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.
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.
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.
Phosphorus (P) deficiency limits crop production worldwide. Crops differ in their ability to acquire and utilise the P available. The aim of this study was to determine root traits (root exudates, ...root system architecture (RSA), tissue-specific allocation of P, and gene expression in roots) that (a) play a role in P-use efficiency and (b) contribute to large shoot zinc (Zn) concentration in Brassica oleracea.
Two B. oleracea accessions (var. sabellica C6, a kale, and var. italica F103, a broccoli) were grown in a hydroponic system or in a high-throughput-root phenotyping (HTRP) system where they received Low P (0.025 mM) or High P (0.25 mM) supply for 2 weeks. In hydroponics, root and shoot P and Zn concentrations were measured, root exudates were profiled using both Fourier-Transform-Infrared spectroscopy and gas-chromatography-mass spectrometry and previously published RNAseq data from roots was re-examined. In HTRP experiments, RSA (main and lateral root number and lateral root length) was assessed and the tissue-specific distribution of P was determined using micro-particle-induced-X-ray emission. The C6 accession had greater root and shoot biomass than the F103 accession, but the latter had a larger shoot P concentration than the C6 accession, regardless of the P supply in the hydroponic system. The F103 accession had a larger shoot Zn concentration than the C6 accession in the High P treatment. Although the F103 accession had a larger number of lateral roots, which were also longer than in the C6 accession, the C6 accession released a larger quantity and number of polar compounds than the F103 accession. A larger number of P-responsive genes were found in the Low P treatment in roots of the F103 accession than in roots of the C6 accession. Expression of genes linked with "phosphate starvation" was up-regulated, while those linked with iron homeostasis were down-regulated in the Low P treatment.
The results illustrate large within-species variability in root acclimatory responses to P supply in the composition of root exudates, RSA and gene expression, but not in P distribution in root cross sections, enabling P sufficiency in the two B. oleracea accessions studied.
MeV-SIMS is an emerging mass spectrometry imaging method, which utilizes fast, heavy ions to desorb secondary molecules. High yields and low fragmentation rates of large molecules, associated with ...the electronic sputtering process, make it particularly useful in biomedical research, where insight into distribution of organic molecules is needed. Since the implementation of MeV-SIMS in to the micro-beam line at the tandem accelerator of Jožef Stefan Institute, MeV-SIMS provided some valuable observations on the distribution of biomolecules in plant tissue, as discussed by Jenčič et al. (Nucl. Inst. Methods Phys. Res. B.
371
, 205–210,
2016
; Nucl. Inst. Methods Phys. Res. B.
404
, 140–145, 2017). However, limited focusing ability of the chlorine ion beam only allowed imaging at the tissue level. In order to surpass shortcomings of the existing method, we introduced a new approach, where we employ a continuous, low-current primary beam. In this mode, we bombard thin samples with a steady chlorine ion flux of approx. 5000 ions/s. After desorbing molecules, chlorine ions penetrate through the thinly cut sample and trigger the time-of-flight “start” signal on a continuous electron multiplier detector, positioned behind the sample. Such bombardment is more effective than previously used pulsing-beam mode, which demanded several orders of magnitude higher primary ion beam currents. Sub-micrometer focusing of low-current primary ion beam allows imaging of biological tissue on a subcellular scale. Simultaneously, new time-of-flight acquisition approach also improves mass resolution by a factor of 5. Within the article, we compare the performance of both methods and demonstrate the application of continuous mode on biological tissue. We also describe the thin sample preparation protocol, necessary for measurements with low primary ion currents.
The metallic-associated adverse local tissue reactions (ALTR) and events accompanying worn-broken implant materials are still poorly understood on the subcellular and molecular level. Current ...immunohistochemical techniques lack spatial resolution and chemical sensitivity to investigate causal relations between material and biological response on submicron and even nanoscale. In our study, new insights of titanium alloy debris-tissue interaction were revealed by the implementation of label-free high-resolution correlative microscopy approaches. We have successfully characterized its chemical and biological impact on the periprosthetic tissue obtained at revision surgery of a fractured titanium-alloy modular neck of a patient with hip osteoarthritis. We applied a combination of photon, electron and ion beam micro-spectroscopy techniques, including hybrid optical fluorescence and reflectance micro-spectroscopy, scanning electron microscopy (SEM), Energy-dispersive X-ray Spectroscopy (EDS), helium ion microscopy (HIM) and micro-particle-induced X-ray emission (micro-PIXE). Micron-sized wear debris were found as the main cause of the tissue oxidative stress exhibited through lipopigments accumulation in the nearby lysosome. This may explain the indications of chronic inflammation from prior histologic examination. Furthermore, insights on extensive fretting and corrosion of the debris on nm scale and a quantitative measure of significant Al and V release into the tissue together with hydroxyapatite-like layer formation particularly bound to the regions with the highest Al content were revealed. The functional and structural information obtained at molecular and subcellular level contributes to a better understanding of the macroscopic inflammatory processes observed in the tissue level. The established label-free correlative microscopy approach can efficiently be adopted to study any other clinical cases related to ALTR.
Increasing the concentration of an element in edible produce (i.e., biofortification) can mitigate the element deficiency in humans. Sprouts are small but popular part of healthy diets providing ...vitamins and essential elements throughout the year. Element composition of sprouts can easily be amended, e.g., by soaking the grains in element-rich solution before germination (grain-priming). In addition, pre-treatment of grains to improve element translocation from the solution into the grain may further enhance the element concentration in the sprout. Cold plasma technique could provide such solution, as it increases wettability and water uptake of grains. Grains of common buckwheat (
Moench) were pre-treated/ untreated with cold plasma and soaked in ZnCl
solution/pure water. Germination tests, α-amylase activity, grain hydrophilic properties and water uptake were assessed. Element composition of grain tissues and of sprouts was assessed by micro-particle-induced-X-ray emission and X-ray fluorescence spectroscopy, respectively. Grain-priming increased Zn concentration in shoots of common buckwheat sprouts more than five-times, namely from 79 to 423 mg Zn kg
dry weight. Cold plasma treatment increased grain wettability and water uptake into the grain. However, cold plasma pre-treatment followed by grain-priming with ZnCl
did not increase Zn concentration in different grain tissues or in the sprouts more than the priming alone, but rather decreased the Zn concentration in sprout shoots (average ± standard error: 216 ± 6.13 and 174 ± 7.57 mg Zn kg
dry weight, respectively). When the fresh weight portion of whole sprouts (i.e., of roots and shoots) was considered, comparable average requirements of Zn, namely 24.5 % and 35 % for adult men and women would be satisfied by consuming cold plasma pre-treated and not pre-treated grains. Potential advantages of cold plasma pre-treatment need to be tested further, mainly to optimize the duration of soaking required to produce Zn-enriched sprouts.
Halophytes are able to tolerate relatively high concentrations of hazardous metals in a growing substrate, what makes them suitable candidates for phytoremediation of metal-contaminated soils. In ...this work, we aimed to study the physiological responses of the halophyte
Sesuvium portulacastrum
L. to Ni, with main focus on Ni localization, compartmentation and ligand environment, to decipher Ni tolerance and toxicity mechanisms. Seedlings were grown in hydroponic nutrient solution containing 0, 25, 50 and 100 μM Ni as NiCl
2
for 3 weeks. Ni localization in leaves was assessed by micro-proton-induced X-ray emission (micro-PIXE). Ni ligand environment was studied by Ni K-edge X-ray absorption near edge structure (XANES). In addition, Ni-soluble, weakly bound/exchangeable and insoluble leaf tissue fractions were determined by sequential extraction. Results show that
S. portulacastrum
is able to tolerate up to ~ 500 μg g
−1
dry weight (DW) of Ni in the shoots without significant growth reduction. At higher Ni concentrations (> 50 μM Ni in nutrient solution), chloroses were observed due to the accumulation of Ni in photosynthetically active chlorenchyma as revealed by micro-PIXE. Water storage tissue represented the main pool for Ni storage. Incorporation of Ni into Ca-oxalate crystals was also observed in some specimens, conferring tolerance to high leaf Ni concentrations. The majority of Ni (> 70%) was found in soluble tissue fraction. Ni K XANES revealed Ni bound mainly to O- (55%) and N-ligands (45%). Ni toxicity at higher Ni levels was associated with Ni binding to amino groups of proteins in cytosol of chlorenchyma and increased level of lipid peroxidation. Proline levels also increased at high Ni exposures and were associated with Ni-induced oxidative stress and alteration of water regime.
The aim was to determine how the nutritional composition of Tartary buckwheat (Fagopyrum tataricum Gaertn.) and wheat (Triticum aestivum L.) sprouts is affected by the mineral composition of ...different waters used during their cultivation. We used tap water (TW) and two mineral-rich waters (MRWs), namely moderately mineral-rich water (MMRW) and extremely mineral-rich water (EMRW) originating from springs that contain naturally present mineral elements. Grain germination was not negatively affected by MRWs, however EMRW impeded radicle growth, and consequently prevented sprout development. In comparison to cultivation in TW, cultivation in MMRW resulted in higher Na, Mg, K and Mn concentrations in both sprouts. There were no water-related effects on distribution of mineral elements within plant species, however there were differences in Ca distribution. In Tartary buckwheat Ca was located in inter-vasculature mesophyll, presumably as oxalate crystals. In wheat Ca predominated in epidermis. Only in Tartary buckwheat cultivation in MMRW resulted in less dietary fibre and catechin and more quercetin. By capturing compositional profiles of mineral elements and bioactive compounds in Tartary buckwheat and wheat sprouts we identified the potential for selective enhancement of MMRW. We suggest further work using different spring MRWs to identify optimal conditions for cultivation of different sprouts.
•Successful sprout development depended on the mineral element (ME) composition of waters.•ME concentration but not distribution was affected by ME composition of water.•Total dietary fibre was affected by ME composition of waters only in Tartary buckwheat sprouts.•Quercetin and catechin, but not rutin concentration depended on ME composition of waters.
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