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

Evidence for a Role of Endocannabinoids, Astrocytes and p38 Phosphorylation in the Resolution of Postoperative Pain

An alarming portion of patients develop persistent or chronic pain following surgical procedures, but the mechanisms underlying the transition from acute to chronic pain states are not fully understood. In general, endocannabinoids (ECBs) inhibit nociceptive processing by stimulating cannabinoid receptors type 1 (CB(1)) and type 2 (CB(2)). We have previously shown that intrathecal administration of a CB(2) receptor agonist reverses both surgical incision-induced behavioral hypersensitivity and associated over-expression of spinal glial markers. We therefore hypothesized that endocannabinoid signaling promotes the resolution of acute postoperative pain by modulating pro-inflammatory signaling in spinal cord glial cells.To test this hypothesis, rats receiving paw incision surgery were used as a model of acute postoperative pain that spontaneously resolves. We first characterized the concentration of ECBs and localization of CB(1) and CB(2) receptors in the spinal cord following paw incision. We then administered concomitant CB(1) and CB(2) receptor antagonists/inverse agonists (AM281 and AM630, 1 mg x kg(-1) each, i.p.) during the acute phase of paw incision-induced mechanical allodynia and evaluated the expression of glial cell markers and phosphorylated p38 (a MAPK associated with inflammation) in the lumbar dorsal horn. Dual blockade of CB(1) and CB(2) receptor signaling prevented the resolution of postoperative allodynia and resulted in persistent over-expression of spinal Glial Fibrillary Acidic Protein (GFAP, an astrocytic marker) and phospho-p38 in astrocytes. We provide evidence for the functional significance of these astrocytic changes by demonstrating that intrathecal administration of propentofylline (50 microg, i.t.) attenuated both persistent behavioral hypersensitivity and over-expression of GFAP and phospho-p38 in antagonist-treated animals.Our results demonstrate that endocannabinoid signaling via CB(1) and CB(2) receptors is necessary for the resolution of paw incision-induced behavioral hypersensitivity and for the limitation of pro-inflammatory signaling in astrocytes following surgical insult. Our findings suggest that therapeutic strategies designed to enhance endocannabinoid signaling may prevent patients from developing persistent or chronic pain states following surgery

Experimental assessment of a new form of scaling law for near-wall turbulence

Scaling laws and intermittency in the wall region of a turbulent flow are addressed by analyzing moderate Reynolds number data obtained by single component hot wire anemometry in the boundary layer of a flat plate. The paper aims in particular at the experimental validation of a new form of refined similarity recently proposed for the shear dominated range of turbulence, where the classical Kolmogorov-Oboukhov inertial range theory is inappropriate. An approach inspired to the extended self-similarity allows for the extraction of the different power laws for the longitudinal structure functions at several wall normal distances. A double scaling regime is found in the logarithmic region, confirming previous experimental results. Approaching the wall, the scaling range corresponding to the classical cascade-dominated range tends to disappear and, in the buffer layer, a single power law is found to describe the available range of scales. The double scaling is shown to be associated with two different forms of refined similarity. The classical form holds below the shear scale L s . The other, originally introduced on the basis of DNS data for a turbulent channel, is experimentally confirmed to set up above L s . Given the experimental diffulties in the evaluation of the instantaneous dissipation rate, some care is devoted to check that its one-dimensional surrogate does not bias the results. The increased intermittency as the wall is approached is experimentally found entirely consistent with the failure of the refined Kolmogorov-Oboukhov similarity and the establishment of its new form near the wall.Comment: 27 pages, 9 figure

Experimental study of Taylor's hypothesis in a turbulent soap film

An experimental study of Taylor's hypothesis in a quasi-two-dimensional turbulent soap film is presented. A two probe laser Doppler velocimeter enables a non-intrusive simultaneous measurement of the velocity at spatially separated points. The breakdown of Taylor's hypothesis is quantified using the cross correlation between two points displaced in both space and time; correlation is better than 90% for scales less than the integral scale. A quantitative study of the decorrelation beyond the integral scale is presented, including an analysis of the failure of Taylor's hypothesis using techniques from predictability studies of turbulent flows. Our results are compared with similar studies of 3D turbulence.Comment: 27 pages, + 19 figure

Toksični učinci olova u profesionalno izložene indijske obitelji

This article describes an entire family manufacturing lead acid batteries who all suffered from lead poisoning. The family of five lived in a house, part of which had been used for various stages of battery production for 14 years. Open space was used for drying batteries. They all drank water from a well located on the premises. Evaluation of biomarkers of lead exposure and/or effect revealed alarming blood lead levels [(3.92±0.94) µmol L-1], 50 % reduction in the activity of δ-aminolevulinic acid dehydratase [(24.67±5.12) U L-1] and an increase in zinc protoporphyrin [(1228±480) µg L-1]. Liver function tests showed an increase in serum alkaline phosphatase [(170.41±41.82) U L-1]. All other liver function test parameters were normal. Renal function tests showed an increase in serum uric acid [(515.81±86.29) µmol L-1] while urea and creatinine were normal. Serum calcium was low [(1.90±0.42) mmol L-1 in women and (2.09±0.12) mmol L-1 in men], while blood pressure was high in the head of the family and his wife and normal in children. Lead concentration in well water was estimated to 180 µg L-1. The family was referred to the National Referral Centre for Lead Poisoning in India, were they were received treatment and were informed about the hazards of lead poisoning. A follow up three months later showed a slight decrease in blood lead levels and a significant increase in haemoglobin. These findings can be attributed to behavioural changes adopted by the family, even though they continued producing lead batteries.Olovo je sveprisutni metal s mnogo namjena, a čovječanstvo ga rabi već više od 6000 godina. Danas je olovo među najrasprostranjenijim toksinima u okolišu, a drugi je na popisu toksičnih metala, odmah iza arsena. Mnogi još nisu svjesni njegova toksičnoga djelovanja te se i dalje izlažu olovu. Ovdje je opisana obitelj koja proizvodi olovne akumulatore i koja je pretrpjela trovanje olovom zahvaljujući svojoj neobaviještenosti. Ova peteročlana obitelj živjela je u jednome kućanstvu čiji je dio namijenjen različitim fazama proizvodnje akumulatora već 14 godina. Akumulatori su se sušili na otvorenome. Na imanju je bio i bunar s pitkom vodom. Mjerenja biopokazatelja izloženosti olovu i njegova djelovanja u svih pet članova obitelji dovela su do alarmantnoga saznanja o razinama olova u krvi [(3,92±0,94) µmol L-1], 50 %-tnom padu aktivnosti dehidrataze δ-aminolevulinske kiseline [(24,67±5,12) U L-1] te povišenom cinkovu protoporfirinu [(1228±480) µg L-1]. Jetrene probe otkrile su povišene razine alkalne fosfataze u serumu [(170,41±41,82) U L-1]. Ostali su parametri jetrene funkcije bili normalni. Testovi funkcije bubrega otkrili su povišene razine mokraćne kiseline u serumu [(515,81±86,29) µmol L-1], dok su razine ureje i kreatinina bile normalne. Također je zabilježen pad razina kalcija u serumu [(1,90±0,42) mmol L-1 u žena te (2,09±0,12) mmol L-1 u muškaraca]. Povišeni krvni tlak zamijećen je u glave obitelji i njegove supruge, dok je u djece bio normalan. Koncentracija olova u bunarskoj vodi bila je izrazito visoka, prema procjeni 180 µg L-1. Obitelj je upućena u indijski Državni referalni centar za otrovanje olovom (National Referral Centre for Lead Poisoning) gdje je primila lijekove i bila upoznata s činjenicama vezanim uz otrovanje olovom. Tromjesečno je praćenje pokazalo blagi pad razina olova u krvi te značajan porast hemoglobina. Ovi se nalazi mogu pripisati promjenama u ponašanju obitelji, bez obzira na to što je nastavila proizvoditi akumulatore

LES-based Study of the Roughness Effects on the Wake of a Circular Cylinder from Subcritical to Transcritical Reynolds Numbers

This paper investigates the effects of surface roughness on the flow past a circular cylinder at subcritical to transcritical Reynolds numbers. Large eddy simulations of the flow for sand grain roughness of size k/D = 0.02 are performed (D is the cylinder diameter). Results show that surface roughness triggers the transition to turbulence in the boundary layer at all Reynolds numbers, thus leading to an early separation caused by the increased momentum deficit, especially at transcritical Reynolds numbers. Even at subcritical Reynolds numbers, boundary layer instabilities are triggered in the roughness sublayer and eventually lead to the transition to turbulence. The early separation at transcritical Reynolds numbers leads to a wake topology similar to that of the subcritical regime, resulting in an increased drag coefficient and lower Strouhal number. Turbulent statistics in the wake are also affected by roughness; the Reynolds stresses are larger due to the increased turbulent kinetic energy production in the boundary layer and separated shear layers close to the cylinder shoulders.We acknowledge “Red Española de Surpercomputación” (RES) for awarding us access to the MareNostrum III machine based in Barcelona, Spain (Ref. FI-2015-2-0026 and FI-2015-3-0011). We also acknowledge PRACE for awarding us access to Fermi and Marconi Supercomputers at Cineca, Italy (Ref. 2015133120). Oriol Lehmkuhl acknowledges a PDJ 2014 Grant by AGAUR (Generalitat de Catalunya). Ugo Piomelli acknowledges the support of the Natural Sciences and Engineering Research Council (NSERC) of Canada under the Discovery Grant Programme (Grant No. RGPIN-2016-04391). Ricard Borrell acknowledges a Juan de la Cierva postdoctoral grant (IJCI-2014-21034). Ivette Rodriguez, Oriol Lehmkuhl, Ricard Borrell and Assensi Oliva acknowledge Ministerio de Economía y Competitividad, Secretaría de Estado de Investigación, Desarrollo e Innovación, Spain (ref. ENE2014-60577-R).Peer ReviewedPostprint (author's final draft

Large-Eddy Simulation: Current Capabilities, Recommended Practices, and Future Research

This paper presents the results of an activity by the Large Eddy Simulation (LES) Working Group of the AIAA Fluid Dynamics Technical Committee to (1) address the current capabilities of LES, (2) outline recommended practices and key considerations for using LES, and (3) identify future research needs to advance the capabilities and reliability of LES for analysis of turbulent flows. To address the current capabilities and future needs, a survey comprised of eleven questions was posed to LES Working Group members to assemble a broad range of perspectives on important topics related to LES. The responses to these survey questions are summarized with the intent not to be a comprehensive dictate on LES, but rather the perspective of one group on some important issues. A list of recommended practices is also provided, which does not treat all aspects of a LES, but provides guidance on some of the key areas that should be considered