Cholesterol Slows down the Lateral Mobility of an Oxidized Phospholipid in a Supported Lipid Bilayer

We investigated the mobility and phase-partitioning of the fluorescent oxidized phospholipid analogue 1-palmitoyl-2-glutaroyl-sn-glycero-3-phospho-N-Alexa647-ethanolamine (PGPE-Alexa647) in supported lipid bilayers. Compared to the conventional phospholipid dihexadecanoylphosphoethanolamine (DHPE)-Bodipy we found consistently higher diffusion constants. The effect become dramatic when immobile obstacles were inserted into the bilayer. which essentially blocked the diffusion of DHPE-Bodipy but hardly influenced the movements of PGPE-Alexa647. In a supported lipid bilayer made of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), the differences in probe mobility leveled off with increasing cholesterol content. Using coarse-grained molecular dynamics simulations, we could ascribe this effect to increased interactions between the oxidized phospholipid and the membrane matrix, concomitant with a translation in the headgroup position of the oxidized phospholipid: at zero cholesterol content, its headgroup is shifted to the outside of the DOPC headgroup region, whereas increasing cholesterol concentrations pulls the headgroup into the bilayer plane

LHC Beam Loss Detector Design: Simulations and Measurements

The Beam Loss Monitoring (BLM) system is integrated in the active equipment protection system of the LHC. It determines the number of particles lost from the primary hadron beam by measuring the radiation field of the shower particles outside of the vacuum chamber. The LHC BLM system will use ionization chambers as its standard detectors but in the areas where very high dose rates are expected, the Secondary Emission Monitor (SEM) chambers will be additionally employed because of their high linearity, low sensitivity and fast response.The sensitivity of the SEM was modeled in Geant4 via the Photo-Absorption Ionization module together with custom parameterization of the very low energy secondary electron production. The prototypes were calibrated by proton beams. For the calibration of the BLM system the signal response of the ionization chamber is simulated in Geant4 for all relevant particle types and energies (keV to TeV range). The results are validated by comparing the simulations to measurements using protons, neutrons, photons and mixed radiation fields at various energies and intensities

Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding

UID/Multi/04378/2019 Grant no. BB/P010660/1 grant number BB/M011216/1Saturation-transfer difference (STD) NMR spectroscopy is a fast and versatile method which can be applied for drug-screening purposes, allowing the determination of essential ligand binding affinities (KD). Although widely employed to study soluble proteins, its use remains negligible for membrane proteins. Here the use of STD NMR for KD determination is demonstrated for two competing substrates with very different binding affinities (low nanomolar to millimolar) for an integral membrane transport protein in both detergent-solubilised micelles and reconstituted proteoliposomes. GltPh, a homotrimeric aspartate transporter from Pyrococcus horikoshii, is an archaeal homolog of mammalian membrane transport proteins—known as excitatory amino acid transporters (EAATs). They are found within the central nervous system and are responsible for fast uptake of the neurotransmitter glutamate, essential for neuronal function. Differences in both KD’s and cooperativity are observed between detergent micelles and proteoliposomes, the physiological implications of which are discussed.publishersversionpublishe

Adhesion of Escherichia coli under flow conditions reveals potential novel effects of FimH mutations

FimH-mediated adhesion of Escherichia coli to bladder epithelium is a prerequisite for urinary tract infections. FimH is also essential for blood-borne bacterial dissemination, but the mechanisms are poorly understood. The purpose of this study was to assess the influence of different FimH mutations on bacterial adhesion using a novel adhesion assay, which models the physiological flow conditions bacteria are exposed to. We introduced 12 different point mutations in the mannose binding pocket of FimH in an E. coli strain expressing type 1 fimbriae only (MSC95-FimH). We compared the bacterial adhesion of each mutant across several commonly used adhesion assays, including agglutination of yeast, adhesion to mono- and tri-mannosylated substrates, and static adhesion to bladder epithelial and endothelial cells. We performed a comparison of these assays to a novel method that we developed to study bacterial adhesion to mammalian cells under flow conditions. We showed that E. coli MSC95-FimH adheres more efficiently to microvascular endothelium than to bladder epithelium, and that only endothelium supports adhesion at physiological shear stress. The results confirmed that mannose binding pocket mutations abrogated adhesion. We demonstrated that FimH residues E50 and T53 are crucial for adhesion under flow conditions. The coating of endothelial cells on biochips and modelling of physiological flow conditions enabled us to identify FimH residues crucial for adhesion. These results provide novel insights into screening methods to determine the effect of FimH mutants and potentially FimH antagonists. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10096-016-2820-8) contains supplementary material, which is available to authorized users

Is transcranial sonography useful to distinguish scans without evidence of dopaminergic deficit patients from Parkinson's disease?

BACKGROUND: Approximately 10% of patients clinically diagnosed with early Parkinson's disease (PD) subsequently have normal dopaminergic functional imaging. Transcranial sonography (TCS) has been shown to detect midbrain hyperechogenicity in approximately 90% of Parkinson's disease (PD) patients and 10% of the healthy population. The aim of this study was to investigate the prevalence of midbrain hyperechogenicity in patients with suspected parkinsonism and scans without evidence of dopaminergic deficit (SWEDD), in comparison to PD patients. METHODS: TCS was performed in 14 patients with SWEDD and 19 PD patients. RESULTS: There was a significantly increased area of echogenicity in the PD group (0.24 ± 0.06 cm(2) ), compared to the group of patients with SWEDD (0.13 ± 0.06 cm(2) ; P < 0.001). One (9.1%) of these patients, compared to 14 (82.5%) of the PD patients, was found to have hyperechogenicity (P < 0.001). CONCLUSIONS: We conclude that TCS is useful to distinguish PD patients from patients with suspected parkinsonism and SWEDD

The LHC Beam Loss Measurement System

An unprecedented amount of energy will be stored in the circulating beams of LHC. The loss of even a very small fraction of a beam may induce a quench in the superconducting magnets or cause physical damage to machine components. A fast (one turn) loss of 3 . 10-9 and a constant loss of 3 . 10-12 times the nominal beam intensity can quench a dipole magnet. A fast loss of 3 . 10-6 times nominal beam intensity can damage a magnet. The stored energy in the LHC beam is a factor of 200 (or more) higher than in existing hadron machines with superconducting magnets (HERA, TEVATRON, RHIC), while the quench levels of the LHC magnets are a factor of about 5 to 20 lower than the quench levels of these machines. To comply with these requirements the detectors, ionisation chambers and secondary emission monitors are designed very reliable with a large operational range. Several stages of the acquisition chain are doubled and frequent functionality tests are automatically executed. The failure probabilities of single components were identified and optimised. First measurements show the large dynamic range of the system

The Environment Shapes the Inner Vestibule of LeuT

Human neurotransmitter transporters are found in the nervous system terminating synaptic signals by rapid removal of neurotransmitter molecules from the synaptic cleft. The homologous transporter LeuT, found in Aquifex aeolicus, was crystallized in different conformations. Here, we investigated the inward-open state of LeuT. We compared LeuT in membranes and micelles using molecular dynamics simulations and lanthanide-based resonance energy transfer (LRET). Simulations of micelle-solubilized LeuT revealed a stable and widely open inward-facing conformation. However, this conformation was unstable in a membrane environment. The helix dipole and the charged amino acid of the first transmembrane helix (TM1A) partitioned out of the hydrophobic membrane core. Free energy calculations showed that movement of TM1A by 0.30 nm was driven by a free energy difference of similar to 15 kJ/mol. Distance measurements by LRET showed TM1A movements, consistent with the simulations, confirming a substantially different inward-open conformation in lipid bilayer from that inferred from the crystal structure

Analysis of sequence divergence in mammalian abcgs predicts a structural network of residues that underlies functional divergence

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. The five members of the mammalian G subfamily of ATP-binding cassette transporters differ greatly in their substrate specificity. Four members of the subfamily are important in lipid transport and the wide substrate specificity of one of the members, ABCG2, is of significance due to its role in multidrug resistance. To explore the origin of substrate selectivity in members 1, 2, 4, 5 and 8 of this subfamily, we have analysed the differences in conservation between members in a multiple sequence alignment of ABCG sequences from mammals. Mapping sets of residues with similar patterns of conservation onto the resolved 3D structure of ABCG2 reveals possible explanations for differences in function, via a connected network of residues from the cytoplasmic to transmembrane domains. In ABCG2, this network of residues may confer extra conformational flexibility, enabling it to transport a wider array of substrates

Effects of experimentally added salmon subsidies on resident fishes via direct and indirect pathways

Artificial additions of nutrients of differing forms such as salmon carcasses and analog pellets (i.e. pasteurized fishmeal) have been proposed as a means of stimulating aquatic productivity and enhancing populations of anadromous and resident fishes. Nutrient mitigation to enhance fish production in stream ecosystems assumes that the central pathway by which effects occur is bottom-up, through aquatic primary and secondary production, with little consideration of reciprocal aquatic-terrestrial pathways. The net outcome (i.e. bottom-up vs. top-down) of adding salmon-derived materials to streams depend on whether or not these subsidies indirectly intensify predation on in situ prey via increases in a shared predator or alleviate such predation pressure. We conducted a 3-year experiment across nine tributaries of the N. Fork Boise River, Idaho, USA, consisting of 500-m stream reaches treated with salmon carcasses (n = 3), salmon carcass analog (n = 3), and untreated control reaches (n = 3). We observed 2–8 fold increases in streambed biofilms in the 2–6 weeks following additions of both salmon subsidy treatments in years 1 and 2 and a 1.5-fold increase in standing crop biomass of aquatic invertebrates to carcass additions in the second year of our experiment. The consumption of benthic invertebrates by stream fishes increased 110–140% and 44–66% in carcass and analog streams in the same time frame, which may have masked invertebrate standing crop responses in years 3 and 4. Resident trout directly consumed 10.0–24.0 g·m-2·yr-1 of salmon carcass and \u3c1–11.0 g·m-2·yr-1 of analog material, which resulted in 1.2–2.9 g·m-2·yr-1 and 0.03–1.4 g·m-2·yr-1 of tissue produced. In addition, a feedback flux of terrestrial maggots to streams contributed 0.0–2.0 g·m-2·yr-1 to trout production. Overall, treatments increased annual trout production by 2–3 fold, though density and biomass were unaffected. Our results indicate the strength of bottom-up and top-down responses to subsidy additions was asymmetrical, with top-down forces masking bottom-up effects that required multiple years to manifest. The findings also highlight the need for nutrient mitigation programs to consider multiple pathways of energy and nutrient flow to account for the complex effects of salmon subsidies in stream-riparian ecosystems