Structure of human GABAB receptor in an inactive state.

The human GABAB receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction1. A unique GPCR that is known to require heterodimerization for function2-6, the GABAB receptor has two subunits, GABAB1 and GABAB2, that are structurally homologous but perform distinct and complementary functions. GABAB1 recognizes orthosteric ligands7,8, while GABAB2 couples with G proteins9-14. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail15. Although the VFT heterodimer structure has been resolved16, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABAB receptor, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique 'intersubunit latch' within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

A fluorogenic substrate for the detection of lipid amidases in intact cells.

Lipid amidases of therapeutic relevance include acid ceramidase (AC), N-acylethanolamine-hydrolyzing acid amidase, and fatty acid amide hydrolase (FAAH). Although fluorogenic substrates have been developed for the three enzymes and high-throughput methods for screening have been reported, a platform for the specific detection of these enzyme activities in intact cells is lacking. In this article, we report on the coumarinic 1-deoxydihydroceramide RBM1-151, a 1-deoxy derivative and vinilog of RBM14-C12, as a novel substrate of amidases. This compound is hydrolyzed by AC (appKm = 7.0 µM; appVmax = 99.3 nM/min), N-acylethanolamine-hydrolyzing acid amidase (appKm = 0.73 µM; appVmax = 0.24 nM/min), and FAAH (appKm = 3.6 µM; appVmax = 7.6 nM/min) but not by other ceramidases. We provide proof of concept that the use of RBM1-151 in combination with reported irreversible inhibitors of AC and FAAH allows the determination in parallel of the three amidase activities in single experiments in intact cells.

Distinctive Lipid Characteristics of Colorectal Cancer Revealed through Non-targeted Lipidomics Analysis of Tongue Coating.

The metabolites and microbiota in tongue coating display distinct characteristics in certain digestive disorders, yet their relationship with colorectal cancer (CRC) remains unexplored. Here, we employed liquid chromatography coupled with tandem mass spectrometry to analyze the lipid composition of tongue coating using a nontargeted approach in 30 individuals with colorectal adenomas (CRA), 32 with CRC, and 30 healthy controls (HC). We identified 21 tongue coating lipids that effectively distinguished CRC from HC (AUC = 0.89), and 9 lipids that differentiated CRC from CRA (AUC = 0.9). Furthermore, we observed significant alterations in the tongue coating lipid composition in the CRC group compared to HC/CRA groups. As the adenoma-cancer sequence progressed, there was an increase in long-chain unsaturated triglycerides (TG) levels and a decrease in phosphatidylethanolamine plasmalogen (PE-P) levels. Furthermore, we noted a positive correlation between N-acyl ornithine (NAOrn), sphingomyelin (SM), and ceramide phosphoethanolamine (PE-Cer), potentially produced by members of the Bacteroidetes phylum. The levels of inflammatory lipid metabolite 12-HETE showed a decreasing trend with colorectal tumor progression, indicating the potential involvement of tongue coating microbiota and tumor immune regulation in early CRC development. Our findings highlight the potential utility of tongue coating lipid analysis as a noninvasive tool for CRC diagnosis.

A Mechanistic Basis for Phosphoethanolamine Modification of the Cellulose Biofilm Matrix in Escherichia coli.

Biofilms are communities of self-enmeshed bacteria in a matrix of exopolysaccharides. The widely distributed human pathogen and commensal Escherichia coli produces a biofilm matrix composed of phosphoethanolamine (pEtN)-modified cellulose and amyloid protein fibers, termed curli. The addition of pEtN to the cellulose exopolysaccharide is accomplished by the action of the pEtN transferase, BcsG, and is essential for the overall integrity of the biofilm. Here, using the synthetic co-substrates p-nitrophenyl phosphoethanolamine and ß-d-cellopentaose, we demonstrate using an in vitro pEtN transferase assay that full activity of the pEtN transferase domain of BcsG from E. coli (EcBcsGΔN) requires Zn2+ binding, a catalytic nucleophile/acid-base arrangement (Ser278/Cys243/His396), disulfide bond formation, and other newly uncovered essential residues. We further confirm that EcBcsGΔN catalysis proceeds by a ping-pong bisubstrate-biproduct reaction mechanism and displays inefficient kinetic behavior (kcat/KM = 1.81 × 10-4 ± 2.81 × 10-5 M-1 s-1), which is typical of exopolysaccharide-modifying enzymes in bacteria. Thus, the results presented, especially with respect to donor binding (as reflected by KM), have importantly broadened our understanding of the substrate profile and catalytic mechanism of this class of enzymes, which may aid in the development of inhibitors targeting BcsG or other characterized members of the pEtN transferase family, including the intrinsic and mobile colistin resistance factors.

Investigation of the aquaporin-2 gating mechanism with molecular dynamics simulations.

Aquaporin-2 plays a vital role in the human kidney as a water passage channel. Any disorder with its function can cause water imbalance and consequently disease in humans, especially nephrogenic diabetes insipidus (NDI). For this reason, an accurate understanding of its performance can be useful for therapeutic purposes. In this article, we investigate the gating mechanism induced by spontaneous fluctuations in aquaporin-2's (AQP2) channels in the palmitoyl-oleoyl-phosphatidyl-ethanolamine lipid bilayer by molecular dynamics. Our results show that the selectivity filter (SF) in AQP2 is also a gating site depending on the side-chain conformation of His172. The important role of His172 in modulating the wide and narrow conformations has been further investigated by the simulation of the H172G mutant. The osmotic permeability values of all four monomers are in the range of wide state and the average is very close to that of the wide channel formed by wild-type AQP2. Moreover, by calculating the osmotic permeability and the potential of mean force of each of the AQP2 monomers for wide/narrow states of the SF, it is seen that the SF at its narrow conformation can induce a much larger energy barrier for water molecules permeation, hindering the transport of water molecules remarkably. The reason for the discrepancy among osmotic permeabilities of different monomers of aquaporins is revealed by investigating the osmotic permeability of each monomer through the wide/narrow states of their SF.

Anti-allergic property of 4,8-sphingadienine stereoisomers in vivo and in vitro model.

4,8-Sphingadienines (SD), metabolites of glucosylceramides (GlcCer), are sometimes determined as key mediators of the biological activity of dietary GlcCer, and cis/trans geometries of 4,8-SD have been reported to affect its activity. Since regulating excessive activation of mast cells seems an important way to ameliorate allergic diseases, this study was focused on cis/trans stereoisomeric-dependent inhibitory effects of 4,8-SD on mast cell activation. Degranulation of RBL-2H3 was inhibited by treatment of 4-cis-8-trans- and 4-cis-8-cis-SD, and their intradermal administrations ameliorated ear edema in passive cutaneous anaphylaxis reaction, but 4-trans-8-trans- and 4-trans-8-cis-SD did not. Although the activation of mast cells depends on the bound IgE contents, those stereoisomers did not affect IgE contents on RBL-2H3 cells after the sensitization of anti-TNP IgE. These results indicated that 4-cis-8-trans- and 4-cis-8-cis-SD directly inhibit the activation of mast cells. In conclusion, it was assumed that 4,8-SD stereoisomers with cis double bond at C4-position shows anti-allergic activity by inhibiting downstream pathway after activation by the binding of IgE to mast cells.

Variation in the ratio of curli and phosphoethanolamine cellulose associated with biofilm architecture and properties.

Bacterial biofilms are communities of bacteria entangled in a self-produced extracellular matrix (ECM). Escherichia coli direct the assembly of two insoluble biopolymers, curli amyloid fibers, and phosphoethanolamine (pEtN) cellulose, to build remarkable biofilm architectures. Intense curiosity surrounds how bacteria harness these amyloid-polysaccharide composites to build biofilms, and how these biopolymers function to benefit bacterial communities. Defining ECM composition involving insoluble polymeric assemblies poses unique challenges to analysis and, thus, to comparing strains with quantitative ECM molecular correlates. In this work, we present results from a sum-of-the-parts 13 C solid-state nuclear magnetic resonance (NMR) analysis to define the curli-to-pEtN cellulose ratio in the isolated ECM of the E. coli laboratory K12 strain, AR3110. We compare and contrast the compositional analysis and comprehensive biofilm phenotypes for AR3110 and a well-studied clinical isolate, UTI89. The ECM isolated from AR3110 contains approximately twice the amount of pEtN cellulose relative to curli content as UTI89, revealing plasticity in matrix assembly principles among strains. The two parent strains and a panel of relevant gene mutants were investigated in three biofilm models, examining: (a) macrocolonies on agar, (b) pellicles at the liquid-air interface, and (c) biomass accumulation on plastic. We describe the influence of curli, cellulose, and the pEtN modification on biofilm phenotypes with power in the direct comparison of these strains. The results suggest that curli more strongly influence adhesion, while pEtN cellulose drives cohesion. Their individual and combined influence depends on both the biofilm modality (agar, pellicle, or plastic-associated) and the strain itself.

Packing polymorphism, odd-even alternation and thermotropic phase transitions in N-,O-diacylethanolamines with varying N-acyl chains. A combined experimental and computational study.

N-,O-Diacylethanolamines (DAEs) are derived by simple esterification of bioactive N-acylethanolamines, which are present in plant and animal tissues. In this study, two homologous series of DAEs, namely N-acyl (n = 8-15), O-palmitoylethanolamines (Nn-O16s) and N-acyl (n = 8-14), O-pentadecanoylethanolamines (Nn-O15s) were synthesized and characterized with respect to thermotropic phase transitions, crystal structures and intermolecular interactions. In addition, computational studies were performed to get a molecular level insight into the role of different factors in selective polymorphism in Nn-O16s and Nn-O15s. Differential scanning calorimetric studies revealed that dry Nn-O16s exhibit odd-even alternation in their calorimetric properties, which is absent in Nn-O15s. The 3-dimensional structures of three Nn-O16s (n = 12-14) and two Nn-O15s (n = 12, 14) have been determined by single-crystal X-ray diffraction. Analysis of the molecular packing in these crystals showed the presence of two packing polymorphs (α and ß) in the crystal lattice of Nn-O16s, whereas only the ß polymorph was observed in the Nn-O15s. Further, intermolecular hydrogen bonding interactions (N-H⋯O and C-H⋯O) and dispersion interactions among acyl chains have been found to stabilize the molecular packing observed in the crystal lattice. Molecular dynamics simulations show that the ß polymorph is slightly energetically preferred over the α polymorph in all the systems due to favorable packing of terminal methyl groups at the interlayers. These findings are relevant for understanding the interactions of the DAEs with membrane lipids and proteins.

Modulation of hydrophobic interactions by proximally immobilized ions.

The structure of water near non-polar molecular fragments or surfaces mediates the hydrophobic interactions that underlie a broad range of interfacial, colloidal and biophysical phenomena. Substantial progress over the past decade has improved our understanding of hydrophobic interactions in simple model systems, but most biologically and technologically relevant structures contain non-polar domains in close proximity to polar and charged functional groups. Theories and simulations exploring such nanometre-scale chemical heterogeneity find it can have an important effect, but the influence of this heterogeneity on hydrophobic interactions has not been tested experimentally. Here we report chemical force microscopy measurements on alkyl-functionalized surfaces that reveal a dramatic change in the surfaces' hydrophobic interaction strengths on co-immobilization of amine or guanidine groups. Protonation of amine groups doubles the strength of hydrophobic interactions, and guanidinium groups eliminate measurable hydrophobic interactions in all pH ranges investigated. We see these divergent effects of proximally immobilized cations also in single-molecule measurements on conformationally stable ß-peptides with non-polar subunits located one nanometre from either amine- or guanidine-bearing subunits. Our results demonstrate the importance of nanometre-scale chemical heterogeneity, with hydrophobicity not an intrinsic property of any given non-polar domain but strongly modulated by functional groups located as far away as one nanometre. The judicious placing of charged groups near hydrophobic domains thus provides a strategy for tuning hydrophobic driving forces to optimize molecular recognition or self-assembly processes.

A Bioassay Using a Pentadecanal Derivative to Measure S1P Lyase Activity.

Sphingosine-1-phosphate (S1P) is a unique lipid ligand binding to S1P receptors to transduce various cell survival or proliferation signals via small G proteins. S1P lyase (S1PL) is the specific enzyme that degrades S1P to phosphoethanolamine and (2E)-hexadecenal and therefore regulates S1P levels. S1PL also degrades dihydrosphingosine-1-phosphate (Sa1P), with a higher affinity to produce hexadecanal. Here, we developed a newly designed assay using a C17-Sa1P substrate that degrades into pentadecanal and phosphoethanolamine. For higher sensitivity in pentadecanal analysis, we developed a quantitative protocol as well as a 5,5-dimethyl cyclohexanedione (5,5-dimethyl CHD) derivatization method. The derivatization conditions were optimized for the reaction time, temperature, and concentrations of the 5,5-dimethyl CHD reagent, acetic acid, and ammonium acetate. The S1PL reaction in the cell lysate after spiking 20 µM of C17-Sa1P for 20 min was linear to the total protein concentrations of 50 µg. The S1PL levels (4 pmol/mg/min) were readily detected in this HPLC with fluorescence detection (λex = 366 nm, λem = 455 nm). The S1PL-catalyzed reaction was linear over 30 min and yielded a Km value of 2.68 µM for C17-Sa1P. This new method was validated to measure the S1PL activity of mouse embryonal carcinoma cell lines of the standard cell (F9-0), S1PL knockdown cells (F9-2), and S1PL-overexpressed cells (F9-4). Furthermore, we treated F9-4 cells with different S1PL inhibitors such as FTY720, 4-deoxypyridoxine (DOP), and the deletion of pyridoxal-5-phosphate (P5P), an essential cofactor for S1PL activity, and observed a significant decrease in pentadecanal relative to the untreated cells. In conclusion, we developed a highly sensitive S1PL assay using a C17-Sa1P substrate for pentadecanal quantification for application in the characterization of S1PL activity in vitro.

Ultramicronized Palmitoylethanolamide in the Management of Sepsis-Induced Coagulopathy and Disseminated Intravascular Coagulation.

Disseminated intravascular coagulation (DIC) is a severe condition characterized by the systemic formation of microthrombi complicated with bleeding tendency and organ dysfunction. In the last years, it represents one of the most frequent consequences of coronavirus disease 2019 (COVID-19). The pathogenesis of DIC is complex, with cross-talk between the coagulant and inflammatory pathways. The objective of this study is to investigate the anti-inflammatory action of ultramicronized palmitoylethanolamide (um-PEA) in a lipopolysaccharide (LPS)-induced DIC model in rats. Experimental DIC was induced by continual infusion of LPS (30 mg/kg) for 4 h through the tail vein. Um-PEA (30 mg/kg) was given orally 30 min before and 1 h after the start of intravenous infusion of LPS. Results showed that um-PEA reduced alteration of coagulation markers, as well as proinflammatory cytokine release in plasma and lung samples, induced by LPS infusion. Furthermore, um-PEA also has the effect of preventing the formation of fibrin deposition and lung damage. Moreover, um-PEA was able to reduce the number of mast cells (MCs) and the release of its serine proteases, which are also necessary for SARS-CoV-2 infection. These results suggest that um-PEA could be considered as a potential therapeutic approach in the management of DIC and in clinical implications associated to coagulopathy and lung dysfunction, such as COVID-19.

Potato Peels Mediated Synthesis of Cu(II)-nanoparticles from Tyrosinase Reacted with bis-(N-aminoethylethanolamine) (Tyr-Cu(II)-AEEA NPs) and Their Cytotoxicity against Michigan Cancer Foundation-7 Breast Cancer Cell Line.

The synthesis of nanoparticles is most important in the context of cancer therapy, particularly copper nanoparticles, which are widely used. In this work, copper(II)-tyrosinase was isolated from potato peel powder. Copper nanoparticles (Tyr-Cu(II)-AEEA NPs) were synthesized via the reaction of tyrosinase with N-aminoethylethanolamine to produce Cu(II)-NPs and these were characterized by means of FT-IR, UV-Spectroscopy, XRD, SEM, TEM and a particle size analyzer. These Tyr-Cu(II)-AEEA NPs were tested as anticancer agents against MCF-7 breast cancer cells. Fluorescence microscopy and DNA fragmentation were also performed, which revealed the inhibiting potentials of Cu(II)-AEEA NPs and consequent cell death; Tyr-Cu(II)-AEEA NPs show potential cytotoxicity activity and this nano material could be contemplated as an anticancer medicament in future investigations.

Evaluation of Phosphoethanolamine Cellulose Production among Bacterial Communities Using Congo Red Fluorescence.

Bacterial biofilms are surface-associated communities of bacterial cells enmeshed in an extracellular matrix (ECM). The biofilm lifestyle results in physiological heterogeneity across the community, promotes persistence, and protects cells from external insults such as antibiotic treatment. Escherichia coli was recently discovered to produce a chemically modified form of cellulose, phosphoethanolamine (pEtN) cellulose, which contributes to the formation of its extracellular matrix and elaboration of its hallmark wrinkled macrocolony architectures. Both pEtN cellulose and unmodified cellulose bind dyes such as calcofluor white and Congo red (CR). Here, we present the use of CR fluorescence to distinguish between pEtN cellulose and unmodified cellulose producers. We demonstrate the utility of this tool in the evaluation of a uropathogenic E. coli clinical isolate that appeared to produce curli and a cellulosic component but did not exhibit macrocolony wrinkling. We determined that lack of macrocolony wrinkling was attributed to a single-nucleotide mutation and introduction of a stop codon in bcsG, abrogating production of BcsG, the pEtN transferase. Thus, this work underscores the important contribution of the pEtN cellulose modification to the E. coli agar-based macrocolony wrinkling phenotype and introduces a facile approach to distinguish between modified and unmodified cellulose.IMPORTANCEE. coli bacteria produce amyloid fibers, termed curli, and a cellulosic component to assemble biofilm communities. Cellulose is the most abundant biopolymer on Earth, and we recently discovered that the cellulosic component in E. coli biofilms was not standard cellulose, but a newly identified cellulosic polymer, phosphoethanolamine cellulose. Studies involving the biological and functional impact of this cellulose modification among E. coli and other organisms are just beginning. Convenient methods for distinguishing pEtN cellulose from unmodified cellulose in E. coli and for estimating production are needed to facilitate further research. Dissecting the balance of pEtN cellulose and curli production by E. coli commensal strains and clinical isolates will improve our understanding of the host microbiome and of factors contributing to bacterial pathogenesis.

Tetracosahexaenoylethanolamide, a novel N-acylethanolamide, is elevated in ischemia and increases neuronal output.

N-acylethanolamines (NAEs) are endogenous lipid-signaling molecules derived from fatty acids that regulate numerous biological functions, including in the brain. Interestingly, NAEs are elevated in the absence of fatty acid amide hydrolase (FAAH) and following CO2-induced ischemia/hypercapnia, suggesting a neuroprotective response. Tetracosahexaenoic acid (THA) is a product and precursor to DHA; however, the NAE product, tetracosahexaenoylethanolamide (THEA), has never been reported. Presently, THEA was chemically synthesized as an authentic standard to confirm THEA presence in biological tissues. Whole brains were collected and analyzed for unesterified THA, total THA, and THEA in wild-type and FAAH-KO mice that were euthanized by either head-focused microwave fixation, CO2 + microwave, or CO2 only. PPAR activity by transient transfection assay and ex vivo neuronal output in medium spiny neurons (MSNs) of the nucleus accumbens by patch clamp electrophysiology were determined following THEA exposure. THEA in the wild-type mice was nearly doubled (P < 0.05) following ischemia/hypercapnia (CO2 euthanization) and up to 12 times higher (P < 0.001) in the FAAH-KO compared with wild-type. THEA did not increase (P > 0.05) transcriptional activity of PPARs relative to control, but 100 nM of THEA increased (P < 0.001) neuronal output in MSNs of the nucleus accumbens. Here were identify a novel NAE, THEA, in the brain that is elevated upon ischemia/hypercapnia and by KO of the FAAH enzyme. While THEA did not activate PPAR, it augmented the excitability of MSNs in the nucleus accumbens. Overall, our results suggest that THEA is a novel NAE that is produced in the brain upon ischemia/hypercapnia and regulates neuronal excitation.

Structural analysis of a plant fatty acid amide hydrolase provides insights into the evolutionary diversity of bioactive acylethanolamides.

N-Acylethanolamines (NAEs) are fatty acid derivatives that in animal systems include the well-known bioactive metabolites of the endocannabinoid signaling pathway. Plants use NAE signaling as well, and these bioactive molecules often have oxygenated acyl moieties. Here, we report the three-dimensional crystal structures of the signal-terminating enzyme fatty acid amide hydrolase (FAAH) from Arabidopsis in its apo and ligand-bound forms at 2.1- and 3.2-Å resolutions, respectively. This plant FAAH structure revealed features distinct from those of the only other available FAAH structure (rat). The structures disclosed that although catalytic residues are conserved with the mammalian enzyme, AtFAAH has a more open substrate-binding pocket that is partially lined with polar residues. Fundamental differences in the organization of the membrane-binding "cap" and the membrane access channel also were evident. In accordance with the observed structural features of the substrate-binding pocket, kinetic analysis showed that AtFAAH efficiently uses both unsubstituted and oxygenated acylethanolamides as substrates. Moreover, comparison of the apo and ligand-bound AtFAAH structures identified three discrete sets of conformational changes that accompany ligand binding, suggesting a unique "squeeze and lock" substrate-binding mechanism. Using molecular dynamics simulations, we evaluated these conformational changes further and noted a partial unfolding of a random-coil helix within the region 531-537 in the apo structure but not in the ligand-bound form, indicating that this region likely confers plasticity to the substrate-binding pocket. We conclude that the structural divergence in bioactive acylethanolamides in plants is reflected in part in the structural and functional properties of plant FAAHs.

Turning autophobic wetting on biomimetic surfaces into complete wetting by wetting additives.

Autophobicity or pseudo partial wetting, a phenomenon of a liquid not spreading on its own monolayer, is characterized by an energy barrier that prevents the growth of a wetting film beyond the monolayer thickness. Applying a molecularly detailed self-consistent field theory we illustrate how autophobic wetting can be overcome by wetting additives. More specifically we use an emulsifier which keeps the interfacial tension between the wetting component and the majority solvent low, and a co-solvent additive which partitions inside the film and then destroys the molecular order in it so that the barrier for film growth is cleared. An application wherein it is believed that autophobic wetting is counteracted by such a set of wetting additives is found in an antidandruff shampoo formulation. We have experimental results that show thick deposits onto hydrophobic hair surfaces by administration of the antidandruff shampoo. The complementary modeling of such a system suggests that the active ingredient plays the role of the co-solvent additive. As significant amounts of the co-solvent additives are needed to approach the completely wet state, the formulation naturally brings large amounts of active ingredient to the root of the hair where its presence is required.

Discovery of New Hydroxyethylamine Analogs against 3CLpro Protein Target of SARS-CoV-2: Molecular Docking, Molecular Dynamics Simulation, and Structure-Activity Relationship Studies.

The novel coronavirus, SARS-CoV-2, has caused a recent pandemic called COVID-19 and a severe health threat around the world. In the current situation, the virus is rapidly spreading worldwide, and the discovery of a vaccine and potential therapeutics are critically essential. The crystal structure for the main protease (Mpro) of SARS-CoV-2, 3-chymotrypsin-like cysteine protease (3CLpro), was recently made available and is considerably similar to the previously reported SARS-CoV. Due to its essentiality in viral replication, it represents a potential drug target. Herein, a computer-aided drug design (CADD) approach was implemented for the initial screening of 13 approved antiviral drugs. Molecular docking of 13 antivirals against the 3-chymotrypsin-like cysteine protease (3CLpro) enzyme was accomplished, and indinavir was described as a lead drug with a docking score of -8.824 and a XP Gscore of -9.466 kcal/mol. Indinavir possesses an important pharmacophore, hydroxyethylamine (HEA), and thus, a new library of HEA compounds (>2500) was subjected to virtual screening that led to 25 hits with a docking score more than indinavir. Exclusively, compound 16 with a docking score of -8.955 adhered to drug-like parameters, and the structure-activity relationship (SAR) analysis was demonstrated to highlight the importance of chemical scaffolds therein. Molecular dynamics (MD) simulation analysis performed at 100 ns supported the stability of 16 within the binding pocket. Largely, our results supported that this novel compound 16 binds with domains I and II, and the domain II-III linker of the 3CLpro protein, suggesting its suitability as a strong candidate for therapeutic discovery against COVID-19.

Anchoring of triethanolamine-Cu(II) complex on magnetic carbon nanotube as a promising recyclable catalyst for the synthesis of 5-substituted 1H-tetrazoles from aldehydes.

The development of heterogenization of copper nanoparticles on conductive supports is very challenging and has received much attention. Here, we synthesize a practical, efficient, and inexpensive heterogeneous catalyst to grow stable metallic copper(II) nanoparticles on the surface of magnetic carbon nanotube (Fe3O4-CNT) catalyst support physically functionalized with triethanolamine (TEA) that acts as a low-cost and non-toxic ligand to capture the copper nanoparticles [Fe3O4-CNT-TEA-Cu(II)]. The as-prepared heterogeneous catalyst was characterized by different techniques, such as Fourier transform infrared spectroscopy, energy-dispersive X-ray spectroscopy, thermogravimetric analysis, vibrating sample magnetometer, X-ray diffraction patterns, field-emission scanning electron microscopy, and atomic absorption spectroscopy analysis. The catalytic behavior of Fe3O4-CNT-TEA-Cu(II) was investigated in the preparation of 5-substituted 1H-tetrazole derivatives via one-pot, three-component reaction between aromatic aldehydes, hydroxylamine, and sodium azide. The low catalyst loading, wide substrate scope, use of inexpensive materials, simple separation of the catalyst from the reaction mixture by an external magnet, short reaction times, easy workup, affordability, and superb yield are some advantages of this protocol.

N-Docosahexaenoylethanolamine Attenuates Neuroinflammation and Improves Hippocampal Neurogenesis in Rats with Sciatic Nerve Chronic Constriction Injury.

Chronic neuropathic pain is a condition that causes both sensory disturbances and a variety of functional disorders, indicating the involvement of various brain structures in pain pathogenesis. One of the factors underlying chronic neuropathic pain is neuroinflammation, which is accompanied by microglial activation and pro-inflammatory factor release. N-docosahexaenoylethanolamine (DHEA, synaptamide) is an endocannabinoid-like metabolite synthesized endogenously from docosahexaenoic acid. Synaptamide exhibits anti-inflammatory activity and improves neurite outgrowth, neurogenesis, and synaptogenesis within the hippocampus. This study aims to evaluate the effects of synaptamide obtained by the chemical modification of DHA, extracted from the Far Eastern raw material Berryteuthis magister on neuroinflammatory response and hippocampal neurogenesis changes during neuropathic pain. The study of microglial protein and cytokine concentrations was performed using immunohistochemistry and ELISA. The brain lipid analysis was performed using the liquid chromatography-mass spectrometry technique. Behavioral experiments showed that synaptamide prevented neuropathic pain-associated sensory and behavioral changes, such as thermal allodynia, impaired locomotor activity, working and long-term memory, and increased anxiety. Synaptamide attenuated microglial activation, release of proinflammatory cytokines, and decrease in hippocampal neurogenesis. Lipid analysis revealed changes in the brain N-acylethanolamines composition and plasmalogen concentration after synaptamide administration. In conclusion, we show here that synaptamide may have potential for use in preventing or treating neuropathic cognitive pain and emotional effects.

Synthesis, Molecular Modeling and Biological Evaluation of Metabolically Stable Analogues of the Endogenous Fatty Acid Amide Palmitoylethanolamide.

Palmitoylethanolamide (PEA) belongs to the class of N-acylethanolamine and is an endogenous lipid potentially useful in a wide range of therapeutic areas; products containing PEA are licensed for use in humans as a nutraceutical, a food supplement, or food for medical purposes for its analgesic and anti-inflammatory properties demonstrating efficacy and tolerability. However, the exogenously administered PEA is rapidly inactivated; in this process, fatty acid amide hydrolase (FAAH) plays a key role both in hepatic metabolism and in intracellular degradation. So, the aim of the present study was the design and synthesis of PEA analogues that are more resistant to FAAH-mediated hydrolysis. A small library of PEA analogues was designed and tested by molecular docking and density functional theory calculations to find the more stable analogue. The computational investigation identified RePEA as the best candidate in terms of both synthetic accessibility and metabolic stability to FAAH-mediated hydrolysis. The selected compound was synthesized and assayed ex vivo to monitor FAAH-mediated hydrolysis and to confirm its anti-inflammatory properties. 1H-NMR spectroscopy performed on membrane samples containing FAAH in integral membrane protein demonstrated that RePEA is not processed by FAAH, in contrast with PEA. Moreover, RePEA retains PEA's ability to inhibit LPS-induced cytokine release in both murine N9 microglial cells and human PMA-THP-1 cells.