The Murine Angiotensin II-Induced Abdominal Aortic Aneurysm Model: Rupture Risk and Inflammatory Progression Patterns

An abdominal aortic aneurysm (AAA) is an enlargement of the greatest artery in the body defined as an increase in diameter of 1.5-fold. AAAs are common in the elderly population and thousands die each year from their complications. The most commonly used mouse model to study the pathogenesis of AAA is the angiotensin II (Ang II) infusion method delivered via osmotic mini-pump for 28 days. Here, we studied the site-specificity and onset of aortic rupture, characterized three-dimensional (3D) images and flow patterns in developing AAAs by ultrasound imaging, and examined macrophage infiltration in the Ang II model using 65 apolipoprotein E-deficient mice. Aortic rupture occurred in 16 mice (25%) and was nearly as prevalent at the aortic arch (44%) as it was in the suprarenal region (56%) and was most common within the first 7 days after Ang II infusion (12 of 16; 75%). Longitudinal ultrasound screening was found to correlate nicely with histological analysis and AAA volume renderings showed a significant relationship with AAA severity index. Aortic dissection preceded altered flow patterns and macrophage infiltration was a prominent characteristic of developing AAAs. Targeting the inflammatory component of AAA disease with novel therapeutics will hopefully lead to new strategies to attenuate aneurysm growth and aortic rupture

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

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

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

Complete Acid Ceramidase ablation prevents cancer-initiating cell formation in melanoma cells

Acid ceramidase (AC) is a lysosomal cysteine hydrolase that catalyzes the conversion of ceramide into fatty acid and sphingosine. This reaction lowers intracellular ceramide levels and concomitantly generates sphingosine used for sphingosine-1-phosphate (S1P) production. Since increases in ceramide and consequent decreases of S1P reduce proliferation of various cancers, AC might offer a new target for anti-tumor therapy. Here we used CrispR-Cas9-mediated gene editing to delete the gene encoding for AC, ASAH1, in human A375 melanoma cells. ASAH1-null clones show significantly greater accumulation of long-chain saturated ceramides that are substrate for AC. As seen with administration of exogenous ceramide, AC ablation blocks cell cycle progression and accelerates senescence. Importantly, ASAH1-null cells also lose the ability to form cancer-initiating cells and to undergo self-renewal, which is suggestive of a key role for AC in maintaining malignancy and self-renewal of invasive melanoma cells. The results suggest that AC inhibitors might find therapeutic use as adjuvant therapy for advanced melanoma

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

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

The endogenous cannabinoid system in the gut of patients with inflammatory bowel disease

Activation of cannabinoid receptors (CBs) by endocannabinoids impacts on a number of gastrointestinal functions. Recent data indicate that CB1 agonists improve 2,4-dinitrobenzene sulfonic acid-induced colitis in mice, thus suggesting a role for the endocannabinoid agonist anandamide (AEA) in protecting the gut against inflammation. We here examined the gut endocannabinoid system in inflammatory bowel disease (IBD) patients, and investigated the ex vivo and in vitro effects of the non-hydrolysable AEA analog methanandamide (MAEA) on the mucosal proinflammatory response. The content of AEA, but not of 2-arachidonoyl-glycerol and N-palmitoylethanolamine, was significantly lower in inflamed than uninflamed IBD mucosa, and this was paralleled by lower activity of the AEA-synthesizing enzyme N-acyl-phosphatidylethanolamine-specific phospholipase D and higher activity of the AEA-degrading enzyme fatty acid amide hydrolase. MAEA significantly downregulated interferon-γ and tumor necrosis factor-α secretion by both organ culture biopsies and lamina propria mononuclear cells. Although these results are promising, further studies are needed to determine the role of cannabinoid pathways in gut inflammation. © 2011 Society for Mucosal Immunology