Identification of a Widespread Palmitoylethanolamide Contamination in Standard Laboratory Glassware.

Introduction: Fatty acid ethanolamides (FAEs) are a family of lipid mediators that participate in a host of biological functions. Procedures for the quantitative analysis of FAEs include organic solvent extraction from biological matrices (e.g., blood), followed by purification and subsequent quantitation by liquid chromatography-mass spectrometry (LC/MS) or gas chromatography-mass spectrometry. During the validation process of a new method for LC/MS analysis of FAEs in biological samples, we observed unusually high levels of the FAE, palmitoylethanolamide (PEA), in blank samples that did not contain any biological material. Materials and Methods: We investigated a possible source of this PEA artifact via liquid chromatography coupled to tandem mass spectrometry, as well as accurate mass analysis. Results: We found that high levels of a contaminant indistinguishable from PEA is present in new 5.75″ glass Pasteur pipettes, which are routinely used by laboratories to carry out lipid extractions. This artifact might account for discrepancies found in the literature regarding PEA levels in human blood serum and other tissues. Conclusions: It is recommended to take into account this pitfall by analyzing potential contamination of the disposable glassware during the validation process of any method used for analysis of FAEs

On the large-eddy simulation of transitional wall-bounded flows

The structure of the subgrid scale fields in plane channel flow has been studied at various stages of the transition process to turbulence. The residual stress and subgrid scale dissipation calculated using velocity fields generated by direct numerical simulations of the Navier-Stokes equations are significantly different from their counterparts in turbulent flows. The subgrid scale dissipation changes sign over extended areas of the channel, indicating energy flow from the small scales to the large scales. This reversed energy cascade becomes less pronounced at the later stages of transition. Standard residual stress models of the Smagorinsky type are excessively dissipative. Rescaling the model constant improves the prediction of the total (integrated) subgrid scale dissipation, but not that of the local one. Despite the somewhat excessive dissipation of the rescaled Smagorinsky model, the results of a large eddy simulation of transition on a flat-plate boundary layer compare quite well with those of a direct simulation, and require only a small fraction of the computational effort. The inclusion of non-dissipative models, which could lead to further improvements, is proposed

Dynamic subfilter-scale stress model for large-eddy simulations

We present a modification of the integral length-scale approximation (ILSA) model originally proposed by Piomelli et al. [Piomelli et al., J. Fluid Mech. 766, 499 (2015)] and apply it to plane channel flow and a backward-facing step. In the ILSA models the length scale is expressed in terms of the integral length scale of turbulence and is determined by the flow characteristics, decoupled from the simulation grid. In the original formulation the model coefficient was constant, determined by requiring a desired global contribution of the unresolved subfilter scales (SFSs) to the dissipation rate, known as SFS activity; its value was found by a set of coarse-grid calculations. Here we develop two modifications. We de-fine a measure of SFS activity (based on turbulent stresses), which adds to the robustness of the model, particularly at high Reynolds numbers, and removes the need for the prior coarse-grid calculations: The model coefficient can be computed dynamically and adapt to large-scale unsteadiness. Furthermore, the desired level of SFS activity is now enforced locally (and not integrated over the entire volume, as in the original model), providing better control over model activity and also improving the near-wall behavior of the model. Application of the local ILSA to channel flow and a backward-facing step and comparison with the original ILSA and with the dynamic model of Germano et al. [Germano et al., Phys. Fluids A 3, 1760 (1991)] show better control over the model contribution in the local ILSA, while the positive properties of the original formulation (including its higher accuracy compared to the dynamic model on coarse grids) are maintained. The backward-facing step also highlights the advantage of the decoupling of the model length scale from the mesh

Nested Dissection Method on Transputer

Nested dissection method is an elimination method for a set of linear algebraic equations with minimum fillins. Physically it divides a domain into four subdomains, and each subdomain is again divided into four. This procedure is repeated till all nodes are included in some subdomains. Using this characteristic, the authors examine the efficiency of the method on the transputer

Different roles for the acyl chain and the amine leaving group in the substrate selectivity of N-Acylethanolamine acid amidase

N-acylethanolamine acid amidase (NAAA) is an N-terminal nucleophile (Ntn) hydrolase that catalyses the intracellular deactivation of the endogenous analgesic and anti-inflammatory agent palmitoylethanolamide (PEA). NAAA inhibitors counteract this process and exert marked therapeutic effects in animal models of pain, inflammation and neurodegeneration. While it is known that NAAA preferentially hydrolyses saturated fatty acid ethanolamides (FAEs), a detailed profile of the relationship between catalytic efficiency and fatty acid-chain length is still lacking. In this report, we combined enzymatic and molecular modelling approaches to determine the effects of acyl chain and polar head modifications on substrate recognition and hydrolysis by NAAA. The results show that, in both saturated and monounsaturated FAEs, the catalytic efficiency is strictly dependent upon fatty acyl chain length, whereas there is a wider tolerance for modifications of the polar heads. This relationship reflects the relative stability of enzyme-substrate complexes in molecular dynamics simulations

Identification of a Widespread Palmitoylethanolamide Contamination in Standard Laboratory Glassware

Introduction: Fatty acid ethanolamides (FAEs) are a family of lipid mediators that participate in a host of biological functions. Procedures for the quantitative analysis of FAEs include organic solvent extraction from biological matrices (e.g., blood), followed by purification and subsequent quantitation by liquid chromatography-mass spectrometry (LC/MS) or gas chromatography-mass spectrometry. During the validation process of a new method for LC/MS analysis of FAEs in biological samples, we observed unusually high levels of the FAE, palmitoylethanolamide (PEA), in blank samples that did not contain any biological material. Materials and Methods: We investigated a possible source of this PEA artifact via liquid chromatography coupled to tandem mass spectrometry, as well as accurate mass analysis. Results: We found that high levels of a contaminant indistinguishable from PEA is present in new 5.75″ glass Pasteur pipettes, which are routinely used by laboratories to carry out lipid extractions. This artifact might account for discrepancies found in the literature regarding PEA levels in human blood serum and other tissues. Conclusions: It is recommended to take into account this pitfall by analyzing potential contamination of the disposable glassware during the validation process of any method used for analysis of FAEs

N-Acylethanolamine Acid Amidase (NAAA): Mechanism of Palmitoylethanolamide Hydrolysis Revealed by Mechanistic Simulations

The N-terminal cysteine hydrolase N-acylethanolamine acid amidase (NAAA) catalyzes the hydrolytic deactivation of the lipid messenger palmitoylethanolamide (PEA), with optimal activity at acidic pH. Using the crystal structure of human NAAA as a starting point, we investigated the catalytic mechanism of PEA hydrolysis with a multiscale approach based on classic molecular dynamics (MD) and quantum mechanical/molecular mechanics (QM/MM) simulations coupled with enhanced sampling and path-collective variables (PCVs). The proton configuration of the catalytic nucleophile, Cys126, and of the surrounding carboxylates was critical to preserve the active site architecture. A stable Michaelis complex was then used to reconstruct the free-energy surfaces of NAAA acylation and deacylation during PEA hydrolysis. Acylation emerged as the critical step, with Cys126 acting both as an acid, to protonate the ethanolamine leaving group, and as a nucleophile, to attack the PEA carbonyl carbon. The ethanol fragment of PEA did not appear to play an indispensable role in acylation, a result further supported by kinetic experiments showing that NAAA hydrolyzes palmitoyl methyl amide (PMA) with high catalytic efficiency. Our multiscale approach identified a distinctive protonation state and catalytic mechanism for NAAA which accounts for pH-dependent activity, mutagenesis data, and mechanism of covalent inhibitors

N-Acylethanolamine Acid Amidase (NAAA): Structure, Function, and Inhibition

N-Acylethanolamine acid amidase (NAAA) is an N-terminal cysteine hydrolase primarily found in the endosomal-lysosomal compartment of innate and adaptive immune cells. NAAA catalyzes the hydrolytic deactivation of palmitoylethanolamide (PEA), a lipid-derived peroxisome proliferator-activated receptor-α (PPAR-α) agonist that exerts profound anti-inflammatory effects in animal models. Emerging evidence points to NAAA-regulated PEA signaling at PPAR-α as a critical control point for the induction and the resolution of inflammation and to NAAA itself as a target for anti-inflammatory medicines. The present Perspective discusses three key aspects of this hypothesis: the role of NAAA in controlling the signaling activity of PEA; the structural bases for NAAA function and inhibition by covalent and noncovalent agents; and finally, the potential value of NAAA-targeting drugs in the treatment of human inflammatory disorders