New Insights into the Dependence of CPEB3 Ribozyme Cleavage on Mn2+ and Mg2.

CPEB3 ribozyme is a self-cleaving RNA that occurs naturally in mammals and requires divalent metal ions for efficient activity. Ribozymes exhibit preferences for specific metal ions, but the exact differences in the catalytic mechanisms of various metal ions on the CPEB3 ribozyme remain unclear. Our findings reveal that Mn2+ functions as a more effective cofactor for CPEB3 ribozyme catalysis compared to Mg2+, as confirmed by its stronger binding affinity to CPEB3 by EPR. Cleavage assays of CPEB3 mutants and molecular docking analyses further showed that excessive Mn2+ ions can bind to a second binding site near the catalytic site, hindering CPEB3 catalytic efficiency and contributing to the Mn2+ bell-shaped curve. These results implicate a pivotal role for the local nucleobase-Mn2+ interactions in facilitating RNA folding and modulating the directed attack of nucleophilic reagents. Our study provides new insights and experimental evidence for exploring the divalent cation dependent cleavage mechanism of the CPEB3 ribozyme.

Heat sensitive E-helix cut ferritin nanocages for facile and high-efficiency loading of doxorubicin.

Ferritin possesses a stable and uniform cage structure, along with tumor-targeting properties and excellent biocompatibility, making it a promising drug delivery vehicle. However, the current ferritin drug loading strategy involves complex steps and harsh reaction conditions, resulting in low yield and recovery of drug loading, which limits the clinical application prospects of ferritin nanomedicine. In this study, we utilized the high-efficiency heat-sensitivity of the multiple channel switch structures of the E-helix-cut ferritin mutant (Ecut-HFn) and Cu2+ assistance to achieve high-efficiency loading of chemotherapeutic drugs in a one-step process at low temperatures. This method features mild reaction conditions (45 °C), high loading efficiency (about 110 doxorubicin (Dox) per Ecut-HFn), and improved protein and Dox recovery rates (with protein recovery rate around 94 % and Dox recovery rate reaching up to 45 %). The prepared ferritin-Dox particles (Ecut-HFn-Cu-Dox) exhibit a uniform size distribution, good stability, and retain the natural tumor targeting ability of ferritin. Overall, this temperature-controlled drug loading strategy utilizing heat-sensitivity ferritin mutants is energy-saving, environmentally friendly, efficient, and easy to operate, offering a new perspective for scaling up the industrial production of ferritin drug carriers.

Boosting SARS-CoV-2 Enrichment with Ultrasmall Immunomagnetic Beads Featuring Superior Magnetic Moment.

The isolation and enrichment efficiency of SARS-CoV-2 virus in complex biological environments is often relatively low, presenting challenges in direct detection and an increased risk of false negatives, particularly during the early stages of infection. To address this issue, we have developed a novel approach using ultrasmall magnetosome-like nanoparticles (≤10 nm) synthesized via biomimetic mineralization of the Mms6 protein derived from magnetotactic bacteria. These nanoparticles are surface-functionalized with hydrophilic carboxylated polyethylene glycol (mPEG2000-COOH) to enhance water solubility and monodispersity. Subsequently, they are coupled with antibodies targeting the receptor-binding domain (RBD) of the virus. The resulting magnetosome-like immunomagnetic beads (Mal-IMBs) exhibit high magnetic responsiveness comparable to commercial magnetic beads, with a saturation magnetization of 90.6 emu/g. Moreover, their smaller particle size provides a significant advantage by offering a higher specific surface area, allowing for a greater number of RBD single-chain fragment variable (RBD-scFv) antibodies to be coupled, thereby enhancing immune capture ability and efficiency. To validate the practicality of Mal-IMBs, we evaluated their performance in recognizing the RBD antigens, achieving a maximum capture ability of 83 µg/mg per unit mass. Furthermore, we demonstrated the binding capability of Mal-IMBs to SARS-CoV-2 pseudovirus using fluorescence microscopy. The Mal-IMBs effectively enriched the pseudovirus at a low copy concentration of 70 copies/mL. Overall, the small Mal-IMB exhibited excellent magnetic responsiveness and binding efficiency. By employing a multisite virus binding mechanism, it significantly improves the enrichment and separation of SARS-CoV-2 in complex environments, facilitating rapid detection of COVID-19 and contributing to effective measures against its spread.

Screening and characterization of inhibitory vNAR targeting nanodisc-assembled influenza M2 proteins.

Influenza A virus poses a constant challenge to human health. The highly conserved influenza matrix-2 (M2) protein is an attractive target for the development of a universal antibody-based drug. However, screening using antigens with subphysiological conformation in a nonmembrane environment significantly reduces the generation of efficient antibodies. Here, M2(1-46) was incorporated into nanodiscs (M2-nanodiscs) with M2 in a membrane-embedded tetrameric conformation, closely resembling its natural physiological state in the influenza viral envelope. M2-nanodisc generation, an antigen, was followed by Chiloscyllium plagiosum immunization. The functional vNARs were selected by phage display panning strategy from the shark immune library. One of the isolated vNARs, AM2H10, could specifically bind to tetrameric M2 instead of monomeric M2e (the ectodomain of M2 protein). Furthermore, AM2H10 blocked ion influx through amantadine-sensitive and resistant M2 channels. Our findings indicated the possibility of developing functional shark nanobodies against various influenza viruses by targeting the M2 protein.

Towards magnetism in pigeon MagR: Iron- and iron-sulfur binding work indispensably and synergistically.

The ability to navigate long distances is essential for many animals to locate shelter, food, and breeding grounds. Magnetic sense has evolved in various migratory and homing species to orient them based on the geomagnetic field. A highly conserved iron-sulfur cluster assembly protein IscA is proposed as an animal magnetoreceptor (MagR). Iron-sulfur cluster binding is also suggested to play an essential role in MagR magnetism and is thus critical in animal magnetoreception. In the current study, we provide evidence for distinct iron binding and iron-sulfur cluster binding in MagR in pigeons, an avian species that relies on the geomagnetic field for navigation and homing. Pigeon MagR showed significantly higher total iron content from both iron- and iron-sulfur binding. Y65 in pigeon MagR was shown to directly mediate mononuclear iron binding, and its mutation abolished iron-binding capacity of the protein. Surprisingly, both iron binding and iron-sulfur binding demonstrated synergistic effects, and thus appear to be integral and indispensable to pigeon MagR magnetism. These results not only extend our current understanding of the origin and complexity of MagR magnetism, but also imply a possible molecular explanation for the huge diversity in animal magnetoreception.

Synthesis, characterization and application of magnetoferritin nanoparticle by using human H chain ferritin expressed by Pichia pastoris.

Protein-based nanoparticles have developed rapidly in areas such as drug delivery, biomedical imaging and biocatalysis. Ferritin possesses unique properties that make it attractive as a potential platform for a variety of nanobiotechnological applications. Here we synthesized magnetoferritin (P-MHFn) nanoparticles for the first time by using the human H chain of ferritin that was expressed by Pichia pastoris (P-HFn). Western blot results showed that recombinant P-HFn was successfully expressed after methanol induction. Transmission electron microscopy (TEM) showed the spherical cage-like shape and monodispersion of P-HFn. The synthesized magnetoferritin (P-MHFn) retained the properties of magnetoferritin nanoparticles synthesized using HFn expressed by E. coli (E-MHFn): superparamagnetism under ambient conditions and peroxidase-like activity. It is stable under a wider range of pH values (from 5.0 to 11.0), likely due to post-translational modifications such as N-glycosylation on P-HFn. In vivo near-infrared fluorescence imaging experiments revealed that P-MHFn nanoparticles can accumulate in tumors, which suggests that P-MHFn could be used in tumor imaging and therapy. An acute toxicity study of P-MHFn in Sprague Dawley rats showed no abnormalities at a dose up to 20 mg Fe Kg-1 body weight. Therefore, this study shed light on the development of magnetoferritin nanoparticles using therapeutic HFn expressed by Pichia pastoris for biomedical applications.

Experimental Evaluation on the Heating Efficiency of Magnetoferritin Nanoparticles in an Alternating Magnetic Field.

The superparamagnetic substance magnetoferritin is a potential bio-nanomaterial for tumor magnetic hyperthermia because of its active tumor-targeting outer protein shell, uniform and tunable nanosized inner mineral core, monodispersity and good biocompatibility. Here, we evaluated the heating efficiency of magnetoferritin nanoparticles in an alternating magnetic field (AMF). The effects of core-size, Fe concentration, viscosity, and field frequency and amplitude were investigated. Under 805.5 kHz and 19.5 kA/m, temperature rise (ΔT) and specific loss power (SLP) measured on magnetoferritin nanoparticles with core size of 4.8 nm at 5 mg/mL were 14.2 °C (at 6 min) and 68.6 W/g, respectively. The SLP increased with core-size, Fe concentration, AMF frequency, and amplitude. Given that: (1) the SLP was insensitive to viscosity of glycerol-water solutions and (2) both the calculated effective relaxation time and the fitted relaxation time were closer to Néel relaxation time, we propose that the heating generation mechanism of magnetoferritin nanoparticles is dominated by the Néel relaxation. This work provides new insights into the heating efficiency of magnetoferritin and potential future applications for tumor magnetic hyperthermia treatment and heat-triggered drug release.

Thermostable iron oxide nanoparticle synthesis within recombinant ferritins from the hyperthermophile Pyrococcus yayanosii CH1.

Thermostable nanoparticles have numerous applications in catalysis and in the oil/gas industry. However, synthesizing these nanoparticles requires expensive polymers. Here, a novel thermostable ferritin named PcFn, originally from the hyperthermophilic archaeon Pyrococcus yayanosii CH1, was overexpressed in Escherichia coli, purified and characterized, which could successfully direct the synthesis of thermostable magnetoferritins (M-PcFn) with monodispersed iron oxide nanoparticles in one step. Transmission electron microscopy and magnetic measurements show that the cores of the M-PcFn have an average diameter of 4.7 nm, are well-crystalline and superparamagnetic. Both the PcFn and M-PcFn can resist temperatures up to 110 °C, which is significantly higher than for human H-chain ferritin (HFn) and M-HFn, and comparable to temperatures previously reported for Pyrococcus furiosus ferritin (PfFn) and M-PfFn. After heating at 110 °C for 30 minutes, PcFn and M-PcFn maintained their secondary structures and PcFn retained 87.4% of its iron uptake activity. This remarkable thermostability of PcFn and M-PcFn suggests potential applications in elevated temperature environments.

Positive magnetic resonance angiography using ultrafine ferritin-based iron oxide nanoparticles.

Iron oxide nanoparticles with good biocompatibility can serve as safe magnetic resonance imaging contrast agents. Herein, we report that ultrafine ferritin-based iron oxide (hematite/maghemite) nanoparticles synthesized by controlled biomimetic mineralization using genetically recombinant human H chain ferritin can be used as a positive contrast agent in magnetic resonance angiography. The synthesized magnetoferritin with an averaged core size of 2.2 ± 0.7 nm (hereafter named M-HFn-2.2) shows a r1 value of 0.86 mM-1 s-1 and a r2/r1 ratio of 25.1 at a 7 T magnetic field. Blood pool imaging on mice using the M-HFn-2.2 nanoparticles that were injected through a tail vein by single injection at a dose of 0.54 mM Fe per kg mouse body weight enabled detecting detailed vascular nets at 3 minutes post-injection; the MR signal intensity continuously enhanced up to 2 hours post-injection, which is much longer than that of the commercial magnevist (Gd-DTPA) contrast. Moreover, biodistribution examination indicates that organs such as liver, spleen and kidney safely cleared the injected nanoparticles within one day after the injection, demonstrating no risk of iron overload in test mice. Therefore, this study sheds light on developing high-performance gadolinium free positive magnetic resonance contrast agents for biomedical applications.

Complete Genome Sequence of Magnetospirillum sp. Strain XM-1, Isolated from the Xi'an City Moat, China.

The magnetotactic bacterium Magnetospirillum sp. strain XM-1 was recently isolated from the Xi'an City moat, China. It belongs to the Rhodospirillaceae family in the Alphaproteobacteria class. Here, we report the complete genome sequence of XM-1. The genome contains a single circular chromosome of 4,825,187 bp and a plasmid of 167,290 bp.