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DapE is a Zn2+-metallohydrolase recognized as a drug target for bacterial control. It is a homodimer that requires the exchange of interface strands by an induced fit essential for catalysis. Identifying novel anti-DapE agents requires greater structural details. Most of the characterized DapEs are from the Gram-negative group. Here, two high-resolution DapE crystal structures from Enterococcus faecium are presented for the first time with novel aspects. A loosened enzyme intermediate between the open and closed conformations is observed. Substrates may bind to loose state, subsequently it closes, where hydrolysis occurs, and finally, the change to the open state leads to the release of the products. Mutation of His352 suggests a role, along with His194, in the oxyanion stabilization in the mono-metalated Zn2+ isoform, while in the di-metalated isoform, the metal center 2 complements it function. An aromatic-π box potentially involved in the interaction of DapE with other proteins, and a peptide flip could determine the specificity in the Gram-positive ArgE/DapE group. Finally, details of two extra-catalytic cavities whose geometry changes depending on the conformational state of the enzyme are presented. These cavities could be a target for developing non-competitive agents that trap the enzyme in an inactive state.

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Resumen Antecedentes: la ingeniería tisular permite obtener órganos como injertos a partir de tejidos descelularizados, regenerados con células autólogas. Objetivo: descelularizar y regenerar tráqueas porcinas. Material y métodos: se descelularizaron tráqueas porcinas colocándolas cada una en el epiplón de cuatro cerdos Yorkshire para su regeneración in vivo. Una tráquea desce-lularizada con tritón (DT), descelularizada con desoxicolato (DD), descelularizada con desoxicolato y reforzada con un polímero y células epiteliales (DDR), y una nativa crio-preservada (NC). Después de 8 días se obtuvieron la DD, NC y DDR; y al día 15, la DT. Se las evaluó mecánica e histológicamente, se realizó el análisis casuístico. Resultados: las tráqueas descelularizadas conservaron la integridad del cartílago, sin diferencias mecánicas, excepto la DDR con mayor rigidez. Las tráqueas regeneradas presentaron menor rigidez, excepto la DDR que además perdió el epitelio y la vascula-ridad. Las DT, DD mostraron epitelio no respiratorio, fibrosis y vasculogénesis con in-flamación. Conclusiones: las matrices conservaron sus características mecánicas. La regenera-ción in vivo ofrece ventajas como la esterilidad, interacción celular, nutrientes; es senci-llo, factible y económico, pero no hay control del crecimiento celular y vascularización, y los tejidos presentaron alteraciones mecánicas e histológicas. El polímero impidió la re-epitelialización y revascularización. Este estudio abre la posibilidad de mejorar las me-todologías de ingeniería tisular aplicadas al tejido traqueal.

Abstract Introduction: tissue engineering makes it possible to obtain organs as grafts from de-cellularized tissues, regenerated with autologous cells.Objective: decellularize and regenerate porcine tracheas.ARTÍCULO ORIGINAL | Respirar, 2023; 15(3): 188-199 | ISSN 2953-3414 | https://doi.org/10.55720/respirar.15.3.5RECIBIDO: 9 agosto 2023ACEP TADO: 31 agosto 2023 Elisa Barrera-Ramírezhttps://orcid.org/0000-0002-2778-0882Rubén Efraín Garrido-Cardonahttps://orcid.org/0000-0001-6083-5403Alejandro Martínez-Martínezhttps://orcid.org/0000-0003-3448-910XLuis Fernando Plenge-Tellecheahttps://orcid.org/0000-0002-1619-5004Edna Rico-Escobarhttps://orcid.org/0000-0002-0933-0220Esta revista está bajo una licencia de Creative Commons Reconocimiento 4.0 Internacional. Respirar 2023; 15 (3): 189ARTÍCULO ORIGINAL / E. Barrera-Ramírez, R.E. Garrido-Cardona, A. Martínez-Martínez, L.F. Plenge-Tellechea, E. Rico-EscobarDescelularización y regeneración de tráqueaISSN 2953-3414Materials and Methods: Porcine tracheas were decellularized by placing each one in the omentum of four Yorkshire pigs for regeneration in vivo. A trachea decellularized with triton (DT), decellularized with deoxycholate (DD), decellularized with deoxycho-late and reinforced with a polymer, and epithelial cells (DDR), and a cryopreserved na-tive (NC). After 8 days, the DD, NC and DDR were obtained; and on day 15, the DT. The evaluation was mechanically and histologically, performing the case analysis.Results: the decellularized tracheas preserved the integrity of the cartilage, with no me-chanical differences, except for the DDR with greater rigidity. The regenerated trache-as presented less rigidity, except the DDR, which also lost the epithelium and vascular-ity. The DT, DD showed non-respiratory epithelium, fibrosis and vasculogenesis with inflammation.Conclusions: the matrices retained their mechanical characteristics, in vivo regenera-tion offers advantages such as sterility, cell interaction, nutrients; it is simple, feasible and economical, but there is no control of cell growth and vascularization, and the tis-sues presented mechanical and histological alterations. The polymer prevented re-epi-thelialization and revascularization. This study opens the possibility of improving tissue engineering methodologies applied to tracheal tissue.

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Inflammation may negatively impact health, particularly that of the central nervous system. Phenolic compounds are bioactive molecules present in fruits and vegetables with potential anti-inflammatory effects. The purpose of the present work is to review the immunomodulatory bioactivities of phenolic compounds in the periphery and in the central nervous system. Results show that various types of phenolics are able to counter diet- or pathogen-induced systemic inflammation (among others) in various models. In vitro data show significant effects of flavonoids and phenolic acids in particular; similar bioactivities were reported in vivo, when administering them as pure compounds or from fruit and vegetable extracts that contain them. In the central nervous system, phenolics counter chronic inflammation and aggressive acute inflammatory processes, such as ischemic events, when administered preemptively and even therapeutically. We therefore conclude that the immunomodulatory potential of phenolic compounds can maintain an adequate immune response; their regular consumption should therefore be prioritized in order to maintain health. PRACTICAL APPLICATIONS: The immune response must be carefully regulated in order to avoid its deleterious effects. The present work highlights how phenolic compounds, dietary components ubiquitous in everyday diet, are able to maintain it within an adequate range. As humans are exposed to more proinflammatory stimuli (inadequate dietary pattern, mental stress, environmental pollution, chronic diseases, etc.), it becomes necessary to counter them, and consuming adequate amounts of foods that contain compounds with this ability is a rather simple strategy. Thus, the present work highlights how fruits and vegetables can help to maintain an adequate immune response that can preserve systemic health and that of the central nervous system. Furthermore, specific compounds contained in them can also be ideal candidates for additional in-depth studies, which can potentially lead to the development of potent, targeted, and safe anti-inflammatory molecules.

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The application of new technologies for treatments against different diseases is increasingly innovative and effective. In the case of nanomedicine, the combination of nanoparticles with biological membranes consists of a "camouflage" technique, which improves biological interaction and minimizes the secondary effects caused by these remedies. In this work, gold nanoparticles synthesized by chemical reduction (Turkevich ≈13 nm) were conjugated with fluorescein isothiocyanate to amplify their optical properties. Fluorescent nanoparticles were deposited onto the surface of hemoglobin-free erythrocytes. Ghost erythrocytes were obtained from red blood cells by density gradient separation in a hypotonic medium and characterized with fluorescence, optical, and electron microscopy; the average size of erythrocyte ghosts was 9 µm. Results show that the functional groups of sodium citrate (COO-) and fluorophore (-N=C=S) adhere by electrostatic attraction to the surface of the hemoglobin-free erythrocyte membrane, forming the membrane-particle-fluorophore. These interactions can contribute to imaging applications, by increasing the sensitivity of measurement caused by surface plasmon resonance and fluorescence, in the context of biological membranes.

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BACKGROUND: Chronic psychological distress is considered today a pandemic due to the modern lifestyle and has been associated with various neurodegenerative, autoimmune, or systemic inflammation-related diseases. Stress is closely related to liver disease exacerbation through the high activity of the endocrine and autonomic nervous systems, and the connection between the development of these pathologies and the physiological effects induced by oxidative stress is not yet completely understood. The use of nootropics, as the cognitive enhancer and antioxidant piracetam, is attractive to repair the oxidative damage. A proteomic approach provides the possibility to obtain an in-depth comprehension of the affected cellular processes and the possible consequences for the body. Therefore, we considered to describe the effect of distress and piracetam on the liver proteome. METHODS: We used a murine model of psychological stress by predatory odor as a distress paradigm. Female Sprague-Dawley rats were distributed into four experimental groups (n = 6 - 7/group) and were exposed or not to the stressor for five days and treated or not with piracetam (600 mg/kg) for six days. We evaluated the liver proteome by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1D-SDS-PAGE) followed by liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Besides, we analyzed the activity of liver antioxidant enzymes, the biochemical parameters in plasma and rat behavior. RESULTS: Our results showed that distress altered a wide range of proteins involved in amino acids metabolism, glucose, and fatty acid mobilization and degradation on the way to produce energy, protein folding, trafficking and degradation, redox metabolism, and its implications in the development of the non-alcoholic fatty liver disease (NAFLD). Piracetam reverted the changes in metabolism caused by distress exposure, and, under physiological conditions, it increased catabolism rate directed towards energy production. These results confirm the possible relationship between chronic psychological stress and the progression of NAFLD, as well as we newly evidenced the controversial beneficial effects of piracetam. Finally, we propose new distress biomarkers in the liver as the protein DJ-1 (PARK7), glutathione peroxidase 1 (GPX), peroxiredoxin-5 (PRDX5), glutaredoxin 5 (GLRX5), and thioredoxin reductase 1 (TXNDR1), and in plasma as biochemical parameters related to kidney function such as urea and blood urea nitrogen (BUN) levels.

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Psychological distress induces oxidative stress and alters mitochondrial metabolism in the nervous and immune systems. Psychological distress promotes alterations in brain metabolism and neurochemistry in wild-type (WT) rats in a similar manner as in Parkinsonian rats lacking endogenous PTEN-induced kinase 1 (PINK1), a serine/threonine kinase mutated in a recessive forms of Parkinson's disease. PINK1 has been extensively studied in the brain, but its physiological role in peripheral tissues and the extent to which it intersects with the neuroimmune axis is not clear. We surmised that PINK1 modulates the bioenergetics of peripheral blood mononuclear cells (PBMCs) under basal conditions or in situations that promote oxidative stress as psychological distress. By using an XF metabolic bioanalyzer, PINK1-KO-PBMCs showed significantly increased oxidative phosphorylation and basal glycolysis compared to WT cells and correlated with motor dysfunction. In addition, psychological distress enhanced the glycolytic capacity in PINK1-KO-PBMCs but not in WT-PBMCs. The level of antioxidant markers and brain-derived neurotrophic factor were altered in PINK1-KO-PBMCs and by psychological distress. In summary, our data suggest that PINK1 is critical for modulating the bioenergetics and antioxidant responses in PBMCs whereas lack of PINK1 upregulates compensatory glycolysis in response to oxidative stress induced by psychological distress.

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Bulk Cu compounds such as Cu(OH)2 are extensively used as pesticides in agriculture. Recent investigations suggest that Cu-based nanomaterials can replace bulk materials reducing the environmental impacts of Cu. In this study, stress responses of alfalfa (Medicago sativa L.) seedlings to Cu(OH)2 nanoparticle or compounds were evaluated. Seeds were immersed in suspension/solutions of a Cu(OH)2 nanoform, bulk Cu(OH)2, CuSO4, and Cu(NO3)2 at 25 and 75 mg/L. Six days later, the germination, seedling growth, and the physiological and biochemical responses of sprouts were evaluated. All Cu treatments significantly reduced root elongation (average = 63%). The ionic compounds at 25 and 75 mg/L caused a reduction in all elements analyzed (Ca, K, Mg, P, Zn, and Mn), excepting for S, Fe and Mo. The bulk-Cu(OH)2 treatment reduced K (48%) and P (52%) at 75 mg/L, but increased Zn at 25 (18%) and 75 (21%) mg/L. The nano-Cu(OH)2 reduced K (46%) and P (48%) at 75 mg/L, and also P (37%) at 25 mg/L, compared with control. Confocal microscopy images showed that all Cu compounds, at 75 mg/L, significantly reduced nitric oxide, concurring with the reduction in root growth. Nano Cu(OH)2 at 25 mg/L upregulated the expression of the Cu/Zn superoxide dismutase gene (1.92-fold), while ionic treatments at 75 mg/L upregulated (∼10-fold) metallothionein (MT) transcripts. Results demonstrated that nano and bulk Cu(OH)2 compounds caused less physiological impairments in comparison to the ionic ones in alfalfa seedlings.

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Chronic psychological stress is an important public health issue which generates behavioral changes, anxiety, immunosuppression and oxidative damage. Piracetam is a cognitive enhancer, at cellular level it protects from oxidative stress. The aim of this study was to evaluate the effect of psychological stress and of piracetam on circulating mononuclear cells by analyzing the biochemical spectrome using Synchrotron Radiation Fourier Transform Infrared Microspectroscopy (SR-µFTIR). Rats were exposed for five days to a stressor (cat odor) under oral administration of piracetam (600 mg/kg). SR-µFTIR analysis showed a decrease in bands associated to the lipids region (2852 cm-1, 2923 cm-1 and 2962 cm-1) and an increase absorption of the amide I band (1654 cm-1) under stress conditions. The principal component analysis showed increase oxidation of lipids (decrease of 3010 cm-1, 2923 cm-1 and 2852 cm-1 bands) as well as proteins denaturation (increase of 1610 cm-1 and 1690 cm-1 bands) under stress. Piracetam provided protection to polyunsaturated lipids (p ≤ 0.001) and lipids/proteins ratio (p ≤ 0.001). Behaviorally, this drug diminished fear and anxiety in stressed animals by the plus maze test (p ≤ 0.002). However, this drug induced oxidative stress in mononuclear cells from unstressed animals and altered their behavior.

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Urease is a nickel-dependent amidohydrolase that catalyses the decomposition of urea into carbamate and ammonia, a reaction that constitutes an important source of nitrogen for bacteria, fungi and plants. It is recognized as a potential antimicrobial target with an impact on medicine, agriculture, and the environment. The list of possible urease inhibitors is continuously increasing, with a special interest in those that interact with and block the flexible active site flap. We show that disulfiram inhibits urease in Citrullus vulgaris (CVU), following a non-competitive mechanism, and may be one of this kind of inhibitors. Disulfiram is a well-known thiol reagent that has been approved by the FDA for treatment of chronic alcoholism. We also found that other thiol reactive compounds (l-captopril and Bithionol) and quercetin inhibits CVU. These inhibitors protect the enzyme against its full inactivation by the thiol-specific reagent Aldrithiol (2,2'-dipyridyl disulphide, DPS), suggesting that the three drugs bind to the same subsite. Enzyme kinetics, competing inhibition experiments, auto-fluorescence binding experiments, and docking suggest that the disulfiram reactive site is Cys592, which has been proposed as a "hinge" located in the flexible active site flap. This study presents the basis for the use of disulfiram as one potential inhibitor to control urease activity.

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Mutations the in human DJ-1 (hDJ-1) gene are associated with early-onset autosomal recessive forms of Parkinson's disease (PD). hDJ-1/parkinsonism associated deglycase (PARK7) is a cytoprotective multi-functional protein that contains a conserved cysteine-protease domain. Given that cysteine-proteases can act on both amide and ester substrates, we surmised that hDJ-1 possessed cysteine-mediated esterase activity. To test this hypothesis, hDJ-1 was overexpressed, purified and tested for activity towards 4-nitrophenyl acetate (pNPA) as µmol of pNPA hydrolyzed/min/mg·protein (U/mg protein). hDJ-1 showed maximum reaction velocity esterase activity (Vmax = 235.10 ± 12.00 U/mg protein), with a sigmoidal fit (S0.5 = 0.55 ± 0.040 mM) and apparent positive cooperativity (Hill coefficient of 2.05 ± 0.28). A PD-associated mutant of DJ-1 (M26I) lacked activity. Unlike its protease activity which is inactivated by reactive oxygen species (ROS), esterase activity of hDJ-1 is enhanced upon exposure to low concentrations of hydrogen peroxide (<10 µM) and plateaus at elevated concentrations (>100 µM) suggesting that its activity is resistant to oxidative stress. Esterase activity of DJ-1 requires oxidation of catalytic cysteines, as chemically protecting cysteines blocked its activity whereas an oxido-mimetic mutant of DJ-1 (C106D) exhibited robust esterase activity. Molecular docking studies suggest that C106 and L126 within its catalytic site interact with esterase substrates. Overall, our data show that hDJ-1 contains intrinsic redox-sensitive esterase activity that is abolished in a PD-associated mutant form of the hDJ-1 protein.

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