1. Investigación

For the first time in the literature, a relationship between the root application of melatonin and the greater capacity for resistance against Psedomonas syringae DC3000 in tomato plants has been established. Root delivered melatonin (100 μM), induced systemic resistance against pathogen reducing disease incidence by 51%. Mechanisms of action used by melatonin were assessing through different physiological, metabolic, and genetic markers. As a physiological marker, photosynthetic efficiency was studied, with a TARGAS 1 portable photosynthesis system. Metabolic markers were analysed on leaf powder collected 1 week after the pathogen challenge. These markers analysed were grouped into those related to the scavenging of Reactive Oxygen Species (ROS) and oxidative stress (ascorbate peroxidase (APX) activity, hydrogen peroxide (H2O2) concentration, malondialdehyde (MDA) concentration, and proline concentration) and those related to defence mechanisms (ß-1,3-glucanase and chitinase). Genetic markers were studied on leaf powder collected 6 h and 10 h after pathogen challenge. For this, the differential expression of the genes PR1, PR2 and PR3 was studied. Upon pathogen challenge, melatonin reverted the negative effects of the pathogen in net photosynthesis rate achieving similar values to healthy plants. Melatonin reduced oxidative stress, according to lower MDA (29%) and H2O2 (46%), improving ROS scavenging potential by enhancing APX activity (83%) and proline concentration (44%). Melatonin simultaneously triggered the salicylic acid (SA)-mediated pathway and the jasmonic acid/ethylene (JA/ET)-mediated pathway as the enzymatic activities ß-1,3-glucanase (Pathogenesis-Related protein 2; PR2; 103%) and chitinase activitiy (Pathogenesis-Related protein 3; PR3; 44%), markers of the first and second pathways respectively, were enhanced. This enhanced activity was consistent with enhanced expression of genes encoding PR2 and PR3. Results obtained indicate that melatonin, a natural plant compound, could be used in tomato cultivation as an economical and ecofriendly chemical agent against biotic stress.

We evaluated the ability of metabolic elicitors extracted from Pseudomonas fluorescens N21.4 to induce systemic resistance (ISR) in Arabidopsis thaliana against the pathogen Pseudomonas syringae DC3000. Metabolic elicitors were obtained from bacteria free culture medium with n-hexane, ethyl acetate and n-butanol in three consecutive extractions. Each extract showed plant protection activity. The n-hexane fraction was the most effective and was used to study the signal transduction pathways involved by evaluating expression of marker genes of the salicylic acid (SA) signalling pathway (NPR1, PR1, ICS and PR2) and the jasmonic acid/ethylene (JA/ET) signalling pathway (PDF1, MYC2, LOX2 and PR3). In addition, the level of oxidative stress was tested by determining the activity of enzymes related to the ascorbate-glutathione cycle. N-hexane extracts stimulated both pathways based on overexpression of ICS, PR1, PR2, PDF1 and LOX2 genes. In addition, activity of the pathogenesis-related proteins glucanase (PR2) and chitinase (PR3), lipoxygenase and polyphenol oxidase was enhanced together with an increased capacity to remove reactive oxygen species (ROS). This was associated with less oxidative stress as indicated by a decrease in malondialdehyde (MDA), suggesting a causative link between defensive metabolism against P. syringae and ROS scavenging.

The phenomenon of today’s ageing population has increased interest in the search for new active substances that delay the onset and development of neurodegenerative diseases. In this respect, the search for natural compounds, mainly phenolic compounds, with neuroprotective activity has become the focus of growing interest. Verbascoside is a phenylethanoid that has already presented several pharmacological activities. The purpose of this study is to isolate and identify verbascoside from Acanthus mollis leaves. Consequently, its neuroprotective ability through enzymatic inhibition and free radical scavenging ability has been analyzed both in vitro and in cell culture assays. The antioxidant capacity of verbascoside was evaluated in vitro through total antioxidant capacity, DPPH , OH, and O2 —scavenging activity assays. The e ect of verbascoside on intracellular reactive oxygen species (ROS) levels of HepG2 and SH-SY5Y cell lines was studied in normal culture and under induced oxidative stress. The inhibitory ability of the phenylethanoid against several enzymes implied in neurodegenerative diseases (tyrosinase, MAO-A, and AChE) was analyzed in vitro. Verbascoside neuroprotective activity is at least in part related to its free radical scavenging ability. The e ect of verbascoside on ROS production suggests its potential in the prevention of harmful cell redox changes and in boosting neuroprotection.

Antibodies and oxidative stress are hallmarks of multiple sclerosis (MS) lesions. We aimed to clarify the relation between them, their role in MS patients and to investigate their specificity, comparing MS with classical neurodegenerative diseases (ND). Brain samples from 14 MS cases, 6 with ND and 9 controls (without neurological diseases). Immunohistochemistry assays were used to detect oxidized lipids (EO6), IgG and IgM, oligodendrocytes (Olig2), axons (NF, neurofilament) and cellular (TUNEL) and axonal damage (APP, amyloid precursor protein). We did not observe EO6 in controls. All samples from MS patients showed EO6 in oligodendrocytes and axons within lesions. We did not detect co-localization between EO6 and antibodies. Neither did we between EO6 and TUNEL or APP. 94.4% of TUNEL-positive cells in normal appearing white matter were also stained for IgG and 75.5% for IgM. IgM, but not IgG, co-localized with APP. EO6 was associated with axonal damage in amyotrophic lateral sclerosis (ALS). We did not observe association between antibodies and cellular or axonal damage in ND patients. MS patients showed a higher number of B cells and plasma cells in the lesions and meninges than controls. The number of B cells and plasma cells was associated with the presence of antibodies and with the activity of the lesions. We observed a main role of B lymphocytes in the development of MS lesions. Antibodies contribute to the oligodendrocyte and axonal damage in MS. Oxidative stress was associated with axonal damage in ALS.

Climate change consequences for agriculture involve an increase of saline soils which results in lower crop yields due to increased oxidative stress in plants. The present study reports the use of Plant Growth Promoting Bacteria (PGPB) as a tool to modulate plant innate mechanisms of adaptation to water stress (salinity and drought) in one year-old olive plantlets var. Arbosana and Arbequina. Integration of external changes in plants involve changes in Reactive Oxygen Species (ROS) that behave as signals to trigger plant adaptative mechanisms; however, they become toxic in high concentrations. For this reason, plants are endowed with antioxidant systems to keep ROS under control. So, the working hypothesis is that specific beneficial strains will induce a systemic response able to modulate oxidative stress and improve plant adaptation to water stress. Ten strains were assayed, evaluating changes in photosynthesis, pigments, ROS scavenging enzymes and antioxidant molecules, osmolytes and malondialdehyde, as oxidative stress marker. Photosynthesis and photosynthetic pigments were the most affected variables. Despite the specific response of each variety, the favorite targets of PGPBs to improve plant fitness were photosynthetic pigments and the antioxidant pools of glutathione and ascorbate. Our results show the potential of PGPBs to improve plant fitness modulating oxidative stress.

Physiological, metabolic, and genetic changes produced by two plant growth promoting rhizobacteria (PGPR) Pseudomonas sp. (internal code of the laboratory: N 5.12 and N 21.24) inoculated in tomato plants subjected to moderate water stress (10% polyethylene glycol-6000; PEG) were studied. Photosynthesis efficiency, photosynthetic pigments, compatible osmolytes, reactive oxygen species (ROS) scavenging enzymes activities, oxidative stress level and expression of genes related to abscisic acid synthesis (ABA; 9-cis-epoxycarotenoid dioxygenase NCDE1 gene), proline synthesis (Pyrroline-5-carboxylate synthase P5CS gene), and plasma membrane ATPase (PM ATPase gene) were measured. Photosynthetic efficiency was compromised by PEG, but bacterial-inoculated plants reversed the effects: while N5.12 increased carbon fixation (37.5%) maintaining transpiration, N21.24 increased both (14.2% and 31%), negatively affecting stomatal closure, despite the enhanced expression of NCDE1 and plasma membrane ATPase genes, evidencing the activation of different adaptive mechanisms. Among all parameters evaluated, photosynthetic pigments and antioxidant enzymes guaiacol peroxidase (GPX) and ascorbate peroxidase (APX) responded differently to both strains. N 5.12 increased photosynthetic pigments (70% chlorophyll a, 69% chlorophyll b, and 65% carotenoids), proline (33%), glycine betaine (4.3%), and phenolic compounds (21.5%) to a greater extent, thereby decreasing oxidative stress (12.5% in Malondialdehyde, MDA). Both bacteria have highly beneficial effects on tomato plants subjected to moderate water stress, improving their physiological state. The use of these bacteria in agricultural production systems could reduce the amount of water for agricultural irrigation without having a negative impact on food production.

The aim of this study was to characterize alterations in vascular structure and mechanics in murine mesenteric arteries from obese non-hypertensive mice, as well as their relationship with adipokines. Four-week old C57BL/6J male mice were assigned either to a control (C, 10% kcal from fat) or a high-fat diet (HFD, 45% kcal from fat) for 32 weeks. HFD animals weighed 30% more than controls (p b 0.001), exhibited similar blood pressure, increased leptin, insulin and superoxide anion (O2 •−) levels, and reduced adiponectin levels and nitric oxide (NO) bioavailability. Arterial structure showed an outward remodeling with an increase in total number of both adventitial and smoothmuscle cells inHFD.Moreover, HFDmice exhibited an increased arterial stiffness assessed by β-values (C=2.4±0.5 vs HFD=5.3±0.8; p b 0.05) and aortic pulse wave velocity (PWV, C=3.4±0.1 vs HFD = 3.9 ± 0.1; p b 0.05). β-Values and PWV positively correlated with leptin, insulin or O2 •− levels, whereas they negatively correlated with adiponectin levels and NO bioavailability (p b 0.01). A reduction in fenestrae number together with an increase in type-I collagen amount (p b 0.05) were observed in HFD. These data demonstrate that HFD accounts for the development of vascular remodeling and arterial stiffness associated with adipokine dysregulation and oxidative stress, independently of hypertension development.

Fetal undernutrition programs cardiometabolic diseases, with higher susceptibility in males. The mechanisms implicated are not fully understood and may be related to sex differences in placental adaptation. To evaluate this hypothesis, we investigated placental oxidative balance, vascularization, glucocorticoid barrier, and fetal growth in rats exposed to 50% global nutrient restriction from gestation day 11 (MUN, n = 8) and controls (n = 8). At gestation day 20 (G20), we analyzed maternal, placental, and fetal weights; oxidative damage, antioxidants, corticosterone, and PlGF (placental growth factor, spectrophotometry); and VEGF (vascular endothelial growth factor), 11 -HSD2, p22phox, XO, SOD1, SOD2, SOD3, catalase, and UCP2 expression (Western blot). Compared with controls, MUN dams exhibited lower weight and plasma proteins and higher corticosterone and catalase without oxidative damage. Control male fetuses were larger than female fetuses. MUN males had higher plasma corticosterone and were smaller than control males, but had similar weight than MUN females. MUN male placenta showed higher XO and lower 11 - HSD2, VEGF, SOD2, catalase, UCP2, and feto-placental ratio than controls. MUN females had similar feto-placental ratio and plasma corticosterone than controls. Female placenta expressed lower XO, 11 -HSD2, and SOD3; similar VEGF, SOD1, SOD2, and UCP2; and higher catalase than controls, being 11 -HSD2 and VEGF higher compared to MUN males. Male placenta has worse adaptation to undernutrition with lower efficiency, associated with oxidative disbalance and reduced vascularization and glucocorticoid barrier. Glucocorticoids and low nutrients may both contribute to programming in MUN males.

Brown adipose tissue (BAT) dissipates energy to produce heat. Thus, it has the potential to regulate body temperature by thermogenesis. For the last decade, BAT has been in the spotlight due to its rediscovery in adult humans. This is evidenced by over a hundred clinical trials that are currently registered to target BAT as a therapeutic tool in the treatment of metabolic diseases, such as obesity or diabetes. The goal of most of these trials is to activate the BAT thermogenic program via several approaches such as adrenergic stimulation, natriuretic peptides, retinoids, capsinoids, thyroid hormones, or glucocorticoids. However, the impact of BAT activation on total body energy consumption and the potential effect on weight loss is still limited. Other studies have focused on increasing the mass of thermogenic BAT. This can be relevant in obesity, where the activity and abundance of BAT have been shown to be drastically reduced. The aim of this review is to describe pathological processes associated with obesity that may influence the correct differentiation of BAT, such as catecholamine resistance, inflammation, oxidative stress, and endoplasmic reticulum stress. This will shed light on the thermogenic potential of BAT as a therapeutic approach to target obesity-induced metabolic diseases.

Congenital malformations are a common adverse outcome in pregnancies complicated by pregestational obesity, although the underlying mechanisms are still unrevealed. Our aim was to study the effect of oxidative stress in obesity-induced teratogenesis. Wistar rats were fed a high-fat diet for 13 weeks, with (OE group) or without (O group) vitamin E supplementation. Then, rats were mated and sacrificed at day 11.5 of gestation. Embryos from O dams presented a 25.9 3.5% rate of malformations (vs. 8.7 3.4% in C rats), which was reduced in the OE group (11.5 2.3%). Pregestational obesity induced hepatic protein and DNA oxidation and a decline in antioxidant enzymes. Importantly, glutathione content was also decreased, limiting the availability of this antioxidant in the embryos. Vitamin E supplementation efficiently maintained glutathione levels in the obese mothers, which could be used in their embryos to prevent oxidation-induced malformations. To test the effect of decreasing glutathione levels alone in a cell culture model of neuroepithelium, murine embryonic stem cells (ESC) were induced to form neuronal precursors and glutathione synthesis was inhibited with the gamma–glutamylcysteine synthesis inhibitor, buthionine sulfoximine (BSO). BSO inhibited the expression of Pax3, a gene required for neural tube closure that is also inhibited by oxidative stress. Taken together, our data indicate that obesity causes malformations through the depletion of maternal glutathione, thereby decreasing glutathione-dependent free radical scavenging in embryos, which can be prevented by vitamin E supplementation.