Reactive oxygen species (ROS) are generated as the result of a number of physiological and pathological processes. Once formed ROS can promote multiple forms of oxidative damage, including protein oxidation, and thereby influence the function of a diverse array of cellular processes. This review summarizes the mechanisms by which ROS are generated in a variety of cell types, outlines the mechanisms which control the levels of ROS, and describes specific proteins which are common targets of ROS. Additionally, this review outlines cellular processes which can degrade or repair oxidized proteins, and ultimately describes the potential outcomes of protein oxidation on cellular homeostasis. In particular, this review focuses on the relationship between elevations in protein oxidation and multiple aspects of cellular metabolism. Together, this review describes a potential role for elevated levels of protein oxidation contributing to cellular dysfunction and oxidative stress via impacts on cellular metabolism.

The gut-brain axis is a bidirectional communication network functionally linking the gut and the central nervous system (CNS). Based on this, the rational manipulation of intestinal microbiota represents a novel attractive therapeutic strategy for the treatment of CNS-associated disorders. In this study, we explored the properties of a probiotic formulation (namely SLAB51) in counteracting brain oxidative damages associated with Alzheimer’s disease (AD). Specifically, transgenic AD mice (3xTg-AD) were treated with SLAB51 and the effects on protein oxidation, neuronal antioxidant defence and repair systems were monitored, with the particular focus on the role of SIRT1-related pathways. We demonstrated that SLAB51 markedly reduced oxidative stress in AD mice brain by activating SIRT1-dependent mechanisms, thus representing a promising therapeutic adjuvant in AD treatment.Electronic supplementary materialThe online version of this article (10.1007/s12035-018-0973-4) contains supplementary material, which is available to authorized users.

We present thick, uniform and rather flat melanin films obtained using spray deposition. The morphology of the films was investigated using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Temperature-dependent electrical resistance of melanin thin films evidenced a semiconductor-like character and a hysteretic behavior linked to an irreversible process of water molecule desorption from the melanin film. X-ray Photoelectron Spectroscopy (XPS) was carried out to analyze the role of the functional groups in the primary and secondary structure of the macromolecule, showing that the contribution of the 5,6-dihydroxyindole-2-carboxylic acid (DHICA) subunit to the molecule is about 35%. Comparison of the optical absorption of the thick (800nm) and thin (80nm) films showed a spectral change when the thickness increases. From in vacuum photoconductivity (PC) measured at controlled temperatures, we suggest that the melanin films exhibit a possible charge transport mechanism by means of delocalized pi states along the stacked planar secondary structure.

Acetoacetyl-CoA thiolase (EC 2.3.1.9), also called thiolase II, condenses two molecules of acetyl-CoA to give acetoacetyl-CoA. This is the first enzymatic step in the biosynthesis of isoprenoids via mevalonate (MVA). In this work, thiolase II from alfalfa (MsAACT1) was identified and cloned. The enzymatic activity was experimentally demonstrated in planta and in heterologous systems. The condensation reaction by MsAACT1 was proved to be inhibited by CoA suggesting a negative feedback regulation of isoprenoid production. Real-time RT-PCR analysis indicated that MsAACT1 expression is highly increased in roots and leaves under cold and salinity stress. Treatment with mevastatin, a specific inhibitor of the MVA pathway, resulted in a decrease in squalene production, antioxidant activity, and the survival of stressed plants. As expected, the presence of mevastatin did not change chlorophyll and carotenoid levels, isoprenoids synthesized via the plastidial MVA-independent pathway. The addition of vitamin C suppressed the sensitive phenotype of plants challenged with mevastatin, suggesting a critical function of the MVA pathway in abiotic stress-inducible antioxidant defence. MsAACT1 over-expressing transgenic plants showed salinity tolerance comparable with empty vector transformed plants and enhanced production of squalene without altering the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity in salt-stress conditions. Thus, acetoacetyl-CoA thiolase is a regulatory enzyme in isoprenoid biosynthesis involved in abiotic stress adaptation.

Objective. To identify the epitopes recognized by autoantibodies targeting platelet-derived growth factor receptor a (PDGFRa) in systemic sclerosis (SSc) and develop novel assays for detection of serum antiPDGFRa autoantibodies.Methods. Epstein-Barr virus-immortalized B cells from 1 patient with SSc (designated PAM) were screened for expression of IgG binding to PDGFRa and induction of reactive oxygen species in fibroblasts. The variable regions of anti-PDGFRa IgG were cloned into an IgG expression vector to generate distinct recombinant human monoclonal autoantibodies (mAb), which were characterized by binding and functional assays. The epitopes of anti-PDGFRa recombinant human mAb were defined by molecular docking, surface plasmon resonance binding assays, screening of a conformational peptide library spanning the PDGFRa extracellular domains, and expression analyses of alanine-scanned PDGFRa mutants. Direct or competitive enzyme-linked immunosorbent assays were established to detect all serum anti-PDGFRa autoantibodies or, selectively, the agonistic ones.Results. Three types of anti-PDGFRa recombinant human mAb, with the same V H but distinct V L chains, were generated. Nonagonistic V H PAM-V k 13B8 recognized 1 linear epitope, whereas agonistic V H PAM-V l 16F4 and V H PAM-V k 16F4 recognized 2 distinct conformational epitopes. Serum anti-PDGFRa antibodies were detected in 66 of 70 patients with SSc, 63 of 130 healthy controls, 11 of 26 patients with primary Raynaud's phenomenon (RP), and 13 of 29 patients with systemic lupus erythematosus (SLE). Serum V H PAM-V k 16F4-like antibodies were found in 24 of 34 patients with SSc, but not in healthy controls, patients with primary RP, or patients with SLE. Peptides composing the V H PAM-V k 16F4 epitope inhibited PDGFRa signaling triggered by serum IgG from SSc patients.Conclusion. Agonistic anti-PDGFRa autoantibodies are enriched in SSc sera and recognize specific conformational epitopes that can be used to discriminate agonistic from nonagonistic antibodies and block PDGFRa signaling in patients with SSc.

Nitrogen is the second most critical factor for crop production after water. In this study, the beneficial rhizobacterium Pseudomonas protegens Pf-5 was genetically modified to fix nitrogen using the genes encoding the nitrogenase of Pseudomonas stutzeri A1501 via the X940 cosmid. Pf-5 X940 was able to grow in L medium without nitrogen, displayed high nitrogenase activity and released significant quantities of ammonium to the medium. Pf-5 X940 also showed constitutive expression and enzymatic activity of nitrogenase in ammonium medium or in nitrogen-free medium, suggesting a constitutive nitrogen fixation. Similar to Pseudomonas protegens Pf-5, Pseudomonas putida, Pseudomonas veronii and Pseudomonas taetrolens but not Pseudomonas balearica and Pseudomonas stutzeri transformed with cosmid X940 showed constitutive nitrogenase activity and high ammonium production, suggesting that this phenotype depends on the genome context and that this technology to obtain nitrogen-fixing bacteria is not restricted to Pf-5. Interestingly, inoculation of Arabidopsis, alfalfa, tall fescue and maize with Pf-5 X940 increased the ammonium concentration in soil and plant productivity under nitrogen-deficient conditions. In conclusion, these results open the way to the production of effective recombinant inoculants for nitrogen fixation on a wide range of crops.

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BackgroundMalaria represents one of the most devastating infectious diseases. The lack of an effective vaccine and the emergence of drug resistance make necessary the development of new effective control methods. The recent identification of bacteria of the genus Asaia, associated with larvae and adults of malaria vectors, designates them as suitable candidates for malaria paratransgenic control.To better characterize the interactions between Asaia, Plasmodium and the mosquito immune system we performed an integrated experimental approach.MethodsQuantitative PCR analysis of the amount of native Asaia was performed on individual Anopheles stephensi specimens. Mosquito infection was carried out with the strain PbGFPCON and the number of parasites in the midgut was counted by fluorescent microscopy.The colonisation of infected mosquitoes was achieved using GFP or DsRed tagged-Asaia strains.Reverse transcriptase-PCR analysis, growth and phagocytosis tests were performed using An. stephensi and Drosophila melanogaster haemocyte cultures and DsRed tagged-Asaia and Escherichia coli strains.ResultsUsing quantitative PCR we have quantified the relative amount of Asaia in infected and uninfected mosquitoes, showing that the parasite does not interfere with bacterial blooming. The correlation curves have confirmed the active replication of Asaia, while at the same time, the intense decrease of the parasite.The ‘in vitro’ immunological studies have shown that Asaia induces the expression of antimicrobial peptides, however, the growth curves in conditioned medium as well as a phagocytosis test, indicated that the bacterium is not an immune-target.Using fluorescent strains of Asaia and Plasmodium we defined their co-localisation in the mosquito midgut and salivary glands.ConclusionsWe have provided important information about the relationship of Asaia with both Plasmodium and Anopheles. First, physiological changes in the midgut following an infected or uninfected blood meal do not negatively affect the residing Asaia population that seems to benefit from this condition. Second, Asaia can act as an immune-modulator activating antimicrobial peptide expression and seems to be adapted to the host immune response. Last, the co-localization of Asaia and Plasmodium highlights the possibility of reducing vectorial competence using bacterial recombinant strains capable of releasing anti-parasite molecules.