Symmetrical protein cages have evolved to fulfil diverse roles in nature, including compartmentalization and cargo delivery
, and have inspired synthetic biologists to create novel protein assemblies ...via the precise manipulation of protein-protein interfaces. Despite the impressive array of protein cages produced in the laboratory, the design of inducible assemblies remains challenging
. Here we demonstrate an ultra-stable artificial protein cage, the assembly and disassembly of which can be controlled by metal coordination at the protein-protein interfaces. The addition of a gold (I)-triphenylphosphine compound to a cysteine-substituted, 11-mer protein ring triggers supramolecular self-assembly, which generates monodisperse cage structures with masses greater than 2 MDa. The geometry of these structures is based on the Archimedean snub cube and is, to our knowledge, unprecedented. Cryo-electron microscopy confirms that the assemblies are held together by 120 S-Au
-S staples between the protein oligomers, and exist in two chiral forms. The cage shows extreme chemical and thermal stability, yet it readily disassembles upon exposure to reducing agents. As well as gold, mercury(II) is also found to enable formation of the protein cage. This work establishes an approach for linking protein components into robust, higher-order structures, and expands the design space available for supramolecular assemblies to include previously unexplored geometries.
•Dopaminergic neurons dominate effective transverse relaxation in nigrosome 1.•Ion beam microscopy reveals highest iron concentrations in dopaminergic neurons.•Developed biophysical model links MRI ...parameters to cellular iron content.•Ferritin- and neuromelanin-bound iron impact MRI parameters differently.•Quantitative MRI provides a potential biomarker of iron in dopaminergic neurons.
In Parkinson’s disease, the depletion of iron-rich dopaminergic neurons in nigrosome 1 of the substantia nigra precedes motor symptoms by two decades. Methods capable of monitoring this neuronal depletion, at an early disease stage, are needed for early diagnosis and treatment monitoring. Magnetic resonance imaging (MRI) is particularly suitable for this task due to its sensitivity to tissue microstructure and in particular, to iron. However, the exact mechanisms of MRI contrast in the substantia nigra are not well understood, hindering the development of powerful biomarkers. In the present report, we illuminate the contrast mechanisms in gradient and spin echo MR images in human nigrosome 1 by combining quantitative 3D iron histology and biophysical modeling with quantitative MRI on post mortem human brain tissue. We show that the dominant contribution to the effective transverse relaxation rate (R2*) in nigrosome 1 originates from iron accumulated in the neuromelanin of dopaminergic neurons. This contribution is appropriately described by a static dephasing approximation of the MRI signal. We demonstrate that the R2* contribution from dopaminergic neurons reflects the product of cell density and cellular iron concentration. These results demonstrate that the in vivo monitoring of neuronal density and iron in nigrosome 1 may be feasible with MRI and provide directions for the development of biomarkers for an early detection of dopaminergic neuron depletion in Parkinson’s disease.
Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and photo-thermal therapy, but without enough evidence on their potentially adverse immunological effects. ...Using a model of human dendritic cells (DCs), we showed that 10 nm- and 50 nm-sized GNPs (GNP10 and GNP50, respectively) were internalized predominantly via dynamin-dependent mechanisms, and they both impaired LPS-induced maturation and allostimulatory capacity of DCs, although the effect of GNP10 was more prominent. However, GNP10 inhibited LPS-induced production of IL-12p70 by DCs, and potentiated their Th2 polarization capacity, while GNP50 promoted Th17 polarization. Such effects of GNP10 correlated with a stronger inhibition of LPS-induced changes in Ca2+ oscillations, their higher number per DC, and more frequent extra-endosomal localization, as judged by live-cell imaging, proton, and electron microscopy, respectively. Even when released from heat-killed necrotic HEp-2 cells, GNP10 inhibited the necrotic tumor cell-induced maturation and functions of DCs, potentiated their Th2/Th17 polarization capacity, and thus, impaired the DCs' capacity to induce T cell-mediated anti-tumor cytotoxicity in vitro. Therefore, GNP10 could potentially induce more adverse DC-mediated immunological effects, compared to GNP50.
Information about the surface topography is very important to achieve the correct elemental concentrations from the 2D X-ray intensity maps in micro-PIXE. Previously, an innovative approach of ...stereo-PIXE has been introduced to reconstruct the surface topography using two X-ray spectrometers in the lateral direction of the detector positions. Here, we developed the stereo-PIXE method to the four-segment Silicon drift detector (SDD), mounted at the Jožef Stefan Institute (JSI) microprobe, by considering a set of stereovision maps acquired by orthogonal segments to reconstruct the surface topography both in the lateral and longitudinal directions of the scanned area. To this aim, X-ray intensity disparity from the opposing segments was simulated for an ideal flat inclined model of the sample to infer the inclination angle across the opposing segments. Then, the obtained two gradient components of the surface topography from the inclination angles, along the axes that connect the two opposing segments, were reoriented along the scanned area with a proper rotation angle. Thus, to reconstruct the 3-D sample surface topography profile, the integration of the surface topography gradient was numerically performed in the two dimensions along the sample surface. The feasibility of the method is demonstrated on the pure Ti sample with scratched structures.
The increased modularity in the total hip arthroplasty (THA) allows adapting the prosthesis to the specific anatomical characteristics of each patient. However, the advantages of the modular THA are ...shadowed by the increased number of prosthesis failures observed in patients. The presence of junction in modular THA may increase its risk of mechanical failure. Moreover, the micro movements between neck and stem could lead to the production of metallic debris which may cause tissue inflammation and unsealing of prosthesis due to osseous dissolution. It is necessary to understand the mechanism of dispersion of the metal particles from the prosthesis into the tissue. Techniques currently applied in hospitals, such as X-ray scans or optical tissue microscopies, are able to distinguish metal particles, but unable to identify their specific metallic origin. In this work, within the TissueMaps project, we have proved that Proton Induced X-ray Emission (PIXE) is able to provide the distribution and elemental composition of particles from the prosthesis into the pseudo capsular tissue samples (near the femoral head) and identify the features observed under optical microscopy, in a case of broken neck prosthesis.
Porous tantalum has been extensively used in orthopaedic surgery, including uncemented total knee arthroplasty (TKA). Favourable results were reported with earlier monobloc tibial components and the ...design evolved to modular implants. We aimed to analyse possible causes for extensive medial tibia bone loss, resulting in modular porous tantalum tibia baseplate fracture after primary TKA. Retrieved tissue samples were scanned with 3 MeV focused proton beam for Proton-Induced X-ray Emission (micro-PIXE) elemental analysis. Fractographic and microstructural analysis were performed by stereomicroscopy. A full 3D finite-element model was made for numerical analysis of stress-strain conditions of the tibial baseplate. Histological examination of tissue underneath the broken part of the tibial baseplate revealed dark-stained metal debris, which was confirmed by micro-PIXE to consist of tantalum and titanium. Fractographic analysis and tensile testing showed that the failure of the tibial baseplate fulfilled the criteria of a typical fatigue fracture. Microstructural analysis of the contact surface revealed signs of bone ingrowth in 22.5% of the surface only and was even less pronounced in the medial half of the tibial baseplate. Further studies are needed to confirm the responsibility of metal debris for an increased bone absorption leading to catastrophic tibial tray failure.
We report on the analysis of the onset and the intensity of metastable secondary ions, which are desorbed from the target material as a result of bombarding organic samples with MeV primary ions. The ...bimodal time-of-flight mass spectrometer, which can analyze secondary ion in linear and reflectron modes, was used for detection and characterization of such ions. The use of the bimodal mass spectrometer for this specific purpose is demonstrated on amino acid arginine, where three main fragments were detected. We have also analyzed the influence of the primary ion beam on the intensity of metastable ion signal. Results from chlorine ion beams with energies between 3 and 10 MeV have exhibited the importance of electronic sputtering on the product/precursor ion peak intensity ratio, which is significantly decreased when using primary ions with higher energy.
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•Fragmented metastable ions from MeV-SIMS can be observed as long tails of molecular peaks in mass spectra.•Quick characterization of fragments is obtained through linear mode, and more precise identification by reflectron mode.•Fragments with intensities fragment/parent >0.01 can be detected.•Higher energy primary ion beams induce less metastable secondary ions.•Fragmentation pathways are not affected by the primary ion beam energy.
Genetic biofortification requires knowledge on natural variation and the underlying mechanisms of micronutrient accumulation. We therefore studied diversity in grain micronutrient concentrations and ...spatial distribution in barley (Hordeum vulgare), a genetically tractable model cereal and an important crop with widespread cultivation.
We assembled a diverse collection of barley cultivars and landraces and analysed grain micronutrient profiles in genebank material and after three independent cultivations. Lines with contrasting grain zinc (Zn) accumulation were selected for in-depth analysis of micronutrient distribution within the grain by micro-proton-induced X-ray emission (l-PIXE). Also, we addressed association with grain cadmium (Cd) accumulation.
The analysis of > 120 lines revealed substantial variation, especially in grain Zn concentrations. A large fraction of this variation is due to genetic differences. Grain dissection and l-PIXE analysis of contrasting lines showed that differences in grain Zn accumulation apply to all parts of the grain including the endosperm. Cd concentrations exceeded the Codex Alimentarius threshold in most of the representative barley lines after cultivation in a Cd-contaminated agricultural soil.
Two important conclusions for biofortification are: first, high-Zn grains contain more Zn also in the consumed parts of the grain; and second, higher micronutrient concentrations are strongly associated with higher Cd accumulation.
In the present work we have investigated the chemical sensitivity of a mass spectrometry imaging method MeV – SIMS. Primary ion beam within the MeV energy range domain was employed to bombard samples ...of two organic compounds; amino acid arginine, and peptide hormone angiotensin II (human), with average molecular weights of 174.2 u and 1046.2 u respectively. Secondary ion yield was measured as a function of number of molecules per area unit, and the detection limit was determined. For both molecular compounds and two different energies of primary 35Cl ion beam, the secondary ion yield exhibited a significant decrease below the area density of 1015 molecules/cm2, while the density of 1013 molecules/cm2 resulted in molecular peak / background ratio being lesser than 3, which is below the commonly used sensitivity threshold in other techniques. Other experiment related drawbacks to sensitivity were also discussed.
Human hair absorbs numerous biomolecules from the body during its growth. This can act as a fingerprint to determine substance intake of an individual, which can be useful in forensic studies. The ...cocaine concentration profile along the growth axis of hair indicates the time evolution of the metabolic incorporation of cocaine usage. It could be either assessed by chemical extraction and further analysis of hair bundels, or by direct single hair fibre analysis with mass spectroscopy imaging (MSI). Within this work, we analyzed the cocaine distribution in individual hair samples using MeV-SIMS. Unlike conventional surface analysis methods, we demonstrate high yields of nonfragmented molecular ions from the surface of biological materials, resulting in high chemical sensitivity and non-destructive characterisation. Hair samples were prepared by longitudinally cutting along the axis of growth, leaving half-cylindrical shape to access the interior structure of the hair by the probing ion beam, and attached to the silicon wafer. A focused 5.8 MeV 35Cl6+ beam was scanned across the intact, chemically pristine hair structure. A non-fragmented protonated M+ H+ cocaine molecular peak at m/z = 304 was detected and localized along the cross-section of the hair. Its intensity exhibits strong fluctuations along the direction of the hair's growth, with pronounced peaks as narrow as 50 micrometres, corresponding to a metabolic incorporation time of approx. three hours.
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