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In the present work, a structure-based design approach was used for the generation of a novel variant of synthetic glutathione transferase (PvGmGSTU) with higher sensitivity towards pesticides. Molecular modelling studies revealed Phe117 as a key residue that contributes to the formation of the hydrophobic binding site (H-site) and modulates the affinity of the enzyme towards xenobiotic compounds. Site-saturation mutagenesis of position Phe117 created a library of PvGmGSTU variants with altered kinetic and binding properties. Screening of the library against twenty-five different pesticides, showed that the mutant enzyme Phe117Ile displays 3-fold higher catalytic efficiency and exhibits increased affinity towards α-endosulfan, compared to the wild-type enzyme. Based on these catalytic features the mutant enzyme Phe117Ile was explored for the development of an optical biosensor for α-endosulfan. The enzyme was entrapped in alkosixylane sol-gel system in the presence of two pH indicators (bromocresol purple and phenol red). The sensing signal was based on the inhibition of the sol-gel entrapped GST, with subsequent decrease of released [H+] by the catalytic reaction, measured by sol–gel entrapped indicators. The assay response at 562 nm was linear in the range pH = 4–7. Linear calibration curves were obtained for α-endosulfan in the range of 0–30 μΜ. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N = 28). The method was successfully applied to the determination of α-endosulfan in real water samples without sample preparation steps.

Summary Concerted alteration of immune and metabolic homeostasis underlies several inflammation-related pathologies, ranging from metabolic syndrome to infectious diseases. Here, we explored the coordination of nucleic acid-dependent inflammatory responses and metabolic homeostasis. We reveal that the STING (stimulator of interferon genes) protein regulates metabolic homeostasis through inhibition of the fatty acid desaturase 2 (FADS2) rate-limiting enzyme in polyunsaturated fatty acid (PUFA) desaturation. STING ablation and agonist-mediated degradation increased FADS2-associated desaturase activity and led to accumulation of PUFA derivatives that drive thermogenesis. STING agonists directly activated FADS2-dependent desaturation, promoting metabolic alterations. PUFAs in turn inhibited STING, thereby regulating antiviral responses and contributing to resolving STING-associated inflammation. Thus, we have unveiled a negative regulatory feedback loop between STING and FADS2 that fine-tunes inflammatory responses. Our results highlight the role of metabolic alterations in human pathologies associated with aberrant STING activation and STING-targeting therapies., Graphical abstract, Highlights • STING inhibits FADS2-dependent desaturation of PUFAs and LC-PUFAs • STING activation leads to upregulation of FADS2-associated desaturase activity • STING agonists activate FADS2-dependent PUFA and LC-PUFA desaturation • PUFAs inhibit STING-dependent inflammatory responses, The stimulator of interferon genes (STING) is a central regulator of nucleic acid-associated inflammatory responses. Here, Vila et al. discover that STING regulates polyunsaturated fatty acid (PUFA) metabolism, and in turn, PUFAs inhibit STING-dependent inflammation. This cross-regulation is central to the maintenance of metabolic homeostasis.

In the present study, a structure-based approach was exploited for the in silico design of a nucleotide coenzyme mimetic ligand. The enzyme formate dehydrogenase (FDH) was employed as a model in our study. The biomimetic ligand was designed and synthesized based on a tryptamine/3-aminopropylphosphonic acid bi-substituted 1,3,5-triazine (Trz) scaffold (Tra-Trz-3APP), which potentially mimics the interactions of NAD+-FDH complex. Molecular docking studies of the biomimetic ligand predicted that it can occupy the same binding site as the natural coenzyme. Molecular modeling and dynamics simulations revealed that the ligand binds in an energetically more stable pose in the FDH binding site, as it adopts a more twisty conformation, compared to the natural coenzyme. Study of the FDH/Tra-Trz-3APP-Sepharose interaction, through adsorption equilibrium studies and site-directed mutagenesis of selected FDH coenzyme binding residues, provided additional experimental evidences of the specificity of the interaction. The Tra-Trz-3APP-Sepharose biomimetic adsorbent was further evaluated towards a range of different dehydrogenases and was exploited for the development of a single-step purification protocol for FDH. The protocol afforded enzyme with high yield and purity, suitable for analytical and industrial purposes.

Obesity and thus, lipotoxicity, is a major health risk factor. Modern exposure to environmental chemicals has contributed significantly to the obesity epidemic. The purpose of this study was to assess, via telemedicine and using bioelectrical impedance analysis (BIA) in schools, the levels of adiposity and other body composition parameters of Greek adolescents in relation with their metabolic syndrome (MetS) characteristics. A representative sample (1575 adolescents, 14.4 ± 1.7 years-old) of the Attica region population, underwent body composition assessment of fat mass (FM), fat-free mass (FFM), and total body water (TBW) and was evaluated for anthropometric and MetS characteristics. Males demonstrated higher FFM% and TBW% but lower FM% than females. Adolescents with abdominal obesity/MetS (n = 149/n = 40) demonstrated significantly (P Findings suggest that early “osteosarcopenic” elements of abdominal obesity/MetS may exist even in adolescence. The application of BIA, incorporated in the new approach methodology of telemedicine in schools, identified adolescents at risk for obesity complications.

Extracellular form of Francisella is able to cross various cell barriers and invade multiple organs, such as skin, liver, lung and central nervous system. Transient adhesion of Francisella to endothelial cells may trigger the process of translocation. In this report, we showed that Francisella tularensis subsp. holarctica ( Fth ) is able to adhere to the endothelial cells, while ICAM-1 may serve as an adhesion molecule for Fth . Pull down and af fi nity ligand binding assays indicated that the PilE4 could be the probable ligand for ICAM-1. Further deciphering of this ligand:receptor interaction revealed that PilE4 interacts with Ig-like C2-type 1 domain of ICAM-1. To corroborate the role of PilE4 and ICAM-1 interaction in adhesion of extracellular form of Fth to endothelial cells, ICAM-1 was blocked with monoclonal anti-ICAM-1 antibody prior to the incubation with Fth and numbers of adherent bacteria were counted. Blocking of the ICAM-1 signi fi cantly reduced (500 e fold, P < 0.05) number of adherent Fth compared to unblocked cells. PilE4:ICAM-1 interaction unfolded here may provide a new perspective on molecules involved in the adhesion of extracellular form of Francisella to endothelial cells and probably its translocation across endothelial barriers.

Microalgae are unicellular microorganisms indispensible for environmental stability and life on earth, because they produce approximately half of the atmospheric oxygen, with simultaneously feeding on the harmful greenhouse gas carbon dioxide. Using gene fusion analysis, a series of five fusion/fission events was identified, that provided the basis for critical insights to their evolutionary history. Moreover, the three-dimensional structures of both the fused and the component proteins were predicted, allowing us to envisage putative protein-protein interactions that are invaluable for the efficient usage, handling and exploitation of microalgae. Collectively, our proposed approach on the five fusion/fission alga protein events contributes towards the expansion of the microalgae knowledgebase, bridging protein evolution of the ancient microalgal species and the rapidly evolving, modern, bioinformatics field. (C) 2013 Elsevier B.V. All rights reserved.

This study pertains to the regulatory network of neurogenin3 (NGN3, approved symbol: NEUROG3), the main regulator of insulin producing cells’ formation.In silico regulatory region analyses of known and novel targets of NGN3 revealed the presence of two variants of a regulatory module that appeared conserved at the most phylogenetically distant species with pancreas. Both variants of this module contained binding sites of six transcription factors implicated in pancreas development. Nevertheless, an additional factor was found only into the module of the down-regulated by NGN3 genes. Whole genome analyses confirmed the statistical significance of these regulatory modules. Investigation of protein–protein interactions among the factors bound into these sequences indicated the formation of alternative protein complexes resulting into the up- or down-regulation of the respective genes.Subsequently, an NGN3-guided regulatory network, was modeled, describing the interactions among the analyzed genes with their transcriptional regulators, leading into the differentiation of cells capable of producing insulin.

Poly(A)-specific ribonuclease (PARN) is a 3′-exoribonuclease that efficiently degrades poly(A) tails and regulates, in part, mRNA turnover rates. We have previously reported that adenosine- and cytosine-based glucopyranosyl nucleoside analogues with adequate tumour-inhibitory effect could effectively inhibit PARN. In the present study we dissect the mechanism of a more drastic inhibition of PARN by novel glucopyranosyl analogues bearing uracil, 5-fluorouracil or thymine as the base moiety. Kinetic analysis showed that three of the compounds are competitive inhibitors of PARN with Ki values in the low μM concentration and significantly lower (11- to 33-fold) compared to our previous studies. Detailed kinetic analysis of the most effective inhibitor, the uracil-based nucleoside analogue (named U1), revealed slow-binding behaviour. Subsequent molecular docking experiments showed that all the compounds which inhibited PARN can efficiently bind into the active site of the enzyme through specific interactions. The present study dissects the inhibitory mechanism of this novel uracil-based compound, which prolongs its inhibitory effect through a slow-binding and slow-release mode at the active site of PARN, thus contributing to a more efficient inhibition. Such analogues could be used as leading compounds for further rationale design and synthesis of efficient and specific therapeutic agents. Moreover, our data reinforce the notion that human PARN can be established as a novel molecular target of potential anti-cancer agents through lowering mRNA turnover rates.