Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor.

Cell-free expression represents an attractive method to produce large quantities of selectively labeled protein for NMR applications. Here, cell-free expression was used to label specific regions of the growth hormone secretagogue receptor (GHSR) with NMR-active isotopes. The GHSR is a member of the class A family of G protein-coupled receptors.

Towards the molecular architecture of the peroxisomal receptor docking complex.

Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo.

The Dynamics of the Neuropeptide Y Receptor Type 1 Investigated by Solid-State NMR and Molecular Dynamics Simulation.

We report data on the structural dynamics of the neuropeptide Y (NPY) G-protein-coupled receptor (GPCR) type 1 (Y1R), a typical representative of class A peptide ligand GPCRs, using a combination of solid-state NMR and molecular dynamics (MD) simulation. First, the equilibrium dynamics of Y1R were studied using N-NMR and quantitative determination of H-C order parameters through the measurement of dipolar couplings in separated-local-field NMR experiments. Order parameters reporting the amplitudes of the molecular motions of the C-H bond vectors of Y1R in DMPC membranes are 0.

Reduced lipolysis in lipoma phenocopies lipid accumulation in obesity.

Background: Elucidation of lipid metabolism and accumulation mechanisms is of paramount importance to understanding obesity and unveiling therapeutic targets. In vitro cell models have been extensively used for these purposes, yet, they do not entirely reflect the in vivo setup. Conventional lipomas, characterized by the presence of mature adipocytes and increased adipogenesis, could overcome the drawbacks of cell cultures.

Binding of the small-molecule kinase inhibitor ruxolitinib to membranes does not disturb membrane integrity.

Ruxolitinib is a small-molecule protein kinase inhibitor, which is used as a therapeutic agent against several diseases. Due to its anti-inflammatory impact, ruxolitinib has also been considered recently for usage in the treatment of Covid-19. While the specific effects of ruxolitinib on Janus kinases (JAK) is comparatively well investigated, its (unspecific) impact on membranes has not been studied in detail so far.

Serotonin Alters the Phase Equilibrium of a Ternary Mixture of Phospholipids and Cholesterol.

Unsaturated and saturated phospholipids tend to laterally segregate, especially in the presence of cholesterol. Small molecules such as neurotransmitters, toxins, drugs etc. possibly modulate this lateral segregation.

A fully automated high-throughput workflow for 3D-based chemical screening in human midbrain organoids.

Three-dimensional (3D) culture systems have fueled hopes to bring about the next generation of more physiologically relevant high-throughput screens (HTS). However, current protocols yield either complex but highly heterogeneous aggregates ('organoids') or 3D structures with less physiological relevance ('spheroids'). Here, we present a scalable, HTS-compatible workflow for the automated generation, maintenance, and optical analysis of human midbrain organoids in standard 96-well-plates.

Membrane Interaction of Ibuprofen with Cholesterol-Containing Lipid Membranes.

Deciphering the membrane interaction of drug molecules is important for improving drug delivery, cellular uptake, and the understanding of side effects of a given drug molecule. For the anti-inflammatory drug ibuprofen, several studies reported contradictory results regarding the impact of ibuprofen on cholesterol-containing lipid membranes. Here, we investigated membrane localization and orientation as well as the influence of ibuprofen on membrane properties in POPC/cholesterol bilayers using solid-state NMR spectroscopy and other biophysical assays.

Phytochemical Analysis, In Vitro Anti-Inflammatory and Antimicrobial Activity of Piliostigma thonningii Leaf Extracts from Benin.

The leaves of are used in traditional medicine in Benin to treat inflammatory skin diseases and infections. So far, pharmacological studies of the anti-inflammatory and anti-infective effects of phytochemically characterized extracts of have been very limited. Therefore, we investigated the anti-inflammatory and antimicrobial effect of leaf extracts and analyzed the phytochemical composition of extracts of different polarities (water, 50% ethanol, and -hexane).

De novo synthesis of phospholipids and sphingomyelin in multipotent stromal cells - Monitoring studies by mass spectrometry.

Musculoskeletal diseases are extremely widespread and a significant burden on the health systems of the industrialized countries. The use of mesenchymal stromal cells is a promising approach to cure cartilage and tendon injuries, which often also occur in younger people as consequences of sport accidents. Although particular interest is on the collagen and the glycosaminoglycan composition of the tendon and potential alterations compared to healthy tissue, there is nowadays also increasing evidence that some selected phospholipids (PL) are potential mediators of tissue regeneration.

Calcium-sensing receptor-mediated NLRP3 inflammasome response to calciprotein particles drives inflammation in rheumatoid arthritis.

Increased extracellular Ca concentrations ([Ca]) trigger activation of the NLRP3 inflammasome in monocytes through calcium-sensing receptor (CaSR). To prevent extraosseous calcification in vivo, the serum protein fetuin-A stabilizes calcium and phosphate into 70-100 nm-sized colloidal calciprotein particles (CPPs). Here we show that monocytes engulf CPPs via macropinocytosis, and this process is strictly dependent on CaSR signaling triggered by increases in [Ca].

Light-induced lipid mixing implies a causal role of lipid splay in membrane fusion.

The fusion of lipid membranes is central to many biological processes and requires substantial structural reorganization of lipids brought about by the action of fusogenic proteins. Previous molecular dynamics simulations have suggested that splayed lipids, whose tails transiently contact the headgroup region of the bilayer, initiate lipid mixing. Here, we explore the lipid splay hypothesis experimentally.

Interaction of the small-molecule kinase inhibitors tofacitinib and lapatinib with membranes.

Lapatinib and tofacitinib are small-molecule kinase inhibitors approved for the treatment of advanced or metastatic breast cancer and rheumatoid arthritis, respectively. So far, the mechanisms which are responsible for their activities are not entirely understood. Here, we focus on the interaction of these drug molecules with phospholipid membranes, which has not yet been investigated before in molecular detail.

Tuberculosis causes highly conserved metabolic changes in human patients, mycobacteria-infected mice and zebrafish larvae.

Tuberculosis is a highly infectious and potentially fatal disease accompanied by wasting symptoms, which cause severe metabolic changes in infected people. In this study we have compared the effect of mycobacteria infection on the level of metabolites in blood of humans and mice and whole zebrafish larvae using one highly standardized mass spectrometry pipeline, ensuring technical comparability of the results. Quantification of a range of circulating small amines showed that the levels of the majority of these compounds were significantly decreased in all three groups of infected organisms.

Structural characteristics of oligomers formed by pyroglutamate-modified amyloid β peptides studied by solid-state NMR.

Neuronal plaques of amyloid β (Aβ) peptides of varying length carrying different posttranslational modifications represent a molecular hallmark of Alzheimer's disease. It is believed that transient oligomeric Aβ assemblies associating in early fibrillation events represent particularly cytotoxic peptide aggregates. Also, N-terminally truncated (in position 3 or 11) and pyroglutamate modified peptides exhibited an increased toxicity compared to the wildtype.

Dual Transport of Active Substances with a Layer-by-Layer-Based Drug Delivery System to Terminate Inflammatory Processes.

Conventional therapies for chronic inflammation with high dose application of active agents are often accompanied with severe side effects so that other therapeutical strategies shall be developed to be less physically demanding but still highly efficient. Locally applied Layer-by-Layer (LbL) microcarriers transporting a low, but efficient dosage of active agents directly into the inflamed tissue offer a gentle therapy option. Here, the inhibition of highly degradative enzyme human neutrophile elastase (HNE) is adressed, which is produced and secreted by neutrophile granulocytes (PMNs) in the progress of inflammation.

Different glycolipids in sperm from different freshwater fishes - A high-performance thin-layer chromatography/electrospray ionization mass spectrometry study.

Rationale: Glycolipids play important roles in many physiological processes - despite their commonly low abundance. This study summarizes selected data on the (glyco)lipid composition of sperm from different fish species.

Methods: Lipid extraction of fish sperm was performed according to the procedure by Bligh and Dyer.

Bicelles Rich in both Sphingolipids and Cholesterol and Their Use in Studies of Membrane Proteins.

How the distinctive lipid composition of mammalian plasma membranes impacts membrane protein structure is largely unexplored, partly because of the dearth of isotropic model membrane systems that contain abundant sphingolipids and cholesterol. This gap is addressed by showing that phingomyelin and hlesterol-ich (SCOR) lipid mixtures with phosphatidylcholine can be cosolubilized by -dodecyl-β-melibioside to form bicelles. Small-angle X-ray and neutron scattering, as well as cryo-electron microscopy, demonstrate that these assemblies are stable over a wide range of conditions and exhibit the bilayered-disc morphology of ideal bicelles even at low lipid-to-detergent mole ratios.

Comparative toxicometabolomics of perfluorooctanoic acid (PFOA) and next-generation perfluoroalkyl substances.

Owing to environmental health concerns, a number of per- and polyfluoroalkyl substances (PFAS) have been phased-out, and increasingly replaced by various chemical analogs. Most prominent among these replacements are numerous perfluoroether carboxylic acids (PFECA). Toxicity, and environmental health concerns associated with these next-generation PFAS, however, remains largely unstudied.

Deletion of Perilipin 5 Protects Against Hepatic Injury in Nonalcoholic Fatty Liver Disease via Missing Inflammasome Activation.

Nonalcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver diseases with an increasing prevalence due to rising rates of obesity, metabolic syndrome and type II diabetes. Untreated NAFLD may progress to steatohepatitis (NASH) and ultimately liver cirrhosis. NAFLD is characterized by lipid accumulation, and when sufficient excess lipids are obtained, irreversible liver injury may follow.

The European Research Network on Signal Transduction (ERNEST): Toward a Multidimensional Holistic Understanding of G Protein-Coupled Receptor Signaling.

G protein-coupled receptors (GPCRs) are intensively studied due to their therapeutic potential as drug targets. Members of this large family of transmembrane receptor proteins mediate signal transduction in diverse cell types and play key roles in human physiology and health. In 2013 the research consortium GLISTEN (COST Action CM1207) was founded with the goal of harnessing the substantial growth in knowledge of GPCR structure and dynamics to push forward the development of molecular modulators of GPCR function.

Vesiculation of Red Blood Cells in the Blood Bank: A Multi-Omics Approach towards Identification of Causes and Consequences.

Microvesicle generation is an integral part of the aging process of red blood cells in vivo and in vitro. Extensive vesiculation impairs function and survival of red blood cells after transfusion, and microvesicles contribute to transfusion reactions. The triggers and mechanisms of microvesicle generation are largely unknown.