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Ferritin is a multisubunit protein with a hollow interior interface and modifiable surfaces. In this study, the manothermosonication (MTS) technology was applied to apo-red bean seed ferritin (apoRBF) to produce the MTS-treated apoRBF (MTFS). MTS treatment (200 kPa, 50 °C, and 40 s) maintained the spherical morphology of apoRBF (12 nm), but reduced the content of α-helix structure and increased the content of random coil structure, and correspondingly decreased the ferritin stability. The MTS treatment also affected the ferritin's iron storage function by decreasing its iron oxidative deposition activity and increasing the iron release activity. Importantly, the disassembly and reassembly properties of the MTFS induced by pH changes were retained, which facilitated its usage in encapsulation of tea polyphenol-epigallocatechin gallate (EGCG) into the ferritin by a relatively benign pH conversion routine (pH 3.0/6.8). In addition, the water solubility of the MTFS was increased, leading to the improved encapsulation efficiency of the EGCG molecules. This study will facilitate the ferritin modification and functionalization by MTS to design a protein variant to be used as new scaffold for iron and bioactive compounds.

Phosphorylated p53 proteins are biomarkers with clinical utility for early diagnosis of cancer, but difficult to quantify. An inductively coupled plasma mass spectrometry (ICP-MS) based immunoassay is described here that uses uniform lanthanide nanoparticles (NPs) as elemental tags for the simultaneous determination of two phosphorylated p53 proteins. Apoferritin templated europium (Eu) phosphate (AFEP) NPs and apoferritin templated lutetium (Lu) phosphate (AFLP) NPs with 8 nm in diameter were used to label two phosphorylated p53 proteins at serine 15 and serine 392 sites (p-p53 15 and p-p53 392 ), respectively. The assay has a sandwich format, and p-p53 15 and p-p53 392 were first captured and then recognized by AFEP and AFLP NPs labelled antibodies, respectively. The Eu and Lu were then released from the immune complexes under acidic condition for ICP-MS measurement. The limits of detection for p-p53 15 and p-p53 392 are 200 and 20 pg·mL -1 , with linear ranges of 0.5-20 and 0.05-20 ng·mL -1 , respectively. The method was further applied to study the response of p-p53 15 and p-p53 392 in SCC-7 cells exposed to the natural carcinogen arsenite. A significant up-regulation of p-p53 15 and p-p53 392 can be observed when cells were exposed to arsenite at 5 μmol·L -1 level for 24 h. Graphical abstract Schematic presentation of the ICP-MS immunoassay using apoferritin templated europium (III) and lutetium (III) phosphate nanoparticles as labels for the simultaneous determination of two phosphorylated p53 proteins. Europium (Eu) phosphate nanoparticles (blue) and lutetium (Lu) phosphate nanoparticles (pink) were synthesized in the size-restricted cavity of apoferritin. They were further coupled with antibodies to prepare Eu and Lu labelled probes for p-p53 15 (blue) and p-p53 392 (pink), respectively. After formation of a a sandwich, the labelled Eu and Lu were dissociated in acid and then introduced to ICP-MS for the simultaneous determination of two phosphorylated p53 proteins p-p53 15 (blue) and p-p53 392 (pink).

Simultaneous and sensitive detection of various antibiotic residues in one sample is essential to evaluation of food safety status. Herein, a multiplexed electrochemical aptasensor for multiplex antibiotics detection, with kanamycin (KANA) and ampicillin (AMP) as representative analytes, was designed by using metal ions encoded apoferrtin probes and double stirring bars-assisted target recycling for signal amplification. The encoded probes were prepared by apoferritin loading Cd 2+ and Pb 2+ ions and labeling with duplex DNAs (aptamers corresponding to KANA and AMP hybrid with its complementary DNA sequence), respectively. In the presence of KANA and AMP, the targets can recurrently react with the probes on the bars, and then replace a lot of Apo-Mencoded signal tags into supernatant. The peak currents of Cd 2+ and Pb 2+ from the tags corresponding with the concentrations of KANA and AMP were detected by square wave voltammetry in one run. As a result, KANA and AMP can be detected simultaneously within the range from 0.05 pM to 50 nM. And the detection limits were 18 fM KANA and 15 fM AMP (S/N = 3). The assay was testified to detect KANA and AMP residues with consistent results of ELISA in samples, e.g. milks and fishes. The assay was highly-sensitive, selective, cost-effective and easy-to-operate due to Apo-M encoded probes with high loading capacity of signal source substances. Moreover, double stirring bar-assisted target recycling, which was enzyme-free and could overcome matrix interference, was fabricated for signal amplification. Thus, the assay showed potential advantages for sensitively screening of antibiotic residues in foods., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Neuroferritinopathy is a rare, adult-onset, dominantly inherited movement disorder caused by mutations in the ferritin gene. A ferritin light-chain variant related to neuroferritinopathy, in which alanine 96 is replaced with threonine (A96T), was expressed in Escherichia coli, purified, and characterized. The circular dichroism, analytical ultracentrifugation, and small-angle X-ray scattering studies have shown that both the subunit structure and the assembly of A96T are the same as those of wild-type human ferritin light chain (HuFTL). The iron-incorporation ability was also comparable to that of HuFTL. Although the structural stability against heat, acid, and denaturant was reduced, the structure was sufficiently stable under physiological conditions. The most remarkable defects observed for A96T were a lower refolding efficiency and a stronger propensity to aggregate. The possible relationship between folding deficiency and disease is discussed.

Infectious pancreatic necrosis virus (IPNV) has been identified as a viral pathogen for many fish diseases that have become a huge hurdle for the growing fishing industry. Thus, in this work, we report a label-free impedance biosensor to quantify IPNV in real fish samples at point-of-care (POC) level. High specificity IPNV sensor with a detection limit of 2.69 TCID 50 /mL was achieved by conjugating IPNV antibodies to portable Au disk electrode chips using human heavy chain apoferritin (H-AFN) nanoprobes as a binding agent. H-AFN probes were bioengineered through PCR by incorporating pET-28b(+) resulting in 24 subunits of 6 × his-tag and protein-G units on its outer surface to increase the sensitivity of the IPNV detection. The biosensor surface modifications were characterized by differential pulse voltammetry (DPV) and EIS methods for each modification step. The proposed nanoprobe based sensor showed three-fold enhancement in charge transfer resistance toward IPNV detection in comparison with the traditional linker approach when measured in a group of similar virus molecules. The portable sensor exhibited a linear range of 100-10000 TCID 50 /mL and sensitivity of 5.40 × 10 -4 TCID 50 /mL in real-fish samples. The performance of the proposed IPNV sensor was fully validated using an enzyme-linked immunosorbent assay (ELISA) technique with a sensitivity of 3.02 × 10 -4 TCID 50 /mL. Results from H-AFN nanoprobe based IPNV sensor indicated high selectivity, sensitivity, and stability could be a promising platform for the detection of similar fish viruses and other biological molecules of interest.

Volta Phase Plate (VPP) has become an invaluable tool for cryo-EM structural determination of small protein complexes by increasing image contrast. Currently, the standard protocol of VPP usage periodically changes the VPP position to a fresh spot during data collection. Such a protocol was to target the phase shifts to a relatively narrow range (around 90°) based on the observations of increased phase shifts and image blur associated with more images taken with a single VPP position. Here, we report a 2.87 Å resolution structure of apoferritin reconstructed from a dataset collected using only a single position of VPP. The reconstruction resolution and map density features are nearly identical to the reconstruction from the control dataset collected with periodic change of VPP positions. Further experiments have verified that similar results, including a 2.5 Å resolution structure, could be obtained with a full range of phase shifts, different spots of variable phase shift increasing rates, and at different ages of the VPP post-installation. Furthermore, we have found that the phase shifts at low resolutions, probably related to the finite size of the Volta spots, could not be correctly modeled by current CTF model using a constant phase shift at all frequencies. In dataset III, severe beam tilt issue was identified but could be computationally corrected with iterative refinements. The observations in this study may provide new insights into further improvement of both the efficiency and robustness of VPP, and to help turn VPP into a plug-and-play device for high-resolution cryo-EM., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Although ellipticine (Elli) is an efficient anticancer agent, it exerts several adverse effects. One approach to decrease the adverse effects of drugs is their encapsulation inside a suitable nanocarrier, allowing targeted delivery to tumour tissue whereas avoiding healthy cells. We constructed a nanocarrier from apoferritin (Apo) bearing ellipticine, ApoElli, and subsequently characterized. The nanocarrier exhibits a narrow size distribution suggesting its suitability for entrapping the hydrophobic ellipticine molecule. Ellipticine was released from ApoElli into the water environment under pH 6.5, but only less than 20% was released at pH 7.4. The interaction of ApoElli with microsomal membrane particles containing cytochrome P450 (CYP) biotransformation enzymes accelerated the release of ellipticine from this nanocarrier making it possible to be transferred into this membrane system even at pH 7.4 and facilitating CYP-mediated metabolism. Reactive metabolites were formed not only from free ellipticine, but also from ApoElli, and both generated covalent DNA adducts. ApoElli was toxic in UKF-NB-4 neuroblastoma cells, but showed significantly lower cytotoxicity in non-malignant fibroblast HDFn cells. Ellipticine either free or released from ApoElli was concentrated in the nuclei of neuroblastoma cells, concentrations of which being significantly higher in nuclei of UKF-NB-4 than in HDFn cells. In HDFn the higher amounts of ellipticine were sequestrated in lysosomes. The extent of ApoElli entering the nuclei in UKF-NB-4 cells was lower than that of free ellipticine and correlated with the formation of ellipticine-derived DNA adducts. Our study indicates that the ApoElli form of ellipticine seems to be a promising tool for neuroblastoma treatment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human transferrin receptor 1 (CD71) guarantees iron supply by endocytosis upon binding of iron-loaded transferrin and ferritin. Arenaviruses and the malaria parasite exploit CD71 for cell invasion and epitopes on CD71 for interaction with transferrin and pathogenic hosts were identified. Here, we provide the molecular basis of the CD71 ectodomain-human ferritin interaction by determining the 3.9 Å resolution single-particle cryo-electron microscopy structure of their complex and by validating our structural findings in a cellular context. The contact surfaces between the heavy-chain ferritin and CD71 largely overlap with arenaviruses and Plasmodium vivax binding regions in the apical part of the receptor ectodomain. Our data account for transferrin-independent binding of ferritin to CD71 and suggest that select pathogens may have adapted to enter cells by mimicking the ferritin access gate.

The effects of encapsulating the cytotoxic dinuclear trithiolato-bridged arene ruthenium complex [(η 6 -p-MeC 6 H 4 iPr) 2 Ru 2 (μ 2 -S-p-C 6 H 4 tBu) 3 ]Cl (DiRu-1) within the apoferritin (AFt) nanocage were investigated. The DiRu-1-AFt nanocarrier was characterized by UV/Vis spectroscopy, ICP-MS, CD and X-ray crystallography. In contrast to previously reported Au- and Pt-based drug-loaded AFt carriers, we found no evidence of direct interactions between DiRu-1 and AFt. DiRu-1-AFt is cytotoxic toward immortalized murine BALB/c-3T3 fibroblasts transformed with SV40 virus (SVT2) and human epidermoid carcinoma A431 malignant cells, and exhibits moderate selectivity for these cancer cells over normal BALB/c-3T3 cells. DiRu-1-AFt triggers the production of reactive oxygen species, depolarization of mitochondrial membrane potential, and induces cell death via p53-mediated apoptosis. Comparison between our data and previous results suggests that the presence of specific interactions between a metal-based drug and AFt within the protein cage is not essential for drug encapsulation., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Ferritin is a globular intracellular protein that acts as the main reservoir for iron. Malignancies are associated with increased plasma ferritin concentrations. A number of studies show that tumor cells express high levels of transferrin receptors (TfR). Increased TfR expression was observed in prostate carcinoma. Apoferritin (APO) can be used as a protein nanotransporter into which a suitable medicinal substance can be encapsulated. Nanoparticles increase the permeability of tumor cells to nanotransporters and have a photothermal effect. The aim of this study was to encapsulate doxorubicin (DOX) into APO and to modify the resulting APO/DOX with gold (AuNPs) and silver nanoparticles prepared by green synthesis (AgNPsGS)., Methods: APO was characterized using 10% sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) - 120 V, 60 min, 24 mM Tris, 0.2 M glycine, 3 mM SDS. DOX fluorescence (Ex 480 nm; Em 650 nm) was observed, with a typical absorption maximum at 560 nm. Electrochemical measurement was performed in Brdicka solution (three-electrode setup). AgNPsGS were prepared by green synthesis using clover (Trifolium pratense L.)., Results: An electrophoretic study of APO and APO/DOX (5-100 μg/mL) was performed and the behavior of APO and APO/DOX (10 μM) as a function of pH was monitored. In an acidic environment, APO forms subunits of about 20 kDa; in an alkaline medium, it forms a globular protein of about 450 kDa. A change in APO/DOX mobility (about by 10%) was observed. A film of gold nanoparticles was applied to the APO/DOX surface. APO/DOX-AuNPs were washed with ultra-pure water. pH-dependent release of DOX a was monitored. The amount of DOX analyzed was increased by up to 50%. Furthermore, an AgNPsGS-DOX complex (1 mg AgNPsGS/100 μM DOX) was generated and prepared. Subsequently, the AgNPsGS-DOX complex was encapsulated into APO. To further improve therapeutic efficacy, the APO/AgNPsGS-DOX complex was coated with an Au layer. APO/AgNPsGS-DOX/AuNPs were stable and DOX was released from the complex after physical parameters had changed., Conclusion: APO nanocomplexes were prepared and modified to increase therapeutic efficacy against tumors. Tumor cell targeting was achieved by binding to TfR and via increased tumor cell permeability and retention. Release of the drug was made possible due to a pH change and photothermal activation that will now be tested. This work was supported by COST European Cholangiocarcinoma Network CA18122 and International Collaboration Project of The European Technology Platform for Nanomedicine. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 21. 3. 2019 Accepted: 14. 5. 2019.

Cancer is one of the major causes of mortality worldwide. Therefore, it is necessary to provide an effective method of tumor therapy. Herein, we designed a new type of composite particle, apoferritin (APO) encapsulated doxorubicin (DOX), and the surface of APO was modified with Au nanoshell. As a nanocarrier, APO can carry chemotherapy drug DOX (APODOX) and release drug under acidic and high temperature conditions to reduce side effects of anticancer drugs. After covering Au nanoshell (APODOX-Au), the photothermal effect can be produced because of the unique surface plasmon resonance properties of gold nanoshell. This nanoplatform also provides the multi-stimuli responsive drug release system, which can achieve drug release in different conditions and have great potential in biomedical applications. Our investigation has demonstrated that APODOX-Au has good stability, high dispersibility and biocompatibility in vitro. The strong near-infrared absorption and good photothermal effect make sure the quick response to environmental changes (pH, temperature) to achieve drug release. These findings indicate that these nanoparticles have a potential application value in cancer treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Ferritin, a soluble and highly robust protein with subunits packed into well-defined helices, is a key component of the iron regulatory system in the brain and thus is widely recognized as a crucial protein for iron metabolism, but may also bear possible implications in some neurodegenerative disorders. Here, we present evidence of how human recombinant apoferritin can convert into an unusual structure from its folded native state; that is, amyloid fibrils analogue to those found in pathological disorders such as Alzheimer's and Parkinson's diseases. An extensive combination of advanced microscopy, spectroscopy and scattering techniques concur to reveal that apoferritin fibrils possess a common double stranded twisted ribbon structure which can result in a mesoscopic right-handed chirality. We highlight a direct connection between the chirality and morphology of the resulting amyloid fibrils, and the initial protein subunits composition, advancing our understanding on the possible role of misfolding in some ferritin-related pathologies and posing new bases for the design of chiral 1D functional nanostructures.

Reactive oxygen species (ROS) cause oxidative stress, which involves in the pathogenesis of many serious diseases. Apoferittin containing gold-silver nanoparticles (Au-Ag-AFT) was designed and evaluated as a nanozyme for scavenging the ROS. The nanozyme consisting of silver-gold nanohybrid in apoferittin cage represents superoxide dismutase, catalase and peroxidase mimetic activities. The Au-Ag-AFT nanozyme was characterized by spectroscopy, FESEM, TEM and dynamic light scattering. The inhibition process for pyrogallol autoxidation was used for assaying the superoxide dismutase mimetic activity and measuring the kinetic parameters of Au-Ag-AFT nanozyme. Additionally, Aebi method and standard protocol was used for evaluating the catalase and peroxidase mimetic activity. The k cat values for superoxide dismutase, catalase and peroxidase mimetics activity were 1.4 × 10 6 , 0.1 and 9 × 10 3  s -1 respectively. These values indicated that Au-Ag-AFT nanozyme could act as a suitable ROS scavenger. Additionally, Au-Ag-AFT nanozyme was examined as a protective agent for human sperm against oxidative stress induced during the cryopreservation process. Presence of the nanozyme in the sperm media significantly increased the motility and viability of the cells and also decreased the ROS, apoptosis and necrosis (P < 0.05) compare to the control group., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cancer stem-like cells (CSCs) are proposed to be responsible for tumor metastasis, resistance and relapse after therapy, but are unable to be eliminated by many current therapies. Herein, we report that the apoferritin nanocages loading cytotoxic mertansine (M-AFN) can significantly improve their uptake in CSCs-enriched tumorspheres and effectively eradicate CSCs in tumorspheres for anticancer therapy. M-AFN were uniformly nanocage structures with the mean diameter of 11.26 ± 2.58 nm and the loading capacity of 0.62%. In the CSCs-enriched tumorsphere model, M-AFN could be preferentially internalized by tumorsphere cells and the average half-inhibitory concentration (IC 50 ) of M-AFN was obviously reduced by 5.46-fold when comparing to the parent 4T1 breast cancer cells. Moreover, both the already existing tumorspheres and the formation of secondary tumorspheres were drastically disrupted by M-AFN, but barely impacted by mertansine alone. The flow cytometer analysis showed the CSCs fractions in tumorspheres were considerably reduced by the M-AFN treatment. Therefore, the apoferritin nanocages represent an encouraging nanoplatform to eradicate CSCs for effective anticancer therapy., (Copyright © 2018. Published by Elsevier B.V.)

Cationized magnetoferritin is used for development of a simple, efficient, and fast delivery of short interference RNA into cells using combination of magnetophoresis for pre-concentration of siRNA-magnetoferritin complex on the surface of plated cells with subsequent application of nanosecond laser pulses producing stress waves in transfection chamber, which permeabilize cell membrane for the facilitated delivery of siRNA into the cell interior. As has been quantified using siRNA inducing cell death assay, by combination of these two physical factors we have obtained high efficiency for tested three different human carcinoma cells. Proposed method of gene silencing based on cationized magnetoferritin is a versatile and easily accessible platform with many possible applications in gene therapy.

188 Re-magnetoferritin nanoparticles (NPs) provide an attractive platform for localized radiation therapy due to their magnetic targeting capability while enhancing contrast in magnetic resonans imaging (MRI) signals. In this study, cellular uptake, in vitro cytotoxicity, apoptotic potential of a non-radioactive isotope of rhenium in the form of 187 Re-magnetoferritin NPs were evaluated in both human normal mammary epithelial and breast metastatic adenocarcinoma cell lines. The results showed that, NP administration into the cells is through receptor mediated endocytosis and cancer cells displayed significantly higher uptake and cytotoxicity compared to normal cells. IC 50 values of nanoparticles were calculated as 0.96 mg/mL for cancer and 1.73 mg/mL for normal cells. Annexin V/ Propidium Iodide (PI) staining also showed that, NPs induced higher apoptotic rates in cancer cells compared to normal cells. Gene expression analyses confirming the results showed that, pro-apoptotic PUMA and BAX genes were significantly up-regulated while anti-apoptotic BCL-2 and SURVIVIN genes were down-regulated in cancer cells compared to normal cells. Overall, these in vitro results suggest that, 187 Re-magnetoferritin NPs have a promising potential for cancer therapy and can be used for imaging and diagnostic purposes for breast cancer at concentrations lower than 0.96 mg/mL. At concentrations above 1 mg/mL, NPs induce apoptosis which can also be used for cancer treatments., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The 3DEM map challenge provided an opportunity to test different algorithms and workflows for processing single particle cryo-EM data. We were interested in testing whether we could use the standard Appion workflow with minimal manual intervention to achieve similar or better resolution than other challengers. Another question we were interested in testing was what the influence of particle sorting and elimination would be on the resolution and quality of 3D reconstructions. Since apoferritin is historically a challenging particle for single particle reconstruction and the authors of the original map challenge data used only a fraction of the particles present in the dataset, we focused on the apoferritin dataset for our entry. We submitted a 3.7 Å map from 25,844 particles and a 3.6 Å map from 53,334 particles and after assessment were among the best of the apoferritin maps that were submitted. Here we present the details of our reconstruction strategy and compare our strategy to that of another high-scoring apoferritin map. Altogether, our results suggest that for a relatively conformationally homogeneous particle like apoferritin, including as many particles as possible after elimination of junk leads to the highest resolution, and the choice of parameters for custom mask creation can lead to subtle but significant changes in the resolution of 3D reconstructions., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Optical imaging technologies improve clinical diagnostic accuracy of early gastric cancer (EGC). However, there was a lack of imaging agents exhibiting molecular specificity for EGCs. Here, we employed the dye labeled human heavy-chain ferritin (HFn) as imaging nanoprobe, which recognizes tumor biomarker transferrin receptor 1 (TfR1), to enable specific EGC imaging using confocal laser endomicroscopy (CLE). TfR1 expression was initially examined in vitro in gastric tumor cells and in vivo through whole-body fluorescence and CLE imaging in tumor-bearing mice. Subsequently, dye labeled HFn was topically applied to resected human tissues for EGC detection. CLE analysis of TfR1-targeted fluorescence imaging allowed distinction of neoplastic from non-neoplastic tissues (P < 0.0001), and TfR1 expression level was found to correlate with EGC differentiation degrees (P < 0.0001). Notably, the CLE evaluation correlated well with the immunohistochemical findings (κ-coefficient: 0.8023). Our HFn-nanoprobe-based CLE increases the accuracy of EGC detection and enables visualization of tumor margins and endoscopic resection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Ferritin, a naturally occuring iron-storage protein, plays an important role in nanoengineering and biomedical applications. Upon iron removal, apoferritin was shown to allow the encapsulation of an artificial transfer hydrogenase (ATHase) based on the streptavidin-biotin technology. The third coordination sphere, provided by ferritin, significantly influences the catalytic activity of an ATHase for the reduction of cyclic imines.

Ferritin H-homopolymers have been extensively used as nanocarriers for diverse applications in the targeted delivery of drugs and imaging agents, due to their unique ability to bind the transferrin receptor (CD71), highly overexpressed in most tumor cells. In order to incorporate novel fluorescence imaging properties, we have fused a lanthanide binding tag (LBT) to the C-terminal end of mouse H-chain ferritin, HFt. The HFt-LBT possesses one high affinity Terbium binding site per each of the 24 subunits provided by six coordinating aminoacid side chains and a tryptophan residue in its close proximity and is thus endowed with strong FRET sensitization properties. Accordingly, the characteristic Terbium emission band at 544 nm for the HFt-LBT Tb(III) complex was detectable upon excitation of the tag enclosed at two order of magnitude higher intensity with respect to the wtHFt protein. X-ray data at 2.9 Å and cryo-EM at 7 Å resolution demonstrated that HFt-LBT is correctly assembled as a 24-mer both in crystal and in solution. On the basis of the intrinsic Tb(III) binding properties of the wt protein, 32 additional Tb(III) binding sites, located within the natural iron binding sites of the protein, were identified besides the 24 Tb(III) ions coordinated to the LBTs. HFt-LBT Tb(III) was demonstrated to be actively uptaken by selected tumor cell lines by confocal microscopy and FACS analysis of their FITC derivatives, although direct fluorescence from Terbium emission could not be singled out with conventional, 295-375 nm, fluorescence excitation., Competing Interests: The funder provided support in the form of salaries for authors Trabuco, MC, who is employed at Molirom as a Marie Curie Fellow within the framework of the X-probe EU project and Taciak B., Rygel T and Krol M., who are employed at Cellis within the framework of the National Science Center of Poland. The companies did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Herein, we incorporated dual biotemplates, i.e., cellulose nanocrystals (CNC) and apoferritin, into electrospinning solution to achieve three distinct benefits, i.e., (i) facile synthesis of a WO 3 nanotube by utilizing the self-agglomerating nature of CNC in the core of as-spun nanofibers, (ii) effective sensitization by partial phase transition from WO 3 to Na 2 W 4 O 13 induced by interaction between sodium-doped CNC and WO 3 during calcination, and (iii) uniform functionalization with monodispersive apoferritin-derived Pt catalytic nanoparticles (2.22 ± 0.42 nm). Interestingly, the sensitization effect of Na 2 W 4 O 13 on WO 3 resulted in highly selective H 2 S sensing characteristics against seven different interfering molecules. Furthermore, synergistic effects with a bioinspired Pt catalyst induced a remarkably enhanced H 2 S response ( R air / R gas = 203.5), unparalleled selectivity ( R air / R gas < 1.3 for the interfering molecules), and rapid response (<10 s)/recovery (<30 s) time at 1 ppm of H 2 S under 95% relative humidity level. This work paves the way for a new class of cosensitization routes to overcome critical shortcomings of SMO-based chemical sensors, thus providing a potential platform for diagnosis of halitosis.

Ferritin is a globular protein that consists of 24 subunits forming a hollow nanocage structure that naturally stores iron oxyhydroxides. Elimination of iron atoms to obtain the empty protein called apoferritin is the first step to use this organic shell as a nanoreactor for different nanotechnological applications. Different protocols have been reported for apoferritin formation, but some are time consuming, others are difficult to reproduce and protein recovery yields are seldom reported. Here we tested several protocols and performed a complete material characterization of the apoferritin products using size exclusion chromatography, UV-vis spectroscopy, inductively coupled plasma optical emission spectrometry and dynamic light scattering. Our best method removes more than 99% of the iron from loaded holoferritin, recovering 70-80% of the original protein as monomeric apoferritin nanocages. Our work shows that pH conditions of the reduction step and the presence and nature of chelating agents affect the efficiency of iron removal. Furthermore, process conditions also seem to have an influence on the monomer:aggregate proportion present in the product. We also demonstrate that iron contents markedly increase ferritin absorbance at 280nm. The influence of iron contents on absorbance at 280nm precludes using this simple spectrophotometric measure for protein determination in ferritin‑iron complexes. Apoferritin produced following our protocol only requires readily-available, cheap and biocompatible reagents, which makes this process standardizable, scalable and applicable to be used for in vivo applications of ferritin derivatives as well as nanotechnological and biotechnological uses., (Copyright © 2017. Published by Elsevier Inc.)

Ferritins are ubiquitous and conserved proteins endowed with enzymatic ferroxidase activity, that oxidize Fe(II) ions at the dimetal ferroxidase centre to form a mineralized Fe(III) oxide core deposited within the apo-protein shell. Herein, the in vitro formation of a heterodimetal cofactor constituted by Fe and Mn ions has been investigated in human H ferritin (hHFt). Namely, Mn and Fe binding at the hHFt ferroxidase centre and its effects on Fe(II) oxidation have been investigated by UV-Vis ferroxidation kinetics, fluorimetric titrations, multifrequency EPR, and preliminary Mössbauer spectroscopy. Our results show that in hHFt, both Fe(II) and Mn(II) bind the ferroxidase centre forming a Fe-Mn cofactor. Moreover, molecular oxygen seems to favour Mn(II) binding and increases the ferroxidation activity of the Mn-loaded protein. The data suggest that Mn influences the Fe binding and the efficiency of the ferroxidation reaction. The higher efficiency of the Mn-Fe heterometallic centre may have a physiological relevance in specific cell types (i.e. glia cells), where the concentration of Mn is the same order of magnitude as iron., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The effect of chitosan decoration on the transport of epigallocatechin (EGC)-encapsulated ferritin cage across the Caco-2 cells was investigated. After the encapsulation of EGC in apo-red bean (adzuki) ferritin (apoRBF), the EGC-loaded apoRBF nanoparticle (ER) was fabricated with an encapsulation ratio of 11.6% (w/w). The results indicated that different chitosan molecules (with molecular weights of 980, 4600, 46 000, and 210 000 Da) could attach onto the apoRBF via electrostatic interactions to form ER-chitosan complexes (ERCs) (ERCs980, ERCs4600, ERCs46000, and ERCs210000). ERCs980 and ERCs4600 retained the typical shell-like morphology of ferritin with a size distribution of 12 nm and showed weak negative zeta-potentials at pH 6.7, while ERCs46000 and ERCs210000 significantly aggregated. Furthermore, the transport of EGC in ERCs980 and ERCs4600 across the Caco-2 cells was enhanced by the transferrin receptor 1 (TfR-1)-mediated absorption pathway, demonstrating that chitosan molecules with low molecular weights of 980 and 4600 Da were beneficial to the absorption of EGC based on the ferritin cage. This study will facilitate the application of ferritin-chitosan materials for fabricating the core-shell platform for encapsulation and bioavailability enhancement of bioactive molecules.

Apoferritin (AF) is a natural nontoxic iron carrier and has a natural hollow structure that can be used to deliver small molecules. The surface of AF has many amine functional groups that can be modified to create targeted ligands. We loaded oxaliplatin onto AF, which was then used as a template to conjugate with panitumumab via a polyethylene glycol linker. The oxaliplatin-loaded AF conjugated with panitumumab (AFPO) was designed to specifically target cell lines expressing epidermal growth factor receptor (EGFR). AFPO efficiently released oxaliplatin and suppressed tumor cell growth. Furthermore, the novel AFPO nanocages showed significant inhibition and greater accumulation in tumor models with high EGFR expression in vivo. Our study revealed that combining panitumumab and oxaliplatin into one formulation (AFPO nanocage) could be a promising shortcut in clinical applications.

For the purpose of this article, experimental phasing is understood to mean the determination of macromolecular structures by exploiting small intensity differences of Friedel opposites and possibly of reflections measured at different wavelengths or for heavy-atom derivatives, without the use of specific structural models. The SHELX programs provide a robust and efficient route for routine structure solution by the SAD, MAD and related methods, but involve a number of simplifying assumptions that may limit their applicability in borderline cases. The substructure atoms (i.e. those with significant anomalous scattering) are first located by direct methods, and the experimental data are then used to estimate phase shifts that are added to the substructure phases to obtain starting phases for the native reflections. These are then improved by density modification and, if the resolution of the data and the type of structure permit, polyalanine tracing. A number of extensions to the tracing algorithm are discussed; these are designed to improve its performance at low resolution. Given native data to 2.5 Å resolution or better, a correlation coefficient greater than 25% between the structure factors calculated from such a trace and the native data is usually a good indication that the structure has been solved.

Anesthetics interact with a broad range of different targets, including both soluble and membrane-bound proteins. Understanding these interactions at the molecular level requires detailed structural knowledge of anesthetic-protein complexes, and one of the most productive routes to such knowledge is X-ray crystallography. In this chapter we discuss the application of this technique to the analysis of complexes of anesthetics with soluble proteins. The model protein apoferritin is highlighted, and protocols are presented for obtaining diffraction-quality crystals of this protein in complex with different general anesthetics., (© 2018 Elsevier Inc. All rights reserved.)

Epirubicin (Epr) is an effective chemotherapeutic drug; however, the clinical amenability of Epr is limited by its highly toxic interaction with normal cells. This toxicity can be decreased by utilizing nanocarriers and targeted drug delivery systems. This work describes an approach for the delivery of Epr via encapsulation in the horse spleen apoferritin (HsAFr) cavity. The encapsulation was achieved by the disassembling of apoferritin into subunits at pH 2 followed by its reformation at pH 7.4 in the presence of Epr. The surface of HsAFr-encapsulated Epr was modified with folic acid (FA) for optimal targeting of breast cancer cells (MCF-7). The use of FA to functionalize HsAFr could enhance the cellular uptake efficiency via FA-receptor-mediated endocytosis. UV-vis spectroscopy, fluorescence spectroscopy, circular dichroism (CD) and transmission electron microscopy (TEM) were utilized for structural characterization of the HsAFr-Epr and HsAFr-Epr-FA complexes. The comparison of the anti-cancer activities across the HsAFr-Epr-FA complex and the free Epr drug was performed using the MTT viability assay on MCF-7.

Current immunoassays are in general performed through time-consuming multi-step procedures that depend on the use of premade signal-producing reporters and often cause assay inaccuracy. Here we report an advanced immunoassay technology that resolves the delayed, complex, and inaccurate assay problems of conventional immunoassays. We have developed an accurate, rapid, simple, and label-free one-step-immunoassay based on the self-enhancement of sensitive immunoassay signals in an assay solution. The nano-scale protein particles (hepatitis B virus capsid and human ferritin heavy chain particles) were genetically engineered to present many well-oriented antibody (or antigen) probes and multi-copies of poly-histidine peptides on their surface, resulting in the construction of 3-dimensional (3D) bioprobes that chemisorb gold ions via coordination bonding and sensitively detect both antigen and antibody analytes. Systematic numerical and experimental analyses show that the signal self-enhancement happens through two coupled reactions under reducing conditions: (1) 3D bioprobe-based sensitive immuno-detection of analytes and (2) coordinated assembly of free and chemisorbed gold nanoparticles around the 3D bioprobe-analyte-associated complexes, which is followed by the quick generation of apparent optical signals. This advanced one-step-immunoassay was successfully applied to diagnostic assays requiring high accuracy and/or speed, i.e. diagnosis of acute myocardial infarction and hepatitis C through detecting a cardiac protein (troponin I) and anti-hepatitis C virus antibodies in patient sera, indicating that it is applicable to the accurate and rapid detection of both antigen and antibody markers of a wide range of diseases.

Ferritins are ubiquitous iron storage proteins where Fe(II) sequestration prevents not only its spontaneous oxidation to Fe(III) but also production of toxic free radicals. Recently, scientists have subverted these nature functions and used ferritin cage structures of nanometer dimensions for encapsulation of guest molecules such as anti-cancer drugs or bioactive nutrients based on pH induced ferritin disassembly and reassembly property. However, prior to this study, ferritin nanocage was required to disassemble only under harsh pH conditions (≤2.0 or ≥11.0), followed by reassembly at near neutral pH. Such harsh conditions can cause protein or guest molecules damage to a great extent during this pH-induced unfolding-refolding process. Here, we provide evidence demonstrating that the apoferritin shell is flexible rather than rigid. Indeed, we found that two large complex molecules, uranyl acetate dihydrate and phosphotungstic acid, can reach the cavity of both plant and animal apoferritin followed by mineralization. Moreover, large organic compound such as curcumin and doxorubicin can also be encapsulated within ferritin cavity by its mixing with protein. This strategy will increase the use of ferritin in nanotechnology, and could be also applicable to other shell-like proteins as templates to prepare nanomaterials., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

An ideal brain-targeted nanocarrier must be sufficiently potent to penetrate the blood-brain barrier (BBB) and sufficiently competent to target the cells of interest with adequate optimized physiochemical features and biocompatibility. However, it is an enormous challenge to the researchers to organize the above-mentioned properties into a single nanocarrier particle. New frontiers in nanomedicine are advancing the research of new biomaterials. Herein, we demonstrate a straightforward strategy for brain targeting by encapsulating doxorubicin (DOX) into a naturally available and unmodified apoferritin nanocage (DOX-loaded APO). APO can specifically bind to cells expressing transferrin receptor 1 (TfR1). Because of the high expression of TfR1 in both brain endothelial and glioma cells, DOX-loaded APO can cross the BBB and deliver drugs to the glioma with TfR1. Subsequent research demonstrated that the DOX-loaded APO had good physicochemical properties (particle size of 12.03 ± 0.42 nm, drug encapsulation efficiency of 81.8 ± 1.1%) and significant penetrating and targeting effects in the coculture model of bEnd.3 and C6 cells in vitro. In vivo imaging revealed that DOX-loaded APO accumulated specifically in brain tumor tissues. Additionally, in vivo tumor therapy experiments (at a dosage of 1 mg/kg DOX) demonstrated that a longer survival period was observed in mice that had been treated with DOX-loaded APO (30 days) compared with mice receiving free DOX solution (19 days).

Reports on drug delivery systems capable of overcoming multiple biological barriers are rare. We introduce a nanoparticle-based drug delivery technology capable of rapidly penetrating both lung tumor tissue and the mucus layer that protects airway tissues from nanoscale objects. Specifically, human ferritin heavy-chain nanocages (FTn) were functionalized with polyethylene glycol (PEG) in a unique manner that allows robust control over PEG location (nanoparticle surface only) and surface density. We varied PEG surface density and molecular weight to discover PEGylated FTn that rapidly penetrated both mucus barriers and tumor tissues in vitro and in vivo. Upon inhalation in mice, PEGylated FTn with optimized PEGylation rapidly penetrated the mucus gel layer and thus provided a uniform distribution throughout the airways. Subsequently, PEGylated FTn preferentially penetrated and distributed within orthotopic lung tumor tissue, and selectively entered cancer cells, in a transferrin receptor 1-dependent manner, which is up-regulated in most cancers. To test the potential therapeutic benefits, doxorubicin (DOX) was conjugated to PEGylated FTn via an acid-labile linker to facilitate intracellular release of DOX after cell entry. Inhalation of DOX-loaded PEGylated FTn led to 60% survival, compared with 10% survival in the group that inhaled DOX in solution at the maximally tolerated dose, in a murine model of malignant airway lung cancer. This approach may provide benefits as an adjuvant therapy combined with systemic chemo- or immunotherapy or as a stand-alone therapy for patients with tumors confined to the airways., Competing Interests: Conflict of interest statement: J.H. is a founder of Kala Pharmaceuticals and serves as a consultant. J.H. and Johns Hopkins own company stock; J.H.’s relationship with Kala Pharmaceuticals is subject to certain restrictions under University policy. The terms of this arrangement are being managed by Johns Hopkins University in accordance with its conflict of interest policies.

To develop a multifunctional nanomaterial for dual-mode imaging and synergetic chemotherapy, curcumin (CUR) was physically entrapped into hollow upconversion NaGdF 4 nanomaterial, then apoferritin (AFn) loaded with doxorubicin (DOX) was attached to the NaGdF 4 surface. Subsequent modification with the targeting reagent folic acid (FA) led to generation of the CUR/NaGdF 4 -DOX/AFn-FA conjugate for cancer treatment. X-ray diffraction, scanning (SEM) and transmission electron microscopy (TEM), and Fourier transform infrared (FTIR) spectroscopy demonstrated the successful preparation of hexagonal-phase NaGdF 4 and NaGdF 4 -AFn-FA. Moreover, no toxicity was observed for NaGdF 4 -AFn-FA. In vitro and in vivo experiments demonstrated that the two drugs are sequentially released from the nanocomposites. This two-drug system showed strong growth inhibitory effects on MCF-7 cells. Upconversion luminescence imaging and magnetic resonance (MR) imaging of NaGdF 4 -AFn-FA were carried out. The results of this study show that NaGdF 4 -AFn-FA can be used for targeted anticancer drug delivery as well as imaging, a novel multi-pronged theranostic system for tumor treatment., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Poly(ADP-ribose) polymerase (PARP) inhibitors represent a promising strategy toward the treatment of triple-negative breast cancer (TNBC), which is often associated to genomic instability and/or BRCA mutations. However, clinical outcome is controversial and no benefits have been demonstrated in wild type BRCA cancers, possibly due to poor drug bioavailability and low nuclear delivery. In the attempt to overcome these limitations, we have developed H-Ferritin nanoformulated olaparib (HOla) and assessed its anticancer efficacy on both BRCA-mutated and non-mutated TNBC cells. We exploited the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1), and its physiological tropism toward cell nucleus. TNBC cell lines over-expressing TfR-1 were successfully recognized by H-Ferritin, displaying a fast internalization into the cells. HOla induced remarkable cytotoxic effect in cancer cells, exhibiting 1000-fold higher anticancer activity compared to free olaparib (Ola). Accordingly, HOla treatment enhanced PARP-1 cleavage, DNA double strand breaks and Ola delivery into the nuclear compartment. Our findings suggest that H-Ferritin nanoformulation strongly enhances cytotoxic efficacy of Ola as a stand-alone therapy in both BRCA-mutated and wild type TNBC cells, by promoting targeted nuclear delivery.

The aim of this work is to identify the cisplatin binding sites on human H-chain ferritin. High-resolution X-ray crystallography reveals that cisplatin binds four distinct protein sites, that is, the side chains of His136 and Lys68, the side chain of His105, the side chain of Cys90 and the side chain of Cys102. These Pt binding sites are compared with those observed for the adduct that cisplatin forms upon encapsulation within horse spleen L-chain ferritin (87% identity with human L-chain ferritin).

Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

In animals, the iron storage and detoxification protein, ferritin, is composed of two functionally and genetically distinct subunit types, H (heavy) and L (light), which co-assemble in various ratios with tissue specific distributions to form shell-like protein structures of 24 subunits within which a mineralized iron core is stored. The H-subunit possesses a ferroxidase center (FC) that catalyzes Fe(II) oxidation, whereas the L-subunit does not. To assess the role of the L-subunit in iron oxidation and core formation, two human recombinant heteropolymeric ferritins, designated H-rich and L-rich with ratios of ∼20H:4L and ∼22L:2H, respectively, were employed and compared to the human homopolymeric H-subunit ferritin (HuHF). These heteropolymeric ferritins have a composition similar to the composition of those found in hearts and brains (i.e., H-rich) and in livers and spleens (i.e., L-rich). As for HuHF, iron oxidation in H-rich ferritin was found to proceed with a 2:1 Fe(II):O 2 stoichiometry at an iron level of 2 Fe(II) atoms/H-subunit with the generation of H 2 O 2 . The H 2 O 2 reacted with additional Fe(II) in a 2:1 Fe(II):H 2 O 2 ratio, thus avoiding the production of hydroxyl radical. A μ-1,2-peroxo-diFe(III) intermediate was observed at the FC of H-rich ferritin as for HuHF. Importantly, the H-rich protein regenerated full ferroxidase activity more rapidly than HuHF did and additionally formed larger iron cores, indicating dual roles for the L-subunit in facilitating iron turnover at the FC and in mineralization of the core. The L-rich ferritin, while also facilitating iron oxidation at the FC, additionally promoted oxidation at the mineral surface once the iron binding capacity of the FC was exceeded.

The structural stability of magnetoferritin, a synthetic analogue of ferritin, at various pH levels is assessed here. The structural and electrical properties of the complexes were determined by small-angle X-ray scattering (SAXS), dynamic light scattering (DLS) and zeta potential measurements. At pH 3-6 a reduction of electrostatic repulsion on the suspended colloids resulted in aggregation and sedimentation of magnetoferritin. At neutral to slightly alkaline conditions (pH 7-9) the magnetoferritin structure was stable for lower iron loadings. Higher solution pH 10-12 induced destabilization of the protein structure and dissociation of subunits. Increasing the loading factor in the MFer complex leads to decrease of the stability versus pH changes., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Doxorubicin is employed alone or in combination for the treatment of several hematological and solid malignancies; despite its efficacy, there are associated cardiotoxicity limits both in its application in patients with heart disease risk factors and also in its long-term use. HFt-MP-PAS40 is a genetically engineered human ferritin heavy chain (HFt)-based construct able to efficiently entrap and deliver doxorubicin to cancer cells. HF-MP-PAS contains a short motif sequence (defined as MP) responsive to proteolytic cleavage by tumor matrix metalloproteases (MMPs), located between each HFt subunit and a masking polypeptide sequence rich in proline (P), alanine (A), and serine (S) residues (PAS). This carrier displayed excellent therapeutic efficacy in a xenogenic pancreatic cancer model in vivo, leading to a significant increase in overall animal survival in treated mice. Herein, we describe the HFt-MP-PAS40-Dox efficacy against squamous cell carcinomas of the head and neck (HNSCC) with the goal of validating the application of our nano-drug for the treatment of different solid tumors. In addition, a tolerability study in healthy mice was also performed. The results indicate that HFt-MP-PAS40-Dox produced increased anti-tumor effects both in vitro and in vivo in comparison to the free drug in several HNSCC cell lines. In the acute toxicity studies, the maximum tolerated dose (MTD) of HFt-MP-PAS40-Dox was about 3.5 higher than the free drug: 25 mg/kg versus 7 mg/kg doxorubicin equivalents. Importantly, evaluation of heart tissues provided evidence that doxorubicin is less cardio-toxic when encapsulated inside the ferritin carrier. In conclusion, HFt-MP-PAS40-Dox may be administered safely at higher doses compared with the free drug, resulting in superior efficacy to control HNSCC malignancies., Competing Interests: The authors declare no conflict of interest.

Iron is an essential microelement for almost all living organisms, while an excess of iron is toxic, thus maintenance of iron homeostasis is vital. As iron storage protein, ferritin plays an important role in iron metabolism. In the present study, we cloned and characterized the ferritin H subunit from Megalobrama amblycephala, termed as MamFerH. An iron-responsive element (IRE) was predicted in the 5' untranslated region (UTR) of MamFerH, while its bulge structural was different from that of the reported ferritin M subunit (MamFerM). The MamFerH and MamFerM genes exhibited similar expression patterns during early development with specifically high expression post hatching, whereas their tissue expression patterns were different. Specifically, MamFerM was highly expressed in the spleen, liver and kidney, while MamFerH was predominantly expressed in the blood and brain, indicating their different functions. In addition, the expression of the two genes was induced upon Aeromonas hydrophila infection at both transcriptional and translational levels, and MamFerH was more efficient. Immunohistochemistry and immunofluorescence analysis confirmed their significant changes at protein level and distribution in the liver post infection, indicating their participation in host immune response. Furthermore, bacteriostatic experiment revealed that recombinant MamFerH displayed more significant inhibitory effect on the growth of A. hydrophila., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Propofol (PFL, 1-hydroxyl-2,6-diisopropylbenzene) is currently used widely as one of the most well-known intravenous anesthetics to relieve surgical suffering, but its mechanism of action is not yet clear. Previous experimental studies have demonstrated that the hydroxyl group of PFL plays a dominant role in the molecular recognition of PFL with receptors that lead to hypnosis. To further explore the mechanism of anesthesia induced by PFL in the present work, the exact binding features and interaction details of PFL with three important proteins, human serum albumin (HSA), the pH-gated ion channel from Gloeobacter violaceus (GLIC), and horse spleen apoferritin (HSAF), were investigated systematically by using a rigorous three-layer ONIOM (M06-2X/6-31+G*:PM6:AMBER) method. Additionally, to further characterize the possible importance of such hydroxyl interactions, a similar set of calculations was carried out on the anesthetically inactive fropofol (FFL, 1-fluoro-2,6-diisopropylbenzene) in which the fluorine was substituted for the hydroxyl. According to the ONIOM calculations, atoms in molecules (AIM) analyses, and electrostatic potential (ESP) analyses, the significance of hydrogen bond, halogen bond, and hydrophobic interactions in promoting proper molecular recognition was revealed. The binding interaction energies of PFL with different proteins were generally larger than FFL and are a significant determinant of their differential anesthetic efficacies. Interestingly, although the hydrogen-bonding effect of the hydroxyl moiety was prominent in propofol, the substitution of the 1-hydroxyl by a fluorine atom did not prevent FFL from binding to the protein via a halogen-bonding interaction. It therefore became clear that multiple specific interactions rather than just hydrogen or halogen bonds must be taken into account to explain the different anesthesia endpoints caused by PFL and FFL. The contributions of key residues in ligand-receptor binding were also quantified, and the calculated results agreed with many available experimental observations. This work will provide complementary insights into the molecular mechanisms of anesthetic action for PFL from a robust theoretical point of view. This will not only assist in interpreting experimental observations but will also help to develop working hypotheses for further experiments and future drug design.

We previously developed the Magnet system, which consists of two distinct Vivid protein variants, one positively and one negatively charged, designated the positive Magnet (pMag) and negative Magnet (nMag), respectively. These two proteins bind to each other through electrostatic interactions, preventing unwanted homodimerization and providing selective light-induced heterodimerization. The Magnet system enables the manipulation of cellular functions such as protein-protein interactions and genome editing, although the system could be improved further. To enhance the ability of pMagFast2 (a pMag variant with fast kinetics) to bind nMag, we introduced several pMagFast2 modules in tandem into a single construct, pMagFast2(3×). However, the expression level of this construct decreased drastically with increasing number of pMagFast2 molecules integrated into a single construct. In the present study, we applied a new approach to improve the Magnet system based on an assembly domain (AD). Among several ADs, the Ca 2+ /calmodulin-dependent protein kinase IIα association domain (CAD) most enhanced the Magnet system. The present CAD-Magnet system overcame a trade-off issue between the expression level and binding affinity. The CAD-converged 12 pMag photoswitches exhibited a stronger interaction with nMag after blue light irradiation compared with monomeric pMag. Additionally, the CAD played a key role in converging effector proteins as well in a single complex. Owing to these substantial improvements, the CAD-Magnet system combined with Tiam1 allowed us to robustly induce localized formation of vertical ruffles on the apical plasma membrane. The CAD-Magnet system combined with 4D imaging was instrumental in revealing the dynamics of ruffle formation.

The discovery of biogenic magnetic nanoparticles (BMNPs) in the human brain gives a strong impulse to study and understand their origin. Although knowledge of the subject is increasing continuously, much remains to be done for further development to help our society fight a number of pathologies related to BMNPs. This review provides an insight into the puzzle of the physiological origin of BMNPs in organisms of all three domains of life: prokaryotes, archaea, and eukaryotes, including humans. Predictions based on comparative genomic studies are presented along with experimental data obtained by physical methods. State-of-the-art understanding of the genetic control of biomineralization of BMNPs and their properties are discussed in detail. We present data on the differences in BMNP levels in health and disease (cancer, neurodegenerative disorders, and atherosclerosis), and discuss the existing hypotheses on the biological functions of BMNPs, with special attention paid to the role of the ferritin core and apoferritin., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

A signal-on photoelectrochemical (PEC) biosensor based on nano-encapsulant of ascorbic acid-loaded apoferritin-assisted for protease detection is described. The loaded ascorbic acid could be released from the central cavity of apoferritin into the buffer solution as sacrificial electron donor to capture the photo-generated holes of CdTe quantum dots when light is turned on. In this system, the biosensor relied on monitoring the photocurrent intensity as a result of enzymatic substrate proteolysis in the homogeneous aqueous solution. A low detection limit of 2.7ngmL -1 for trypsin in the linear range from 30 to 450ngmL -1 is achieved. We believe that such a sensing system not only holds the potential ability as a probe for trypsin activity assay, but also could be used for the corresponding inhibitor-screening. It might provide a potentially feasible alternative tool for determining trypsin in human serum in a clinical laboratory. The established method could open a different perspective for PEC enzyme detection and provide a new platform for future development of PEC analysis with other clinically important proteins., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Apoferritin caged gold nanoparticles (AuNPs) were assembled into flower-like structures by precise base pairing of the attached DNA molecules. The key step was to use the eight hydrophilic channels through the apoferritin to control the exact number and locations of the DNA molecules that grafted onto the caged AuNP.

Due to many adverse effects of conventional chemotherapy, novel methods of targeting drugs to cancer cells are being investigated. Nanosize carriers are a suitable platform for this specific delivery. Herein, we evaluated the long-term stability of the naturally found protein nanocarrier apoferritin (Apo) with encapsulated doxorubicin (Dox). The encapsulation was performed using Apo's ability to disassemble reversibly into its subunits at low pH (2.7) and reassemble in neutral pH (7.2), physically entrapping drug molecules in its cavity (creating ApoDox). In this study, ApoDox was prepared in water and phosphate-buffered saline and stored for 12 weeks in various conditions (-20°C, 4°C, 20°C, and 37°C in dark, and 4°C and 20°C under ambient light). During storage, a very low amount of prematurely released drug molecules were detected (maximum of 7.5% for ApoDox prepared in PBS and 4.4% for ApoDox prepared in water). Fourier-transform infrared spectra revealed no significant differences in any of the samples after storage. Most of the ApoDox prepared in phosphate-buffered saline and ApoDox prepared in water and stored at -20°C formed very large aggregates (up to 487% of original size). Only ApoDox prepared in water and stored at 4°C showed no significant increase in size or shape. Although this storage caused slower internalization to LNCaP prostate cancer cells, ApoDox (2.5 μM of Dox) still retained its ability to inhibit completely the growth of 1.5×10 4 LNCaP cells after 72 hours. ApoDox stored at 20°C and 37°C in water was not able to deliver Dox inside the nucleus, and thus did not inhibit the growth of the LNCaP cells. Overall, our study demonstrates that ApoDox has very good stability over the course of 12 weeks when stored properly (at 4°C), and is thus suitable for use as a nanocarrier in the specific delivery of anticancer drugs to patients., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

X-ray structures of homopolymeric L-ferritin obtained by freezing protein crystals at increasing exposure times to a ferrous solution showed the progressive formation of a triiron cluster on the inner cage surface of each subunit. After 60 min exposure, a fully assembled (μ 3 -oxo)Tris[(μ 2 -peroxo)(μ 2 -glutamato-κ O :κ O ')](glutamato-κ O )(diaquo)triiron(III) anionic cluster appears in human L-ferritin. Glu60, Glu61, and Glu64 provide the anchoring of the cluster to the protein cage. Glu57 shuttles incoming iron ions toward the cluster. We observed a similar metallocluster in horse spleen L-ferritin, indicating that it represents a common feature of mammalian L-ferritins. The structures suggest a mechanism for iron mineral formation at the protein interface. The functional significance of the observed patch of carboxylate side chains and resulting metallocluster for biomineralization emerges from the lower iron oxidation rate measured in the E60AE61AE64A variant of human L-ferritin, leading to the proposal that the observed metallocluster corresponds to the suggested, but yet unobserved, nucleation site of L-ferritin.

The high stability and strong self-assembly properties made ferritins the most used proteins for nanotechnological applications. Human ferritins are made of 24 subunits of the H- and L-type that coassemble in an almost spherical nanocage 12nm across, delimiting a large cavity. The mechanism and kinetics of ferritin self-assembly and why H/L heteropolymers formation is favored over the homopolymers remain unclarified. In order to study this, we used the Fluorescence Resonance Energy Transfer (FRET) tool by binding multiple donor or acceptor Alexa Fluor fluorophores on the outer surface of human H and L ferritins and then denaturing and reassembling them in different proportions and conditions. The FRET efficiency increase from <0.3 of the disassembled to >0.7 in the assembled allowed to study the assembly kinetics. We found that their assembly was complete in about one hour, and that the initial rate of self-assembly of H/L heteropolymers was slightly faster than that of the H/H homopolymers. Then, by adding various proportions of unlabeled H or L-chains to the FRET system we found that the presence of the L-chains displaced the formation of H-H dimers more efficiently than that of the H-chains. This favored formation of H/L heterodimers, which is the initial step in ferritin self-assembly, contributes to explain the preferred formation of H/L heteropolymers over the H or L homopolymers. Moreover, we found that the H-chains arrange at distant positions on the heteropolymeric shell until they reach a number above eight, when they start to co-localize., (Copyright © 2016 Elsevier B.V. All rights reserved.)