Nitric oxide sensor NsrR is the key direct regulator of magnetosome formation and nitrogen metabolism in Magnetospirillum.

Nitric oxide (NO) plays an essential role as signaling molecule in regulation of eukaryotic biomineralization, but its role in prokaryotic biomineralization is unknown. Magnetospirillum gryphiswaldense MSR-1, a model strain for studies of prokaryotic biomineralization, has the unique ability to form magnetosomes (magnetic organelles). We demonstrate here that magnetosome biomineralization in MSR-1 requires the presence of NsrRMg (an NO sensor) and a certain level of NO. MSR-1 synthesizes endogenous NO via nitrification-denitrification pathway to activate magnetosome formation. NsrRMg was identified as a global transcriptional regulator that acts as a direct activator of magnetosome gene cluster (MGC) and nitrification genes but as a repressor of denitrification genes. Specific levels of NO modulate DNA-binding ability of NsrRMg to various target promoters, leading to enhancing expression of MGC genes, derepressing denitrification genes, and repressing nitrification genes. These regulatory functions help maintain appropriate endogenous NO level. This study identifies for the first time the key transcriptional regulator of major MGC genes, clarifies the molecular mechanisms underlying NsrR-mediated NO signal transduction in magnetosome formation, and provides a basis for a proposed model of the role of NO in the evolutionary origin of prokaryotic biomineralization processes.

Biosynthesis of magnetosome-nanobody complex in Magnetospirillum gryphiswaldense MSR-1 and a magnetosome-nanobody-based enzyme-linked immunosorbent assay for the detection of tetrabromobisphenol A in water.

In this study, two mutant strains, TBC and TBC+, able to biosynthesize a novel functional magnetosome-nanobody (Nb), were derived from the magnetotactic bacteria Magnetospirillum gryphiswaldense MSR-1. The magnetosome-Nbs biosynthesized by TBC+ containing multi-copies of the Nb gene had a higher binding ability to an environmental pollutant, tetrabromobisphenol A (TBBPA), than those biosynthesized by TBC containing only one copy of the Nb gene. The magnetosome-Nbs from TBC+ can effectively bind to TBBPA in solutions with high capacity without being affected by a broad range of NaCl and methanol concentrations as well as pH. Therefore, a magnetosome-Nb-based enzyme-linked immunosorbent assay (ELISA) was developed and optimized for the detection of TBBPA, yielding a half-maximum signal inhibition concentration of 0.23 ng/mL and a limit of detection of 0.025 ng/mL. The assay was used to detect TBBPA in spiked river water samples, giving average recoveries between 90 and 120% and coefficients of variation of 2.5-6.3%. The magnetosome-Nb complex could be reused 4 times in ELISA without affecting the performance of the assay. Our results demonstrate the potential of magnetosome-Nbs produced by TBC+ as cost-effective and environment-friendly reagents for immunoassays to detect small molecules in environmental waters.

Oxidative Amination of Aldehydes with Amines into α-Amino Ketones.

Oxidative amination for the installation of nitrogen functional molecules from nitrogen nucleophiles has always been a very challenging topic in organic synthesis. Here we report a novel conversion of different aldehydes with secondary amines for the synthesis of diversified α-amino ketones. This method can be achieved through oxidative rearrangement of an in situ-generated enamine intermediate promoted by commercially available sodium percarbonate. Furthermore, this one-pot process is also suitable for the functional modification of complex molecules.

Development of a Genetically Encoded Magnetic Platform in Magnetospirillum gryphiswaldense MSR-1 for Downstream Processing of Protein Expression System.

Background: Protein downstream processing remains a challenge in protein production, especially in low yields of products, in spite of ensuring effective disruption of cell and separation of target proteins. It is complicated, expensive and time-consuming. Here, we report a novel nano-bio-purification system for producing recombinant proteins of interest with automatic purification from engineered bacteria. Results: This system employed a complete genetic engineering downstream processing platform for proteins at low expression levels, referred to as a genetically encoded magnetic platform (GEMP). GEMP consists of four elements as follows. (1) A truncated phage lambda lysis cassette (RRz/Rz1) is controllable for lysis of Magnetospirillum gryphiswaldense MSR-1 (host cell). (2) A surface-expressed nuclease (NucA) is to reduce viscosity of homogenate by hydrolyzing long chain nucleic acids. (3) A bacteriogenic magnetic nanoparticle, known as magnetosome, allows an easy separation system in a magnetic field. (4) An intein realizes abscission of products (nanobodies against tetrabromobisphenol A) from magnetosome. Conclusions: In this work, removal of most impurities greatly simplified the subsequent purification procedure. The system also facilitated the bioproduction of nanomaterials. The developed platform can substantially simplify industrial protein production and reduce its cost.

Aqueous C-H aminomethylation of phenols by iodine catalysis.

A transition-metal-free strategy regarding an iodine-sodium percarbonate catalysis to achieve the ortho-aminomethylation of phenols in aqueous media has been developed. This method can effectively broaden a wide range of phenols, tolerate sensitive functional groups, and achieve the late-stage functionalization of ten functional molecules that contain phenolic structures.

Bacterial Magnetosomes Release Iron Ions and Induce Regulation of Iron Homeostasis in Endothelial Cells.

Magnetosomes (MAGs) extracted from magnetotactic bacteria are well-defined membrane-enveloped single-domain magnetic nanoparticles. Due to their superior magnetic and structural properties, MAGs constitute potential materials that can be manipulated via genetic and chemical engineering for use in biomedical and biotechnological applications. However, the long-term effects exerted by MAGs on cells are of concern in the context of in vivo applications. Meanwhile, it remains relatively unclear which mechanisms are employed by cells to process and degrade MAGs. Hence, a better understanding of MAGs' degradation and fundamental signal modulations occurring throughout this process is essential. In the current study, we investigated the potential actions of MAGs on endothelial cells over a 10-day period. MAGs were retained in cells and found to gradually gather in the lysosome-like vesicles. Meanwhile, iron-ion release was observed. Proteomics further revealed a potential cellular mechanism underlying MAGs degradation, in which a group of proteins associated with vesicle biogenesis, and lysosomal enzymes, which participate in protein hydrolysis and lipid degradation, were rapidly upregulated. Moreover, the released iron triggered the regulation of the iron metabolic profiles. However, given that the levels of cell oxidative damage were relatively stable, the released iron ions were handled by iron metabolic profiles and incorporated into normal metabolic routes. These results provide insights into the cell response to MAGs degradation that may improve their in vivo applications.

Recent Advances in Alkenyl sp2 C-H and C-F Bond Functionalizations: Scope, Mechanism, and Applications.

Alkenes and their derivatives are featured widely in a variety of natural products, pharmaceuticals, and advanced materials. Significant efforts have been made toward the development of new and practical methods to access this important class of compounds by selectively activating the alkenyl C(sp2)-H bonds in recent years. In this comprehensive review, we describe the state-of-the-art strategies for the direct functionalization of alkenyl sp2 C-H and C-F bonds until June 2022. Moreover, metal-free, photoredox, and electrochemical strategies are also covered. For clarity, this review has been divided into two parts; the first part focuses on currently available alkenyl sp2 C-H functionalization methods using different alkene derivatives as the starting materials, and the second part describes the alkenyl sp2 C-F bond functionalization using easily accessible gem-difluoroalkenes as the starting material. This review includes the scope, limitations, mechanistic studies, stereoselective control (using directing groups as well as metal-migration strategies), and their applications to complex molecule synthesis where appropriate. Overall, this comprehensive review aims to document the considerable advancements, current status, and emerging work by critically summarizing the contributions of researchers working in this fascinating area and is expected to stimulate novel, innovative, and broadly applicable strategies for alkenyl sp2 C-H and C-F bond functionalizations in the coming years.

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.

Self-Confirming Magnetosomes for Tumor-Targeted T1 /T2 Dual-Mode MRI and MRI-Guided Photothermal Therapy.

Nanomaterials as T1 /T2 dual-mode magnetic resonance imaging (MRI) contrast agents have great potential in improving the accuracy of tumor diagnosis. Applications of such materials, however, are limited by the complicated chemical synthesis process and potential biosafety issues. In this study, the biosynthesis of manganese (Mn)-doped magnetosomes (MagMn) that not only can be used in T1 /T2 dual-mode MR imaging with self-confirmation for tumor detection, but also improve the photothermal conversion efficiency for MRI-guided photothermal therapy (PTT) is reported. The MagMn nanoparticles (NPs) are naturally produced through the biomineralization of magnetotactic bacteria by doping Mn into the ferromagnetic iron oxide crystals. In vitro and in vivo studies demonstrated that targeting peptides functionalized MagMn enhanced both T1 and T2 MRI signals in tumor tissue and significantly inhibited tumor growth by the further MRI-guided PTT. It is envisioned that the biosynthesized multifunctional MagMn nanoplatform may serve as a potential theranostic agent for cancer diagnosis and treatment.

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.

A comprehensive assessment of the biocompatibility of Magnetospirillum gryphiswaldense MSR-1 bacterial magnetosomes in vitro and in vivo.

Bacterial magnetosomes (BMs) are iron oxide nanoparticles synthesized naturally by magnetotactic bacteria, made up of nano-sized inorganic crystals enclosed by a lipid bilayer membrane. Due to several superior characteristics, such as the narrow size distribution, uniform morphology, high purity and crystallinity, single magnetic domain as well as easy surface modification, increasing biomedical and biotechnological applications of BMs have been developed. The attracted wide attentions raise the urge for the evaluation of safety and toxicity. In this work, we performed a rather comprehensive and systematic assessment of in vitro and in vivo toxicity of BMs from MSR-1, including the cytotoxicity, mice bodyweights, blood test, organ coefficients, inflammation, and hemocompatibility study. We found that BMs have good biocompatibility except for influences on the immune response as demonstrated by enhanced activation of the complement system and inhibition of lymphocyte proliferation when used with an excessive concentration. BMs induced the production of reactive oxygen species (ROS) in macrophages at a dose-dependent manner but did not cause cell membrane damage and cell cycle arrest until the concentration is approximately 40 times the clinical dosage. We anticipate our work will guide modifications of BMs and expand their future applications.

Water-Tolerant ortho-Acylation of Phenols.

A metal-free, water-tolerant, and one-pot process for ortho-acylation of phenols promoted by the iodine source/hydrogen peroxide system has been developed. This transformation undergoes ether formation, iodocyclization, C-C bond cleavage, and oxidative hydrolysis in a one-step manner, which is supported by control experiments.

Biocompatibility of Bacterial Magnetosomes as MRI Contrast Agent: A Long-Term In Vivo Follow-Up Study.

Derived from magnetotactic bacteria (MTB), magnetosomes consist of magnetite crystals enclosed within a lipid bilayer membrane and are known to possess advantages over artificially synthesized nanoparticles because of the narrow size distribution, uniform morphology, high purity and crystallinity, single magnetic domain, good biocompatibility, and easy surface modification. These unique properties have increasingly attracted researchers to apply bacterial magnetosomes (BMs) in the fields of biology and medicine as MRI imaging contrast agents. Due to the concern of biosafety, a long-term follow-up of the distribution and clearance of BMs after entering the body is necessary. In this study, we tracked changes of BMs in major organs of mice up to 135 days after intravenous injection using a combination of several techniques. We not only confirmed the liver as the well-known targeted organs of BMs, but also found that BMs accumulated in the spleen. Besides, two major elimination paths, as well as the approximate length of time for BMs to be cleared from the mice, were revealed. Together, the results not only confirm that BMs have high biocompatibility, but also provide a long-term in-vivo assessment which may further help to forward the clinical applications of BMs as an MRI contrast agent.

Fusion expression of nanobodies specific for the insecticide fipronil on magnetosomes in Magnetospirillum gryphiswaldense MSR-1.

BACKGROUND: Magnetic nanoparticles such as magnetosomes modified with antibodies allow a high probability of their interaction with targets of interest. Magnetosomes biomineralized by magnetotactic bacteria are in homogeneous nanoscale size and have crystallographic structure, and high thermal and colloidal stability. Camelidae derived nanobodies (Nbs) are small in size, thermal stable, highly water soluble, easy to produce, and fusible with magnetosomes. We aimed to functionalize Nb-magnetosomes for the analysis of the insecticide fipronil. RESULTS: Three recombinant magnetotactic bacteria (CF, CF+ , and CFFF) biomineralizing magnetosomes with different abundance of Nbs displayed on the surface were constructed. Compared to magnetosomes from the wild type Magnetospirillum gryphiswaldense MSR-1, all of the Nb-magnetosomes biosynthesized by strains CF, CF+ , and CFFF showed a detectable level of binding capability to fipronil-horseradish peroxidase (H2-HRP), but none of them recognized free fipronil. The Nb-magnetosomes from CFFF were oxidized with H2O2 or a glutathione mixture consisting of reduced glutathione and oxidized glutathione in vitro and their binding affinity to H2-HRP was decreased, whereas that to free fipronil was enhanced. The magnetosomes treated with the glutathione mixture were employed to develop an enzyme-linked immunosorbent assay for the detection of fipronil in water samples, with average recoveries in a range of 78-101%. CONCLUSIONS: The economical and environmental-friendly Nb-magnetosomes biomineralized by the bacterial strain MSR-1 can be potentially applied to nanobody-based immunoassays for the detection of fipronil or nanobody-based assays in general.

An efficient method for the synthesis of 2-pyridones via C-H bond functionalization.

A simple and practical method to access N-substituted 2-pyridones via a formal [3+3] annulation of enaminones with acrylates based on RhIII-catalyzed C-H functionalization was developed. Control and deuterated experiments led to a plausible mechanism involving C-H bond cross-coupling and aminolysis cyclization. This strategy provides a short synthesis of structural motifs of N-substituted 2-pyridones.

Decarboxylative C-H Alkylation of Heteroarene N-Oxides by Visible Light/Copper Catalysis.

This paper reports a highly site-selective alkylation of heteroarene N-oxides using hypervalent iodine(III) carboxylates to serve as an alkylating agent in the presence of a cheap copper catalyst under visible light conditions. This mild method proceeds at room temperature in an air atmosphere and can withstand various heteroarene N-oxides as well as various primary, secondary, and tertiary alkyl carboxylic acids. It also provides a practical method for enabling the rapid conversion of commercially available raw materials into medically relevant "drug-like" molecules.

Dichloroacetophenone Derivatives: A Class of Bioconjugation Reagents for Disulfide Bridging.

A mild and biocompatible method for the construction of disulfide bridging in peptides using dichloroacetophenone derivatives is developed. This method is highly selective (chemo, diastereo, regio, etc.) and atom economic and works under biocompatible reaction conditions (metal-free, water, pH 7, rt, etc.).

Drug-internalized bacterial swimmers for magnetically manipulable tumor-targeted drug delivery.

Tumor-targeted drug carriers are becoming attractive for precise drug delivery in anti-tumor therapy. However, a lot of the reported drug delivery systems are complicatedly designed and their destiny in vivo is beyond our control, which limited their clinical applications. Hence, it is urgently needed to develop spatio-manipulable self-propelled nanosystems for drug delivery in a facile way. Here, we have successfully constructed drug-internalized bacterial swimmers, whose movement can be manually controlled by an external magnetic field (MF). We demonstrate that the swimmers maintain the mobility to align and swim along MF lines. Further studies reveal that the doxorubicin (DOX-) internalized bacterial swimmers are able to navigate toward tumor sites under the guidance of MF, rendering enhanced anti-tumor efficacy compared with that of dead ones and free DOX. Therefore, the MF-guided bacterial swimmers hold great promise for spatio-manipulable drug delivery in precision medicine.