Disposable MMP-9 sensor based on the degradation of peptide cross-linked hydrogel films using electrochemical impedance spectroscopy

Barts and The London Charity and Queen Mary Innovation Lt

Hypersatellite Kα Production in Trapped Ar Ions at KK Trielectronic Recombination Energies

Funding Information: W.B.-N. acknowledges DSC Grant MNiSzW Nr 7150/E-338/M/2018, the GET_INvolved Programme at FAIR/GSI (www.fair-center.eu/get_involved) and the JIPhD program through contract POWR.03.05.00-00-Z309/17-00. D.S.L.M. acknowledges the Kosciuszko Foundation. P.A and F.G acknowledge the FCT through project number UID/04559/2020 (LIBPhys). Publisher Copyright: © 2023 by the authors.We report measurements of hypersatellite radiation of argon ions in the electron energy region of 5200 eV to 7500 eV. Here, we observed a strong enhancement of this hypersatellite  (Formula presented.)  production. Trielectronic recombination (TR) is discussed as a possible channel for  (Formula presented.)  production leading to this enhancement where main TR resonances are expected to occur. Data analysis was mainly based on the extracted intensity ratio of hypersatellite  (Formula presented.)  to  (Formula presented.)  lines ((Formula presented.)). In addition, the collisional excitation and the collisional ionisation of the K-shell ions were modeled as main background processes of the K (Formula presented.)  X-ray production. The  (Formula presented.) / (Formula presented.)  intensity ratio shows a significant rise around 6500 eV electron energy by a factor of about two above the background level. This observation is compared with calculations of the expected electron energies for the resonant  (Formula presented.)  emission due to the KK TR process. The observed rise as a function of the electron collision energy, which occurs in the vicinity of the predicted TR resonances, is significantly stronger and energetically much wider than the results of theoretical calculations for the TR process. However, the experimental evidence of this process is not definitive.publishersversionpublishe

Chemically induced protein cage assembly with programmable opening and cargo release

Engineered protein cages are promising tools that can be customized for applications in medicine and nanotechnology. A major challenge is developing a straightforward strategy for endowing cages with bespoke, inducible disassembly. Such cages would allow release of encapsulated cargoes at desired timing and location. Here, we achieve such programmable disassembly using protein cages, in which the subunits are held together by different molecular cross-linkers. This modular system enables cage disassembly to be controlled in a condition-dependent manner. Structural details of the resulting cages were determined using cryo–electron microscopy, which allowed observation of bridging cross-linkers at intended positions. Triggered disassembly was demonstrated by high-speed atomic force microscopy and subsequent cargo release using an encapsulated Förster resonance energy transfer pair whose signal depends on the quaternary structure of the cage

A Specific Activity-Based Probe to Monitor Family GH59 Galactosylceramidase, the Enzyme Deficient in Krabbe Disease

Galactosylceramidase (GALC) is the lysosomal β-galactosidase responsible for the hydrolysis of galactosylceramide. Inherited deficiency in GALC causes Krabbe disease, a devastating neurological disorder characterized by accumulation of galactosylceramide and its deacylated counterpart, the toxic sphingoid base galactosylsphingosine (psychosine). We report the design and application of a fluorescently tagged activity-based probe (ABP) for the sensitive and specific labeling of active GALC molecules from various species. The probe consists of a β-galactopyranose-configured cyclophellitol-epoxide core, conferring specificity for GALC, equipped with a BODIPY fluorophore at C6 that allows visualization of active enzyme in cells and tissues. Detection of residual GALC in patient fibroblasts holds great promise for laboratory diagnosis of Krabbe disease. We further describe a procedure for in situ imaging of active GALC in murine brain by intra-cerebroventricular infusion of the ABP. In conclusion, this GALC-specific ABP should find broad applications in diagnosis, drug development, and evaluation of therapy for Krabbe disease

Gating of aquaporins by heavy metals in Allium cepa L. epidermal cells

Changes in the water permeability, aquaporin (AQP) activity, of leaf cells were investigated in response to different heavy metals (Zn2+, Pb2+, Cd2+, Hg2+). The cell pressure probe experiments were performed on onion epidermal cells as a model system. Heavy metal solutions at different concentrations (0.05 μM–2 mM) were used in our experiments. We showed that the investigated metal ions can be arranged in order of decreasing toxicity (expressed as a decrease in water permeability) as follows: Hg>Cd>Pb>Zn. Our results showed that β-mercaptoethanol treatment (10 mM solution) partially reverses the effect of AQP gating. The magnitude of this reverse differed depending on the metal and its concentration. The time course studies of the process showed that the gating of AQPs occurred within the first 10 min after the application of a metal. We also showed that after 20–40 min from the onset of metal treatment, the water flow through AQPs stabilized and remained constant. We observed that irrespective of the metal applied, the effect of AQP gating can be recorded within the first 10 min after the administration of metal ions. More generally, our results indicate that the toxic effects of investigated metal ions on the cellular level may involve AQP gating

Structure and Stability of the Spinach Aquaporin SoPIP2;1 in Detergent Micelles and Lipid Membranes

Background: SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1. Methodology/Principal Finding: We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-beta-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly a-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58 degrees C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70 degrees C. Conclusion/Significance: The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications

FIB patterning of stainless steel for the development of nano-structured stent surfaces for cardiovascular applications

Stent implantation is a percutaneous interventional procedure that mitigates vessel stenosis, providing mechanical support within the artery and as such a very valuable tool in the fight against coronary artery disease. However, stenting causes physical damage to the arterial wall. It is well accepted that a valuable route to reduce in-stent re-stenosis can be based on promoting cell response to nano-structured stainless steel (SS) surfaces such as by patterning nano-pits in SS. In this regard patterning by focused ion beam (FIB) milling offers several advantages for flexible prototyping. On the other hand FIB patterning of polycrystalline metals is greatly influenced by channelling effects and redeposition. Correlative microscopy methods present an opportunity to study such effects comprehensively and derive structure–property understanding that is important for developing improved patterning. In this chapter we present a FIB patterning protocol for nano-structuring features (concaves) ordered in rectangular arrays on pre-polished 316L stainless steel surfaces. An investigation based on correlative microscopy approach of the size, shape and depth of the developed arrays in relation to the crystal orientation of the underlying SS domains is presented. The correlative microscopy protocol is based on cross-correlation of top-view scanning electron microscopy, electron backscattering diffraction, atomic force microscopy and cross-sectional (serial) sectioning. Various FIB tests were performed, aiming at improved productivity by preserving nano-size accuracy of the patterned process. The optimal FIB patterning conditions for achieving reasonably high throughput (patterned rate of about 0.03 mm2/h) and nano-size accuracy in dimensions and shapes of the features are discussed as well

"A Theoretical Perspective on Multi-level Systems in Europe: Constitutional Power and Partisan Conflict"

types: ArticleThis article distinguishes three constitutionally defined categories of multi-level systems – confederations, federal arrangements and regionalized arrangements, which differ in whether their lower-level governments enjoy constitutional protection and whether we find a constitutional hierarchy between central and lower levels of government. We argue that the constitutional category a multi-level system belongs to systematically shapes first, the dominant mode of day-to-day intergovernmental coordination, second, the mode of formal competence (re)allocation; and third, the relative impact of party (in)congruence across central and lower-level governments on these coordination processes, respectively. The article then specifies the indicators used to test the hypotheses across the range of case studies. It finally shows how the multi-level systems covered in this special issue span the confederal – federal – regionalized spectrum and thus allow for an encompassing comparative assessment of multi-level dynamics and their long-term evolution.ESR