Revisiting the role of 3-nitrotyrosine residues in the formation of alpha-synuclein oligomers and fibrils.

Nitration of tyrosine residues in alpha-synuclein (a-syn) has been detected in different synucleinopathies, including Parkinson's disease. The potential role of 3-nitrotyrosine formation in a-syn, as an oxidative post-translational modification, is still elusive. In this work, we generated well-characterized tyrosine nitrated a-syn monomers and studied their capability to form oligomers and fibrils. We constructed tyrosine to phenylalanine mutants, containing a single tyrosine residue, a-syn mutant Y(125/133/136)F and Y(39/125/133)F) and assessed the impact in a-syn biophysical properties. Nitrated wild-type a-syn and the Y-F mutants, with one 3-nitrotyrosine residue in either the protein's N-terminal or C-terminal region, showed inhibition of fibril formation but retained the capacity of oligomer formation. The inhibition of a-syn fibrillation occurs even when an important amount of unmodified a-syn is still present. We characterized oligomers from both nitrated and non-nitrated forms of the wild-type protein and the mutant forms obtained. Our results indicate that the formation of 3-nitrotyrosine in a-syn could induce an off-pathway oligomer formation which may have an important impact in the development of synucleinopathies.

Photochemically-induced protein tyrosine nitration in vitro and in cellula by 5-methyl-1,4-dinitro-1H-imidazole (DNI): synthesis and biochemical characterization.

The photochemical nitrating agent 5-methyl-1,4-dinitro-1H-imidazole (DNI) has been recently described as an effective tool for nitrating tyrosine residues in proteins under 390 nm irradiation (Long T. et al., 2021). Herein, we describe the one-step synthesis of DNI from the precursor 4-methyl-5-nitro-1H-imidazole with good yield (66%) and high purity (>99%). Spectral analysis of DNI reveals two maximum peaks (228 and 290 nm) with maximum nitration yields and kinetics occurring at 290 nm. Electron paramagnetic resonance (EPR)- and mass spectrometry (MS)- spin trapping analysis evidenced the formation of nitrogen dioxide (•NO2) upon irradiation of DNI, implying the homolysis of the N-N bond in the DNI molecule. Irradiation of DNI at 290, 390 nm, or UVA light (315-400 nm), produced tyrosine nitration, with yields approaching ca. 30% with respect to DNI at 290 nm exposure. Indeed, using alpha-synuclein as a model protein, the main protein post-translational modification triggered by DNI was the generation of 3-nitrotyrosine as shown by MS analysis. Additionally, the formation of di-tyrosine was also observed. Finally, intracellular •NO2 production upon DNI photolysis in bovine aortic endothelial cells was evidenced by the nitration of the tyrosine analog probe p-hydroxyphenylacetic acid (PHPA) and cellular protein tyrosine nitration.

Garbage in, garbage out: how reliable training data improved a virtual screening approach against SARS-CoV-2 MPro.

Introduction: The identification of chemical compounds that interfere with SARS-CoV-2 replication continues to be a priority in several academic and pharmaceutical laboratories. Computational tools and approaches have the power to integrate, process and analyze multiple data in a short time. However, these initiatives may yield unrealistic results if the applied models are not inferred from reliable data and the resulting predictions are not confirmed by experimental evidence. Methods: We undertook a drug discovery campaign against the essential major protease (MPro) from SARS-CoV-2, which relied on an in silico search strategy -performed in a large and diverse chemolibrary- complemented by experimental validation. The computational method comprises a recently reported ligand-based approach developed upon refinement/learning cycles, and structure-based approximations. Search models were applied to both retrospective (in silico) and prospective (experimentally confirmed) screening. Results: The first generation of ligand-based models were fed by data, which to a great extent, had not been published in peer-reviewed articles. The first screening campaign performed with 188 compounds (46 in silico hits and 100 analogues, and 40 unrelated compounds: flavonols and pyrazoles) yielded three hits against MPro (IC50 ≤ 25 µM): two analogues of in silico hits (one glycoside and one benzo-thiazol) and one flavonol. A second generation of ligand-based models was developed based on this negative information and newly published peer-reviewed data for MPro inhibitors. This led to 43 new hit candidates belonging to different chemical families. From 45 compounds (28 in silico hits and 17 related analogues) tested in the second screening campaign, eight inhibited MPro with IC50 = 0.12-20 µM and five of them also impaired the proliferation of SARS-CoV-2 in Vero cells (EC50 7-45 µM). Discussion: Our study provides an example of a virtuous loop between computational and experimental approaches applied to target-focused drug discovery against a major and global pathogen, reaffirming the well-known "garbage in, garbage out" machine learning principle.

Adaptation and validation of the Adult Dispositional Hope Scale in the Ecuadorian context.

This study aimed to evidence the validity and reliability of the Ecuadorian version of the Adult Dispositional Hope Scale, one of the most studied concepts of positive psychology. The adaptation process included translation and semantic and idiomatic validation. For content validation, an expert review and focus group were conducted. The questionnaire was applied to 2423 workers in Ecuador with a mean age of 37 years (SD = 9.04), and 65.6% were women. A confirmatory factor analysis was conducted to assess the validity of the scale's dimensionality. The reliability and convergent and discriminant validity were also evaluated. In order to investigate the best solution for an Ecuadorian version of the Adult Dispositional Hope Scale, four structural models were assessed. The unidimensional solution was the most adequate structure for the scale. The internal consistency of the scale was adequate. The Adult Dispositional Hope Scale (ADHS) was developed to assess this positive psychological state and has been the most used tool in many contexts. To our knowledge, this study is the first to adapt the Adult Dispositional Hope Scale into the Ecuadorian context and evaluate its validity. The findings support its reliability, factorial, and construct validity in the Ecuadorian context. Furthermore, the results show that dispositional hope acts as a protective factor, promoting work engagement and preventing burnout.

Adaptation and validation of the Adult Dispositional Hope Scale in the Ecuadorian context

Abstract This study aimed to evidence the validity and reliability ofthe Ecuadorian version ofthe Adult Dispositional Hope Scale, one of the most studied concepts of positive psychology. The adaptation process included translation and semantic and idiomatic validation. For content validation, an expert review and focus group were conducted. The questionnaire was applied to 2423 workers in Ecuador with a mean age of 37 years (SD = 9.04), and 65.6% were women. A confirmatory factor analysis was conducted to assess the validity of the scale's dimensionality. The reliability and convergent and discriminant validity were also evaluated. In order to investigate the best solution for an Ecuadorian version ofthe Adult Dispositional Hope Scale, four structural models were assessed. The unidimensional solution was the most adequate structure for the scale. The internal consistency of the scale was adequate. The Adult Dispositional Hope Scale (ADHS) was developed to assess this positive psychological state and has been the most used tool in many contexts. To our knowledge, this study is the first to adapt the Adult Dispositional Hope Scale into the Ecuadorian context and evaluate its validity. The findings support its reliability, factorial, and construct validity in the Ecuadorian context. Furthermore, the results show that dispositional hope acts as a protective factor, promoting work engagement and preventing burnout.

Extracellular Alpha-Synuclein: Mechanisms for Glial Cell Internalization and Activation.

Alpha-synuclein (α-syn) is a small protein composed of 140 amino acids and belongs to the group of intrinsically disordered proteins. It is a soluble protein that is highly expressed in neurons and expressed at low levels in glial cells. The monomeric protein aggregation process induces the formation of oligomeric intermediates and proceeds towards fibrillar species. These α-syn conformational species have been detected in the extracellular space and mediate consequences on surrounding neurons and glial cells. In particular, higher-ordered α-syn aggregates are involved in microglial and oligodendrocyte activation, as well as in the induction of astrogliosis. These phenomena lead to mitochondrial dysfunction, reactive oxygen and nitrogen species formation, and the induction of an inflammatory response, associated with neuronal cell death. Several receptors participate in cell activation and/or in the uptake of α-syn, which can vary depending on the α-syn aggregated state and cell types. The receptors involved in this process are of outstanding relevance because they may constitute potential therapeutic targets for the treatment of PD and related synucleinopathies. This review article focuses on the mechanism associated with extracellular α-syn uptake in glial cells and the consequent glial cell activation that contributes to the neuronal death associated with synucleinopathies.

The Ecuadorian Version of the Burnout Assessment Tool (BAT): Adaptation and Validation.

This study aimed to adapt and show evidence of validity for the Ecuadorian version of the Burnout Assessment Tool (BAT) considering only its "core" dimensions. The adaptation process included its translation and back translation. For content validation, expert reviews and focus groups were carried out. A confirmatory factor analysis was used to identify the psychometric properties and dimensionality of the scale. The reliability of the scale was assessed through the alpha, omega and composite reliability indices. To carry out the study, the questionnaire was applied to a sample of workers with a high level of education in Ecuador. In total, 2237 respondents were considered in the analysis. The results showed that the hierarchical model for BAT-23 and its short version, the BAT-12 scale, is the most adequate structure for analysis of the construct in the Ecuadorian context. The reliability of the general factor of burnout and its dimensions, evaluated by composite reliability, omega and Cronbach's alpha, showed satisfactory indices. The findings obtained provide support for the reliability and validity of the Burnout Assessment Tool for the Ecuadorian context.

Salaried Workers' Self-Perceived Health and Psychosocial Risk in Guayaquil, Ecuador.

Self-perceived health is an important indicator of occupational health. This research explored the relationship between poor self-perceived health and exposure to psychosocial risk factors, taking into account potential socio-demographic, occupational, and employment determinants. Using data from the First Survey of Occupational Safety and Health Conditions, covering 1049 salaried workers in Guayaquil, Ecuador, descriptive and stratified binary logistic regression analyses (odds ratios with corresponding 95% confidence intervals) were carried out. A significant relationship was found between exposure to psychosocial risk factors and the probability of presenting poor self-perceived health by socio-demographic, occupational, and employment characteristics. Occupational exposure factors to psychosocial risks were predictors of self-perceived ill health and were related to the variables analyzed; the most frequently expressed factors among the respondents were cognitive demands (DCOG) and job insecurity (IL). The results have implications in terms of designing effective workplace interventions pursuant to ensuring the health and well-being of employees.

Nitroalkylation of α-Synuclein by Nitro-Oleic Acid: Implications for Parkinson's Disease.

α-Synuclein (α-syn) represents the main component of the amyloid aggregates present in Parkinson's disease and other neurodegenerative disorders, collectively named synucleinopathies. Although α-syn is considered a natively unfolded protein, it shows great structural flexibility which allows the protein to adopt highly rich beta-sheet structures like protofibrils, oligomers and fibrils. In addition, this protein can adopt alpha-helix rich structures when interacts with fatty acids or acidic phospholipid vesicle membranes. When analyzing the toxicity of α-syn, protein oligomers are thought to be the main neurotoxic species by mechanisms that involve modification of intracellular calcium levels, mitochondrial and lysosomal function. Extracellular fibrillar α-syn promotes intracellular protein aggregation and shows many toxic effects as well. Nitro-fatty acids (nitroalkenes) represent novel pleiotropic anti-inflammatory signaling mediators that could interact with α-syn to exert unraveling actions. Herein, we demonstrated that nitro-oleic acid (NO2-OA) nitroalkylate α-syn, forming a covalent adduct at histidine-50. The nitroalkylated-α-syn exhibited strong affinity for phospholipid vesicles, moving the protein to the membrane compartment independent of composition of the membrane phospholipids. Moreover, NO2-OA-modified α-syn showed a reduced capacity to induce α-syn fibrillization compared to the non-nitrated oleic acid. From this data we hypothesize that nitroalkenes, in particular NO2-OA, may inhibit α-syn fibril formation exerting protective actions in Parkinson's disease.

Impact of monomeric, oligomeric and fibrillar alpha-synuclein on astrocyte reactivity and toxicity to neurons.

Synucleinopathies are a group of neurodegenerative disorders characterized by the presence of aggregated and fibrillar forms of alpha-synuclein (α-syn). Here, we analyze the effect of different species of α-syn, including monomeric, oligomeric and fibrillar forms of the protein, on rat astrocytes. Astrocytes treated with these distinct forms of α-syn showed an increase in long and thin processes and glial fibrillary acidic protein expression, indicating cell activation, high levels of intracellular oxidants and increased expression of cytokines. Moreover, astrocytes incubated with the different species induced hippocampal neuronal death in co-culture, and cytotoxicity was particularly enhanced by exposure to fibrillar α-syn. Further exploration of the mechanisms behind astrocyte activation and cytotoxicity revealed differences between the assessed α-syn species. Only oligomers induced mitochondrial dysfunction in astrocytes and significantly increased extracellular hydrogen peroxide production by these cells. Besides, TNF-α and IL-1ß (interleukin 1ß) expression presented different kinetics and levels depending on which species induced the response. Our data suggest that α-syn species (monomeric, oligomeric and fibrillar) induce astrocyte activation that can lead to neuronal death. Nevertheless, the tested α-syn species act through different preferential mechanisms and potency. All together these results help to understand the effect of α-syn species on astrocyte function and their potential impact on the pathogenesis of Parkinson's disease and related α-synucleinopathies.

Oxidation and nitration of α-synuclein and their implications in neurodegenerative diseases.

Synucleinopathies include Parkinson's disease, dementia with Lewy bodies, Lewy body variant of Alzheimer's disease and multiple system atrophy, among the most relevant diseases. All of these diseases are characterized by the presence of amyloid inclusions in neurons, which are rich in the aggregate α-synuclein protein. What is the biological mechanism concerned in the gain-of-function that implicates the participation of α-synuclein in these diseases? Post-translational modifications of α-synuclein induced by nitroxidative stress are a relevant hypothesis that may explain many of the experimental data. We will review the biophysical and biochemical properties of α-synuclein, methionine residues oxidation, nitration and oxidation of tyrosine residues in α-synuclein, and modifications of α-synuclein mediated by proteins and lipids under nitroxidative stress conditions. The biological consequences of these modifications are analyzed in terms of the properties of α-synuclein oligomerization and fibrillation, degradation of α-synuclein and the implications in the immunological response.

Microwave-assisted synthesis of hydroxyphenyl nitrones with protective action against oxidative stress.

Oxidative stress plays an important role in neuronal death in neurodegenerative disorders such as Parkinson's disease (PD). Hydroxyphenyl nitrones, derivatives of the nitrone spin trap alpha-phenyl-N-tert-butylnitrone (PBN), were synthesized and their antioxidant, anti-inflammatory and neuroprotective activity in neural cells evaluated. These hydroxyphenyl nitrones 5-7 were synthesized by reaction of the corresponding hydroxybenzaldehyde with N-tert-butyl hydroxylamine under microwave irradiation. They showed good peroxyl free radical scavenger capacities, analyzed by oxygen radical absorbance capacity (ORAC). Also inhibited peroxynitrite-mediated tyrosine nitration of alpha-synuclein in vitro and protected human neuroblastoma (SH-SY5Y) cells against SIN-1 and 6-OHDA toxicity when micromolar concentrations were used. Besides, the hydroxyphenyl nitrones evaluated showed anti-inflammatory activity modulating nitrite production in primary neural cell cultures of astrocytes and microglia treated with lipopolysaccharide (LPS), a potent inflammatory agent. These experimental data suggest a potential therapeutic use of these hydroxyphenyl nitrones against oxygen and nitrogen reactive species involved in neurodegenerative pathology.