Priority screening of contaminants of emerging concern in drinking water sources of eastern China: Assessing risks based on exposure-activity ratios (EARs).

Increasing and widely detected contaminants of emerging concern (CECs) pose a threat to drinking water safety. Compared with traditional methods, the exposure-activity ratio (EAR) method based on the ToxCast database may have unique advantages in risk assessment of drinking water sources because it provides massive multi-target high-throughput screening toxicity effect data assessment for chemicals with missing traditional toxicity data. In this study, 112 CECs at 52 sampling sites in drinking water sources in Zhejiang Province of eastern China were investigated. Based on EARs and occurrence, priority chemicals were identified as difenoconazole (priority level 1), dimethomorph (priority level 2), acetochlor, caffeine, carbamazepine, carbendazim, paclobutrazol and pyrimethanil (priority level 3). Different from single observable biological effect in traditional methods, a variety of observable biological effects caused by high-risk targets were explored through adverse outcomes pathways (AOPs), revealing ecological risks as well as human health risks, for example, hepatocellular adenomas and carcinomas. Furthermore, the difference between the maximum EAR for a given chemical in a sample (EARmax) and the toxicity quotient (TQ) in priority screening of CECs was compared. The results show that screening priority CECs based on the EAR method is acceptable and more sensitive, suggesting the difference between in vitro and in vivo toxic effects and the necessity of incorporating the harm degree of biological effects into the EAR method to screen priority chemicals in the future.

In vitro magnetosome remineralization for silver-magnetite hybrid magnetosome biosynthesis and used for healing of the infected wound.

BACKGROUND: Magnetosomes (BMPs) are organelles of magnetotactic bacteria (MTB) that are responsible for mineralizing iron to form magnetite. In addition, BMP is an ideal biomaterial that is widely used in bio- and nano-technological applications, such as drug delivery, tumor detection and therapy, and immunodetection. The use of BMPs to create multifunctional nanocomposites would further expand the range of their applications. RESULTS: In this study, we firstly demonstrate that the extracted BMP can remineralize in vitro when it is exposed to AgNO3 solution, the silver ions (Ag+) were transported into the BMP biomembrane (MM) and mineralized into a silver crystal on one crystal plane of Fe3O4. Resulting in the rapid synthesis of an Ag-Fe3O4 hybrid BMP (BMP-Ag). The synergy between the biomembrane, Fe3O4 crystal, and unmineralized iron enabled the remineralization of BMPs at an Ag+ concentration ≥ 1.0 mg mL-1. The BMP-Ag displayed good biocompatibility and antibacterial activity. At a concentration of 2.0 mg/mL, the BMP-Ag and biomembrane removed Ag-Fe3O4 NPs inhibited the growth of gram-negative and gram-positive bacteria. Thus using BMP-Ag as a wound dressing can effectively enhance the contraction of infected wounds. CONCLUSIONS: This study represents the first successful attempt to remineralize organelles ex vivo, realizing the biosynthesis of hybrid BMP and providing an important advancement in the synthesis technology of multifunctional biological nanocomposites.

Biomanufacturing Biotinylated Magnetic Nanomaterial via Construction and Fermentation of Genetically Engineered Magnetotactic Bacteria.

Biosynthesis provides a critical way to deal with global sustainability issues and has recently drawn increased attention. However, modifying biosynthesized magnetic nanoparticles by extraction is challenging, limiting its applications. Magnetotactic bacteria (MTB) synthesize single-domain magnetite nanocrystals in their organelles, magnetosomes (BMPs), which are excellent biomaterials that can be biologically modified by genetic engineering. Therefore, this study successfully constructed in vivo biotinylated BMPs in the MTB Magnetospirillum gryphiswaldense by fusing biotin carboxyl carrier protein (BCCP) with membrane protein MamF of BMPs. The engineered strain (MSR-∆F-BF) grew well and synthesized small-sized (20 ± 4.5 nm) BMPs and were cultured in a 42 L fermenter; the yield (dry weight) of cells and BMPs reached 8.14 g/L and 134.44 mg/L, respectively, approximately three-fold more than previously reported engineered strains and BMPs. The genetically engineered BMPs (BMP-∆F-BF) were successfully linked with streptavidin or streptavidin-labelled horseradish peroxidase and displayed better storage stability compared with chemically constructed biotinylated BMPs. This study systematically demonstrated the biosynthesis of engineered magnetic nanoparticles, including its construction, characterization, and production and detection based on MTB. Our findings provide insights into biomanufacturing multiple functional magnetic nanomaterials.

Strategy for Avoiding Protein Corona Inhibition of Targeted Drug Delivery by Linking Recombinant Affibody Scaffold to Magnetosomes.

PURPOSE: Nanoparticles (NPs) decorated with functional ligands are promising candidates for cancer diagnosis and treatment. However, numerous studies have shown that chemically coupled targeting moieties on NPs lose their targeting capability in the biological milieu because they are shielded or covered by a "protein corona". Herein, we construct a functional magnetosome that recognizes and targets cancer cells even in the presence of protein corona. METHODS: Magnetosomes (BMPs) were extracted from magnetotactic bacteria, M. gryphiswaldense (MSR-1), and decorated with trastuzumab (TZ) via affibody (RA) and glutaraldehyde (GA). The engineered BMPs are referred to as BMP-RA-TZ and BMP-GA-TZ. Their capacities to combine HER2 were detected by ELISA, the quantity of plasma corona proteins was analyzed using LC-MS. The efficiencies of targeting SK-BR-3 were demonstrated by confocal laser scanning microscopy and flow cytometry. RESULTS: Both engineered BMPs contain up to ~0.2 mg TZ per mg of BMP, while the quantity of HER2 binding to BMP-RA-TZ is three times higher than that binding to BMP-GA-TZ. After incubation with normal human plasma or IgG-supplemented plasma, GA-TZ-containing BMPs have larger hydrated radii and more surface proteins in comparison with RA-TZ-containing BMPs. The TZ-containing BMPs all can be targeted to and internalized in the HER2-overexpressing breast cancer cell line SK-BR-3; however, their targeting efficiencies vary considerably: 50-75% for RA-TZ-containing BMPs and 9-19% for GA-TZ-containing BMPs. BMPs were incubated with plasma (100%) and cancer cells to simulate human in vivo environment. In this milieu, BMP-RA-TZ uptake efficiency of SK-BR-3 reaches nearly 80% (slightly lower than for direct interaction with BMP-RA-TZ), whereas the BMP-GA-TZ uptake efficiency is <17%. CONCLUSION: Application of the RA scaffold promotes and orients the arrangement of targeting ligands and reduces the shielding effect of corona proteins. This strategy improves the targeting capability and drug delivery of NP in a simulated in vivo milieu.

Construction of Immunomagnetic Particles with High Stability in Stringent Conditions by Site-Directed Immobilization of Multivalent Nanobodies onto Bacterial Magnetic Particles for the Environmental Detection of Tetrabromobisphenol-A.

Bacterial magnetic particles (BMPs) are an attractive carrier material for immunoassays because of their nanoscale size, dispersal ability, and membrane-bound structure. Antitetrabromobisphenol-A (TBBPA) nanobodies (Nbs) in the form of monovalence (Nb1), bivalence (Nb2), and trivalence (Nb3) were biotinylated and immobilized onto streptavidin (SA)-derivatized BMPs to construct the complexes of BMP-SA-Biotin-Nb1, -Nb2, and -Nb3, respectively. An increasing order of binding capability of BMP-SA-Biotin-Nb1, -Nb2, and -Nb3 to TBBPA was observed. These complexes showed high resilience to temperature (90 °C), methanol (100%), high pH (12), and strong ionic strength (1.37 M NaCl). A BMP-SA-Biotin-Nb3-based enzyme linked immunosorbent assay (ELISA) for TBBPA dissolved in methanol was developed, showing a half-maximum inhibition concentration (IC50) of 0.42 ng mL-1. TBBPA residues in landfill leachate, sewage, and sludge samples determined by this assay were in a range of

Engineered magnetosomes fused to functional molecule (protein A) provide a highly effective alternative to commercial immunomagnetic beads.

BACKGROUND: Magnetosomes (also called bacterial magnetic nanoparticles; BMPs) are biomembrane-coated nanoparticles synthesized by magnetotactic bacteria (MTB). Engineered BMPs fused to protein A (termed ∆F-BMP-FA) bind antibodies (Abs) automatically, and thus provide a series of potential advantages. However, no report so far has systematically evaluated functional applicability of genetically engineered BMPs. RESULTS: We evaluated properties of ∆F-BMP-FA, and developed/optimized culture methods for host strain Magnetospirillum gryphiswaldense ΔF-FA, ∆F-BMP-FA extraction conditions, conditions for Ab conjugation to ∆F-BMP-FA surface, and procedures for antigen detection using ∆F-BMP-FA/Ab complexes (termed BMP-A-Ab). Fed-batch culture for 36 h in a 42-L fermentor resulted in yields (dry weight) of 2.26 g/L for strain ΔF-FA and 62 mg/L for ∆F-BMP-FA. Optimal wash cycle number for ∆F-BMP-FA purification was seven, with magnetic separation following each ultrasonication step. Fusion of protein A to BMPs resulted in ordered arrangement of Abs on BMP surface. Linkage rate 962 µg Ab per mg ∆F-BMP-FA was achieved. BMP-A-Ab were tested for detection of pathogen (Vibrio parahaemolyticus; Vp) surface antigen and hapten (gentamicin sulfate). Maximal Vp capture rate for BMP-A-Ab was 90% (higher than rate for commercial immunomagnetic beads), and detection sensitivity was 5 CFU/mL. ∆F-BMP-FA also bound Abs from crude mouse ascites to form complex. Lowest gentamicin sulfate detection line for BMP-A-Ab was 0.01 ng/mL, 400-fold lower than that for double Ab sandwich ELISA, and gentamicin sulfate recovery rate for BMP-A-Ab was 93.2%. CONCLUSION: Our findings indicate that engineered BMPs such as ∆F-BMP-FA are inexpensive, eco-friendly alternatives to commercial immunomagnetic beads for detection or diagnostic immunoassays, and have high Ab-conjugation and antigen-adsorption capacity.

Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study.