Apoferritin 20% glycerol, 200kV
Ontology highlight
ABSTRACT:
SUBMITTER: Benjamin Basanta
PROVIDER: EMPIAR-10849 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description: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.
| S-EPMC6119502 | biostudies-literature
Project description:Therapeutic outcome for the treatment of glioma was often limited due to the non-targeted nature of drugs and the physiological barriers, including the blood-brain barrier (BBB) and the blood-brain tumor barrier (BBTB). An ideal glioma-targeted delivery system must be sufficiently potent to cross the BBB and BBTB and then target glioma cells with adequate optimized physiochemical properties and biocompatibility. However, it is an enormous challenge to the researchers to engineer the above-mentioned features into a single nanocarrier particle. New frontiers in nanomedicine are advancing the research of new biomaterials. In this study, we demonstrate a strategy for glioma targeting by encapsulating vincristine sulfate (VCR) into a naturally available apoferritin nanocage-based drug delivery system with the modification of GKRK peptide ligand (GKRK-APO). Apoferritin (APO), an endogenous nanosize spherical protein, can specifically bind to brain endothelial cells and glioma cells via interacting with the transferrin receptor 1 (TfR1). GKRK is a peptide ligand of heparan sulfate proteoglycan (HSPG) over-expressed on angiogenesis and glioma, presenting excellent glioma-homing property. By combining the dual-targeting delivery effect of GKRK peptide and parent APO, GKRK-APO displayed higher glioma localization than that of parent APO. After loading with VCR, GKRK-APO showed the most favorable antiglioma effect in vitro and in vivo. These results demonstrated that GKRK-APO is an important potential drug delivery system for glioma-targeted therapy.
| S-EPMC6058491 | biostudies-literature
Project description:We present the optimization of experimental conditions to yield long, rigid apoferritin protein amyloid fibrils, as well as the corresponding fibrillation pathway. Fibril growth kinetics was followed using atomic force microscopy (AFM), transmission electron microscopy (TEM), dynamic light scattering (DLS), circular dichroism (CD), fourier-transform infrared spectroscopy (FTIR), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Among the morphologies identified, we show that the conditions result in small aggregates, as well as medium and long fibrils. Extended incubation times led to progressive unfolding and hydrolysis of the proteins into very short peptide fragments. AFM, SDS-PAGE, and CD support a universal common fibrillation mechanism in which hydrolyzed fragments play the central role. These collective results provide convincing evidence that protein unfolding and complete hydrolysis of the proteins into very short peptide sequences are essential for the formation of the final apoferritin amyloid-like fibrils.
| S-EPMC8462754 | biostudies-literature
Project description:BackgroundThe benzothiazole structure is important in medicinal chemistry, and 5-fluoro-2-(3,4-dimethoxyphenyl) benzothiazole (GW 610) is of particular interest as it shows outstanding anticancer activity in sensitive breast and colorectal carcinoma cell lines via generation of lethal DNA adducts in sensitive cancer cells. Despite promising activity, poor water solubility limits its applications. The apoferritin (AFt) protein cage has been proposed as a robust and biocompatible drug delivery vehicle.AimsHere, we aim to enhance solubility of GW 610 by developing amino acid prodrug conjugates and utilizing the AFt capsule as drug delivery vessel.Methods and resultsThe potent experimental antitumour agent, GW 610, has been successfully encapsulated within AFt with more than 190 molecules per AFt cage. The AFt-GW 610 complex exhibits dose-dependent growth inhibition and is more potent than GW 610 alone in 5/7 cancer cell lines. To enhance both aqueous solubility and encapsulation efficiency, a series of amino acid esters of GW 608 prodrug were synthesized via N,N'-dicyclohexylcarbodiimide ester coupling to produce molecules with different polarity. A dramatic increase in encapsulation efficiency was achieved, with more than 380 molecules of GW 608-Lys molecules per AFt cage. Release studies show sustained release of the cargo over 12 hours at physiologically relevant pH. The AFt-encapsulated amino acid modified GW 608 complexes are sequestered more rapidly and exhibit more potent anticancer activity than unencapsulated agent.ConclusionThese results indicate that AFt-encapsulation of GW 610 prodrug provides a biocompatible delivery option for this potent, selective experimental antitumour agent and for amino acid-modified GW 608. Of particular interest is the encapsulation efficiency and in vitro antitumour activity of AFt-GW 608-Lys, which warrants further preclinical evaluation.
| S-EPMC7941424 | biostudies-literature
Project description:The application of fluorescent proteins (FPs) in optoelectronics is hindered by the need for effective protocols to stabilize them under device preparation and operational conditions. Factors such as high temperatures, irradiation, and organic solvent exposure contribute to the denaturation of FPs, resulting in a low device performance. Herein, we focus on addressing the photoinduced heat generation associated with FP motion and rapid heat transfer. This leads to device temperatures of approximately 65 °C, causing FP-denaturation and a subsequent loss of device functionality. We present a FP stabilization strategy involving the integration of electrostatically self-assembled FP-apoferritin cocrystals within a silicone-based color down-converting filter. Three key achievements characterize this approach: (i) an engineering strategy to design positively supercharged FPs (+22) without compromising photoluminescence and thermal stability compared to their native form, (ii) a carefully developed crystallization protocol resulting in highly emissive cocrystals that retain the essential photoluminescence features of the FPs, and (iii) a strong reduction of the device's working temperature to 40 °C, leading to a 40-fold increase in Bio-HLEDs stability compared to reference devices.
| S-EPMC10684032 | biostudies-literature