bacteria Vibrio fischeri and freshwater protozoa Tetrahymena thermophila were studied in parallel with REO dopant metals (Co2+, Fe3+, Mn2+, Ni2+, Sr2+). The highest concentrations of REOs tested were 100 mg/L with protozoa in deionized water and 500 mg/L with bacteria in 2% NaCl. Although (i) most REOs produced reactive oxygen species; (ii) all studied soluble REEs were toxic to bacteria (half-effective concentration, EC50 3.5-21 mg metal/L; minimal bactericidal concentration, MBC 6.3-63 mg/L) and to protozoa (EC50 28-42 mg/L); and (iii) also some dopant metals (Ni2+, Fe3+) proved toxic (EC50 ≤ 3 mg/L), no toxicity of REOs to protozoa (EC50 > 100 mg/L) and bacteria (EC50 > 500 mg/L; MBC > 500 mg/L) was observed except for La2NiO4 (MBC 25 mg/L). According to kinetics of V. fischeri bioluminescence, the toxicity of REEs was triggered by disturbing cellular membrane integrity. Fortunately, as REEs and REOs are currently produced in moderate amounts and form in the environment insoluble salts and/or oxides, they apparently present no harm to aquatic bacteria and protozoa.

Potency of (doped) rare earth oxide particles and their constituent metals to inhibit algal growth and induce direct toxic effects.

Use of rare earth elements (REEs) has increased rapidly in recent decades due to technological advances. It has been accompanied by recurring rare earth element anomalies in water bodies. In this work we (i) studied the effects of eight novel doped and one non-doped rare earth oxide (REO) particles (aimed to be used in solid oxide fuel cells and gas separation membranes) on algae, (ii) quantified the individual adverse effects of the elements that constitute the (doped) REO particles and (iii) attempted to find a discernible pattern to relate REO particle physicochemical characteristics to algal growth inhibitory properties. Green algae Raphidocelis subcapitata (formerly Pseudokirchneriella subcapitata) were used as a test species in two different formats: a standard OECD201 algal growth inhibition assay and the algal viability assay (a 'spot test') that avoids nutrient removal effects. In the 24h 'spot' test that demonstrated direct toxicity, algae were not viable at REE concentrations above 1mgmetal/L. 72-hour algal growth inhibition EC50 values for four REE salts (Ce, Gd, La, Pr) were between 1.2 and 1.4mg/L, whereas the EC50 for REO particles ranged from 1 to 98mg/L. The growth inhibition of REEs was presumably the result of nutrient sequestration from the algal growth medium. The adverse effects of REO particles were at least in part due to the entrapment of algae within particle agglomerates. Adverse effects due to the dissolution of constituent elements from (doped) REO particles and the size or specific surface area of particles were excluded, except for La2NiO4. However, the structure of the particles and/or the varying effects of oxide composition might have played a role in the observed effects. As the production rates of these REO particles are negligible compared to other forms of REEs, there is presumably no acute risk for aquatic unicellular algae.

A Highly Active Endo-Levanase BT1760 of a Dominant Mammalian Gut Commensal Bacteroides thetaiotaomicron Cleaves Not Only Various Bacterial Levans, but Also Levan of Timothy Grass.

Bacteroides thetaiotaomicron, an abundant commensal of the human gut, degrades numerous complex carbohydrates. Recently, it was reported to grow on a ß-2,6-linked polyfructan levan produced by Zymomonas mobilis degrading the polymer into fructooligosaccharides (FOS) with a cell surface bound endo-levanase BT1760. The FOS are consumed by B. thetaiotaomicron, but also by other gut bacteria, including health-promoting bifidobacteria and lactobacilli. Here we characterize biochemical properties of BT1760, including the activity of BT1760 on six bacterial levans synthesized by the levansucrase Lsc3 of Pseudomonas syringae pv. tomato, its mutant Asp300Asn, levansucrases of Zymomonas mobilis, Erwinia herbicola, Halomonas smyrnensis as well as on levan isolated from timothy grass. For the first time a plant levan is shown as a perfect substrate for an endo-fructanase of a human gut bacterium. BT1760 degraded levans to FOS with degree of polymerization from 2 to 13. At optimal reaction conditions up to 1 g of FOS were produced per 1 mg of BT1760 protein. Low molecular weight (<60 kDa) levans, including timothy grass levan and levan synthesized from sucrose by the Lsc3Asp300Asn, were degraded most rapidly whilst levan produced by Lsc3 from raffinose least rapidly. BT1760 catalyzed finely at human body temperature (37°C) and in moderately acidic environment (pH 5-6) that is typical for the gut lumen. According to differential scanning fluorimetry, the Tm of the endo-levanase was 51.5°C. All tested levans were sufficiently stable in acidic conditions (pH 2.0) simulating the gastric environment. Therefore, levans of both bacterial and plant origin may serve as a prebiotic fiber for B. thetaiotaomicron and contribute to short-chain fatty acids synthesis by gut microbiota. In the genome of Bacteroides xylanisolvens of human origin a putative levan degradation locus was disclosed.