Fully understanding the cellular uptake and intracellular localization of MoS2 nanosheets (NSMoS2) is a prerequisite for their safe applications. Here, we characterized the uptake profile of NSMoS2... Show moreFully understanding the cellular uptake and intracellular localization of MoS2 nanosheets (NSMoS2) is a prerequisite for their safe applications. Here, we characterized the uptake profile of NSMoS2 by functional coelomocytes of the earthworm Eisenia fetida. Considering that vacancy engineering is widely applied to enhance the NSMoS2 performance, we assessed the potential role of such atomic vacancies in regulating cellular uptake processes. Coelomocyte internalization and lysosomal accumulation of NSMoS2 were tracked by fluorescent labeling imaging. Cellular uptake inhibitors, proteomics, and transcriptomics helped to mechanistically distinguish vacancy-mediated endocytosis pathways. Specifically, Mo ions activated transmembrane transporter and ion-binding pathways, entering the coelomocyte through assisted diffusion. Unlike molybdate, pristine NSMoS2 (P-NSMoS2) induced protein polymerization and upregulated gene expression related to actin filament binding, which phenotypically initiated actin-mediated endocytosis. Conversely, vacancy-rich NSMoS2 (V-NSMoS2) were internalized by coelomocytes through a vesicle-mediated and energy-dependent pathway. Mechanistically, atomic vacancies inhibited mitochondrial transport gene expression and likely induced membrane stress, significantly enhancing endocytosis (20.3%, p < 0.001). Molecular dynamics modeling revealed structural and conformational damage of cytoskeletal protein caused by P-NSMoS2, as well as the rapid response of transport protein to V-NSMoS2. These findings demonstrate that earthworm functional coelomocytes can accumulate NSMoS2 and directly mediate cytotoxicity and that atomic vacancies can alter the endocytic pathway and enhance cellular uptake by reprogramming protein response and gene expression patterns. This study provides an important mechanistic understanding of the ecological risks of NSMoS2. Show less
Carnobacterium divergens is frequently isolated from natural environments and is a predominant species found in refrigerated foods, particularly meat, seafood, and dairy. While there is substantial... Show moreCarnobacterium divergens is frequently isolated from natural environments and is a predominant species found in refrigerated foods, particularly meat, seafood, and dairy. While there is substantial interest in using C. divergens as biopreservatives and/or probiotics, some strains are known to be fish pathogens, and the uncontrolled growth of C. divergens has been associated with food spoilage. Bacteriophages offer a selective approach to identify and control the growth of bacteria; however, to date, few phages targeting C. divergens have been reported. In this study, we characterize bacteriophage cd2, which we recently isolated from minced beef. A detailed host range study reveals that phage cd2 infects certain phylogenetic groups of C. divergens. This phage has a latent period of 60 min and a burst size of ~28 PFU/infected cell. The phage was found to be acid and heat sensitive, with a complete loss of phage activity when stored at pH 2 or heated to 60°C. Electron microscopy shows that phage cd2 is a siphophage, and while it shares the B3 morphotype with a unique cluster of Listeria and Enterococcus phages, a comparison of genomes reveals that phage cd2 comprises a new genus of phage, which we have termed as Carnodivirus. Show less
As antimicrobials, graphene materials (GMs) may have advantages over traditional antibiotics due to their physical mechanisms of action which ensure less chance of development of microbial... Show moreAs antimicrobials, graphene materials (GMs) may have advantages over traditional antibiotics due to their physical mechanisms of action which ensure less chance of development of microbial resistance. However, the fundamental question as to whether the antibacterial mechanism of GMs originates from parallel interaction or perpendicular interaction, or from a combination of these, remains poorly understood. Here, we show both experimentally and theoretically that GMs with high surface oxygen content (SOC) predominantly attach in parallel to the bacterial cell surface when in the suspension phase. The interaction mode shifts to perpendicular interaction when the SOC reaches a threshold of ∼0.3 (the atomic percent of O in the total atoms). Such distinct interaction modes are highly related to the rigidity of GMs. Graphene oxide (GO) with high SOC is very flexible and thus can wrap bacteria while reduced GO (rGO) with lower SOC has higher rigidity and tends to contact bacteria with their edges. Neither mode necessarily kills bacteria. Rather, bactericidal activity depends on the interaction of GMs with surrounding biomolecules. These findings suggest that variation of SOC of GMs is a key factor driving the interaction mode with bacteria, thus helping to understand the different possible physical mechanisms leading to their antibacterial activity. Show less
Sulfur cycling is known to control the speciation and bioavailability of copper in the environment via inorganic sulfur oxidation and reduction. However, it remains unclear how the mineralization... Show moreSulfur cycling is known to control the speciation and bioavailability of copper in the environment via inorganic sulfur oxidation and reduction. However, it remains unclear how the mineralization of organic sulfur and associated microbial processes affect Cu transformations. This study discovered a neglected mechanism that mediates Cu mobility and speciation via cysteine mineralization in a paddy soil. We provide evidence for a pathway of sulfide production from cysteine via indigenous soil microorganisms. The produced sulfide promotes the formation of copper sulfide nanoparticles, constituting an alternative copper sulfide formation mechanism that bypasses sulfate reduction. A bacterium isolated from the soil, named Bacillus sp. TR1, played a role in forming cell-associated copper sulfide nanoparticles. A metagenomics approach was applied to detect genes related to cysteine mineralization (dcyD, CTH, CBS, and sseA) and the associated microbes in the soil. The sseA gene was most abundant, and the microorganisms involved in cysteine mineralization were taxonomically diverse, including members of phyla Proteobacteria, Firmicutes, and Thaumarchaeota. Geobacter, Sulfuriferula, Nitrososphaera, Noviherbaspirillum, and Clostridium were the dominant genera with potential to metabolize cysteine to form copper sulfide nanoparticles. Our study not only provides initial molecular-level insights into the abundance, diversity, and metabolism of cysteine-mineralizing microorganisms but also highlights their important ecological functions in metal and sulfur biogeochemical cycles. Show less
Understanding the defensive strategies of hyperaccumulators in plant–herbivore interactions is essential for their safe applications in phytoremediation. Sedum alfredii plants are widely applied in... Show moreUnderstanding the defensive strategies of hyperaccumulators in plant–herbivore interactions is essential for their safe applications in phytoremediation. Sedum alfredii plants are widely applied in metal-contaminated soil where they were found to be easily damaged by herbivores. Thus, we investigated a comparative analysis of the defensive strategies from the perspective of constitutive leafy volatiles between the hyperaccumulator S. alfredii plant and its non-hyperaccumulator counterpart, along with feeding preference tests. Generalist snails prefer hyperaccumulators with lower content of metals while detested non-hyperaccumulators with relatively high food qualities in saccharides. Further leafy volatile profiles of the two ecotypes showed a total of 34 differentially enriched metabolites with 16 down-regulated organic compounds in the hyperaccumulators. KEGG pathway enrichment analysis showed two down-regulated metabolic pathways in phenylalanine metabolism and metabolic pathways due to the lower production of naphthalene and benzeneacetaldehyde, which are known herbivore deterrents and predator attractants. Hence, the high reliance on metals with the reduced performance of organic defense implied a trade-off between the two defensive strategies in the hyperaccumulator S. alfredii plants. Overall, this increased susceptibility to herbivores due to the lack of metals advocates the concerns and solutions for the safe application of this species in the phytoremediation practice. Show less
The aquatic system is a major sink for engineered nanomaterials released into the environment. Here, we assessed the toxicity of graphene oxide (GO) using the freshwater planarian Dugesia japonica,... Show moreThe aquatic system is a major sink for engineered nanomaterials released into the environment. Here, we assessed the toxicity of graphene oxide (GO) using the freshwater planarian Dugesia japonica, an invertebrate model that has been widely used for studying the effects of toxins on tissue regeneration and neuronal development. GO not only impaired the growth of normal (homeostatic) worms, but also inhibited the regeneration processes of regenerating (amputated) worms, with LC10 values of 9.86 mg/L and 9.32 mg/L for the 48-h acute toxicity test, respectively. High concentration (200 mg/L) of GO killed all the worms after 3 (regenerating) or 4 (homeostasis) days of exposure. Whole-mount in situ hybridization (WISH) and immunofluorescence analyses suggest GO impaired stem cell proliferation and differentiation, and subsequently caused cell apoptosis and oxidative DNA damage during planarian regeneration. Mechanistic analysis suggests that GO disturbed the antioxidative system (enzymatic and non-enzymatic) and energy metabolism in the planarian at both molecular and genetic levels, thus causing reactive oxygen species (ROS) over accumulation and oxidative damage, including oxidative DNA damage, loss of mitochondrial membrane integrity, lack of energy supply for cell differentiation and proliferation leading to retardance of neuron regeneration. The intrinsic oxidative potential of GO contributes to the GO-induced toxicity in planarians. These data suggest that GO in aquatic systems can cause oxidative stress and neurotoxicity in planarians. Overall, regenerated tissues are more sensitive to GO toxicity than homeostatic ones, suggesting that careful handling and appropriate decisions are needed in the application of GO to achieve healing and tissue regeneration. Show less
Once discharged into the environment, plastics debris are unavoidably subjected to natural weathering processes. Unfortunately, the impact of weathering on the aggregation tendency and kinetics of... Show moreOnce discharged into the environment, plastics debris are unavoidably subjected to natural weathering processes. Unfortunately, the impact of weathering on the aggregation tendency and kinetics of nanoplastics in complex environmental matrices is poorly understood. Here, we investigated the influence of weathering as induced by UV and O3 treatments, on the aggregation of polystyrene nanoparticles (PSNPs) in simulated waters containing representative organic molecules (humic acid, lysozyme, and alginate) and in natural waters. Results showed that UV/O3 weathering-induced physicochemical transformations of PSNPs, particularly the formation of oxygen-containing functional groups and the increase in hydrophilicity, altered the aggregation state of PSNPs to different extents. The presence of organic molecules destabilized the UV-aged PSNPs with strength of lysozyme > alginate > humic acid, owing to the decrease of sorption of macromolecules on their surface. Differently, the O3-aged PSNPs displayed strong stability in the absence or presence of organic molecules (except for lysozyme), probably due to steric repulsion arising from the leakage of endogenous organic matters. This work demonstrates that the aggregation behavior of PSNPs is determined by the complex interplays among weathering, natural organic matter, and solution chemistry, and provides significant insights into the fate and transport of PSNPs in realistic scenarios. Show less
To assess the safety of engineered nanomaterials (ENMs) and to evaluate and improve ENMs’ targeting ability for medical application, it is necessary to analyze the fate of these materials in... Show moreTo assess the safety of engineered nanomaterials (ENMs) and to evaluate and improve ENMs’ targeting ability for medical application, it is necessary to analyze the fate of these materials in biological media. This protocol presents a workflow that allows researchers to determine, characterize and quantify metal-bearing ENMs (M-ENMs) in biological tissues and cells and quantify their dynamic behavior at trace-level concentrations. Sample preparation methods to enable analysis of M-ENMs in a single cell, a cell layer, tissue, organ and physiological media (e.g., blood, gut content, hemolymph) of different (micro)organisms, e.g., bacteria, animals and plants are presented. The samples are then evaluated using fit-for-purpose analytical techniques e.g., single-cell inductively coupled plasma mass spectrometry, single-particle inductively coupled plasma mass spectrometry and synchrotron X-ray absorption fine structure, providing a protocol that allows comprehensive characterization and quantification of M-ENMs in biological matrices. Unlike previous methods, the protocol uses no fluorescent dyes or radiolabels to trace M-ENMs in biota and enables analysis of most M-ENMs at cellular, tissue and organism levels. The protocols can be applied by a wide variety of users depending on the intended purpose of the application, e.g., to correlate toxicity with a specific particle form, or to understand the absorption, distribution and excretion of M-ENMs. The results facilitate an understanding of the biological fate of M-ENMs and their dynamic behavior in biota. Performing the protocol may take 7–30 d, depending on which combination of methods is applied. Show less
The current debate on hazards associated with sub-micron sized plastics is hampered by a lack of quantitative data on the uptake and biological fate of plastics in organisms. Analytical methods... Show moreThe current debate on hazards associated with sub-micron sized plastics is hampered by a lack of quantitative data on the uptake and biological fate of plastics in organisms. Analytical methods should be developed to identify, characterize, and quantify sub-micron particulate plastic in biota to understand their biological fate in terms of biodistribution, localization, bioaccumulation and clearance. Here we give a perspective on a promising workflow of sample preparation methods and techniques that could enable analysis of sub-micron plastics in biological matrices and discuss their application for biological fate studies of particulate plastic in organisms. We also expect these methods to be largely transferrable to studies considering sub-micron plastics in food, consumer products, human and some environmental compartments. (c) 2021 Elsevier Ltd. All rights reserved. Show less
Toxicity of ZnO nanoparticles (NPs) are often related to the release of Zn2+ ions due to their dissolution. Studies also suggest that the toxicity of ZnO NPs cannot be solely explained by the... Show moreToxicity of ZnO nanoparticles (NPs) are often related to the release of Zn2+ ions due to their dissolution. Studies also suggest that the toxicity of ZnO NPs cannot be solely explained by the release of Zn2+ ions; however, there is a lack of direct evidence of ZnO particulate effects. This study compared the acute toxicity of ZnO NPs and ZnSO4 following intranasal exposure using a combination of metallomics and metabolomics approaches. Significant accumulation of Zn in the liver was only found in the ZnO NP treatment, with 29% of the newly accumulated Zn in the form of ZnO as revealed by X-ray fine structure spectroscopy (XAFS). This is the first direct evidence suggesting the persistence of ZnO NPs in liver upon intranasal exposure. Although both ZnO NPs and ZnSO4 altered the metabolite profiles, with some overlaps and considerable specificity, of both liver and plasma samples, more and distinct metabolites in the liver and opposite effects in the plasma were altered by ZnO NPs compared with ZnSO4, consistent with no accumulation of Zn detected in liver from ZnSO4. Specifically, a large number of antioxidant-related compounds and energetic substrates were exclusively elevated in the liver of ZnO NP-treated animals. These findings provided direct evidence that persistence of ZnO NPs induced particle-specific effects on the antioxidant systems and energy metabolism pathways. Show less
