Marcos A López Patiño

University of Vigo, Vigo, Galicia, Spain

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Publications (9)23.13 Total impact

  • L. Rivera · M. A. López‐Patiño · D. L. Milton · T. P. Nieto · R. Farto
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    ABSTRACT: AimsThis study aimed to select and validate different methodological strategies to quantify the expression of the virulence genes ascC and ascV by qPCR in Aeromonas salmonicida subsp. salmonicida (A. salmonicida).Methods and resultsUsing the geNorm, Normfinder, and BestKeeper algorithms, reference genes for the qPCR were selected based on their in vitro expression stabilities in three A. salmonicida strains. Gene amplification efficiency was calculated by Real-time PCR Miner and LinReg PCR programs, which have not been used previously in the analysis of bacterial gene expression. The expression of the ascC and ascV virulence genes in a virulent A. salmonicida strain was evaluated by three quantification models, including single (least or most stable) or three most stable reference genes, combined with constant or specific gene amplification efficiency. The most stable reference genes were gyrB, proC, and rpoC, while rpoD and fabD were the least stable. Quantification models showed different expression patterns.Conclusions The optimal strategy to quantify mRNA expression was to use a combination of the three algorithms and the quantification model including the three most stable reference genes. Real-time PCR Miner or LinReg PCR were valuable tools to estimate amplification efficiency.Significance and Impact of the studyThe methods used in this study gave more reliable expression data using qPCR than previously published methods. The quantification and expression dynamics of virulence genes will contribute to a better understanding of how A. salmonicida interacts with its host and the environment, and therefore to the prevention of epizootics due to this pathogen.This article is protected by copyright. All rights reserved.
    Journal of Applied Microbiology 12/2014; 118(4). DOI:10.1111/jam.12740 · 2.48 Impact Factor
  • Manuel Gesto · Marcos A. López‐Patiño · Juan Hernández · José L. Soengas · Jesús M. Míguez
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    ABSTRACT: After an intense acute stressor, fish develop a metabolic and behavioral response that usually lasts for several hours. Brain monoaminergic systems, particularly the serotonergic system, seem to play a key role in the central regulation of the stress response. However, the influence of stressor severity on brain monoaminergic systems and on the induced stress responses is yet poorly understood. We hypothesize that serotonergic system could have a direct role in the integration of sensory information during stressor exposure and in the organization of the subsequent integrated stress response. According to our hypothesis, a low stressor intensity would induce a low response of brain serotonergic system and therefore, stress responses of low magnitude and duration. To test this hypothesis, we exposed fish to handling disturbance for 5 s, 15 s or 3 min. We sampled fish at 0 (controls), 3, 15, 45 and 240 min after the start of the stress protocol. Brain levels of serotonin, dopamine and their respective main oxidative metabolites were quantified, along with plasma levels of stress markers (catecholamines, cortisol, glucose and lactate). Regarding stress markers, the 5 s and 15 s stress protocols induced similar and relatively low elevations in all parameters assessed. As expected, the 3 min protocol induced responses of higher intensity and duration in all plasma parameters. Interestingly, the alterations of brain monoaminergic systems did not follow the same trend. The three stress protocols induced increases in the serotonergic activity in all brain regions analyzed (hypothalamus, telencephalon and medulla oblongata), independently of the duration of the handling disturbance, while the effects on the dopaminergic system were minor and brain region-dependent. These data suggest that the brain serotonergic system, though likely involved in the recognition of the stressor stimuli, is not the only actor determining the magnitude and duration of the acute stress response in trout.This article is protected by copyright. All rights reserved.
    Journal of Neuroendocrinology 12/2014; 27(2). DOI:10.1111/jne.12248 · 3.14 Impact Factor
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    ABSTRACT: Intracerebroventricular (ICV) treatment with oleate or octanoate induced in Brockmann bodies (BB) and liver of rainbow trout changes in several parameters related to fatty acid (FA) sensing through FA metabolism, FA transport through FA translocase (FAT/CD36) and mitochondrial activity. Changes observed in BB were completely different to those observed in previous studies after treatment with the same FA either intraperitoneal (IP) or in vitro. Therefore, FA sensing in BB is apparently direct and not related to previous FA sensing in hypothalamus, but it could be influenced by changes in the levels of peripheral hormones like insulin. In contrast, results obtained after ICV treatment with FA in liver were comparable to those observed after IP treatment but different to those observed after in vitro treatment. Therefore, FA sensing in liver is apparently indirect and be the consequence of previous hypothalamic FA sensing followed by vagal and/or sympathetic outflow.
    Aquaculture Nutrition 05/2014; 21(2). DOI:10.1111/anu.12158 · 1.40 Impact Factor
  • Marta Conde-Sieira · Marcos A López Patiño · Jesús M Míguez · José L Soengas
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    ABSTRACT: To assess whether the glucosensing capacity in peripheral (liver and Brockmann bodies) and central (hypothalamus and hindbrain) locations of rainbow trout displays day-night variations in its response to changes in circulating glucose levels, we evaluated the response of parameters related to glucosensing [glucose, glycogen and glucose 6-phosphate levels, activities of glucokinase (GK), glycogen synthetase (GSase) and pyruvate kinase (PK), and mRNA abundance of GK, glucose transporter 2 (GLUT2), and K(ATP) channel subunits Kir6.x-like and sulfonylurea receptor (SUR)-like] in fish subjected to hyperglycemic treatment under night or day conditions. No day-night significant variations were noticed in the glucosensing capacity of the hypothalamus, hindbrain and Brockmann bodies. In contrast, a clear differential response was noticed in the liver, where glucose levels, GK activity (and mRNA levels) and GSase activity displayed increased values during the day in hyperglycemic fish compared with controls, and lower (GK mRNA levels) or non-existent (glucose, GK and GSase activities, and Kir6.x-like mRNA levels) values during the night. A similar decrease in parameters related to glucosensing in the liver was observed when fish under day conditions were treated with melatonin, suggesting a modulatory role of melatonin in day-night changes of the glucosensing response in the same tissue.
    Journal of Experimental Biology 06/2012; 215(Pt 17):3112-9. DOI:10.1242/jeb.069740 · 2.90 Impact Factor
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    ABSTRACT: As demonstrated in previous studies, the functioning of brain glucosensing systems in rainbow trout is altered under stress conditions in a way that they are unable to respond properly to changes in glucose levels. Melatonin has been postulated as necessary for homeostatic control of energy metabolism in several vertebrate groups, and in fish it has been suggested as an anti-stress molecule. To evaluate the possible effects of melatonin on glucosensing, we have incubated hypothalamus and hindbrains of rainbow trout at different glucose concentrations in the presence of increased doses (0.01, 1, and 100nM) of melatonin assessing whether or not the responses to changes in glucose levels of parameters related to glucosensing (glucose, glycogen and glucose 6-phosphate levels, activities of GK, GSase and PK, and mRNA content of GK, GLUT2, Kir6.x-like, and SUR-like) are modified in the presence of melatonin. While no effects of melatonin were observed in hindbrain, in hypothalamus melatonin treatment up-regulated glucosensing parameters, especially under hypo- and normo-glycaemic conditions. The effects of melatonin in hypothalamus occurred apparently through MT(1) receptors since most effects were counteracted by the presence of luzindole but not by the presence of 4-P-PDOT. Moreover, melatonin treatment induced in hypothalamus increased mRNA expression levels of NPY and decreased mRNA levels of POMC, CART, and CRF. A role of the hormone in daily re-adjustment of hypothalamic glucosensor machinery is discussed.
    General and Comparative Endocrinology 05/2012; 178(1):131-8. DOI:10.1016/j.ygcen.2012.04.011 · 2.47 Impact Factor
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    ABSTRACT: Stress conditions induced in rainbow trout a readjustment in the glucosensing response of the hypothalamus and hindbrain such that those sensors did not respond properly to changes in glucose levels, as demonstrated in previous studies. To evaluate the hypothesis that corticotropin-releasing factor (CRF) could be involved in that response, we have incubated the hypothalamus and hindbrain of rainbow trout at different glucose concentrations in the presence of different concentrations of CRF. Under those conditions, we evaluated whether parameters related to glucosensing [the levels of glucose, glycogen and glucose 6-phosphate, the activities of glucokinase (GK), glycogen synthase (GSase) and pyruvate kinase (PK), and mRNA abundance of transcripts for GK, Glut2, Kir.6-like and sulfonylurea receptor (SUR)-like] are modified in the presence of CRF in a way comparable to that observed under stress conditions. We obtained evidence allowing us to suggest that CRF could be involved in the interaction between stress and glucosensing as CRF treatment of the hypothalamus and hindbrain in vitro induced a readjustment in glucosensing parameters similar to that previously observed under stress conditions in vivo. We had also previously demonstrated that stress elicits alterations in food intake in parallel with the readjustment of glucosensing systems. Here, we provide evidence that the mRNA abundance of several of the neuropeptides involved in the regulation of food intake, such as neuropeptide Y (NPY) or cocaine and amphetamine-regulated transcript (CART), is affected by CRF treatment, in such a way that their expression does not respond to changes in glucose levels in the same way as controls, allowing us to suggest that the food intake response that is integrated by changes in those peptides and known to be reduced by stress could be also mediated by CRF action in glucosensing areas.
    Journal of Experimental Biology 11/2011; 214(Pt 22):3887-94. DOI:10.1242/jeb.061564 · 2.90 Impact Factor
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    ABSTRACT: Living organisms show daily rhythms in physiology, behavior, and gene expression, which are due to the presence of endogenous clocks that synchronize biological processes to the 24-h light/dark cycle. In metazoans, generation of circadian rhythmicity is a consequence of specialized tissues known as "master clocks," having different locations among species. A few studies have described clock-gene expression in fish neural tissues, but none of them assessed clock-gene expression in different discrete regions. The present study was designed to explore the presence/absence of circadian clock-gene expression in the rainbow trout (Oncorhynchus mykiss) retina and hypothalamus. Juvenile fish were acclimated to a 12:12 light (L)-dark (D) cycle. Then, retina and hypothalamus were collected from animals kept under LD conditions or constant darkness (DD) for 24 h. Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assays were performed to quantify expression of the core circadian genes Clock1a, Bmal1, and Per1 as representative members of the circadian oscillator. All clock genes analyzed in the retina and hypothalamus showed circadian fluctuations. However, gene expression peaked in the rainbow trout hypothalamus with a 3-h (Clock1a and Bmal1) or 6-h (Per1) delay relative to that observed in the retina, the latter showing highest expression levels at zeitgeber times 9 (ZT9) for Clock1a and Bmal1, and at ZT21 for Per1. When exposed to DD, the rhythmic gene expression pattern was maintained for all genes in the rainbow trout retina, but only for Clock1a and Per1 in the hypothalamus. Bmal1 failed to cycle under DD, suggesting that hypothalamic clock function might depend on either several clock-gene isoforms or regulation from external inputs. Overall, these data indicate that representative molecular members of the core circadian clock are present in both the retina and hypothalamus of rainbow trout.
    Chronobiology International 05/2011; 28(5):381-9. DOI:10.3109/07420528.2011.566398 · 3.34 Impact Factor
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    ABSTRACT: Information regarding melatonin production in molluscs is very limited. In this study the presence and daily fluctuations of melatonin levels were investigated in hemolymph, retina and nervous system-related structures in the cephalopod Octopus vulgaris. Adult animals were maintained in captivity under natural photoperiod and killed at different times in a regular daily cycle. Levels of melatonin, serotonin (5-HT) and its acid metabolite (5-hydroxyindole acetic acid, 5-HIAA) in the hemolymph, retina, optic lobe, and cerebral ganglion were assayed by HPLC. Melatonin content fluctuated rhythmically in the retina and hemolymph, peaking at night. In the retina, but not in the other neural tissues, the rhythm was opposite to that of 5-HT, which displayed basal levels at night. Also, 5-HIAA levels in the retina were higher during the night, supporting that rhythmic melatonin production could be linked to diurnal changes in 5-HT degradation. The high levels of melatonin found in the retina point to it as the major source of melatonin in octopus; in addition, a large variation of melatonin content was found in the optic lobe with maximal values at night. All these data suggest that melatonin might play a role in the transduction of the light-dark cycle information for adjustment of rhythmic physiological events in cephalopods.
    Journal of Comparative Physiology 04/2011; 197(8):789-97. DOI:10.1007/s00359-011-0641-x · 2.04 Impact Factor
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    ABSTRACT: Arginine vasotocin (AVT) and isotocin (IT) are two neurohypophysial peptide hormones for which a role in adaptation to environmental changes has been suggested in fish. In teleosts, there are only a few available studies about circadian changes of AVT and IT levels, and a role of those peptides in the circadian system has been mainly suggested on the basis of the role of the homologous hormone AVP in mammals. Herein, we evaluated the diurnal rhythms in plasma AVT, pituitary AVT and IT content and the hypothalamic pro-vasotocin (pro-VT) expression in rainbow trout kept under a natural photoperiod, as well as their persistence in constant darkness as a tool for defining circadian dependence. Trout kept under a natural light cycle showed clear diurnal rhythms in both circulating and pituitary AVT levels with peak values around the last hours of the light phase. Hypothalamic pro-VT mRNA was also rhythmically expressed with similar peak characteristics. These rhythms persisted in fish kept under constant darkness for nearly two consecutive days, although peaks were progressively attenuated and phase-advanced. An IT rhythm was also found in pituitary of the trout maintained under a natural photoperiod, but not in those kept under continuous darkness. These results suggest that rhythms of hypothalamic AVT synthesis might be regulated by endogenous circadian mechanisms, and these rhythms contribute to maintain a similar fluctuation in pituitary AVT secretion into the blood. A potential role for AVT in the circadian and seasonal time-keeping system of teleost fish, either as a component of the neural machinery that participates in the adaptation to cyclic environmental changes, or as a circadian/seasonal output signal, is also discussed.
    General and Comparative Endocrinology 02/2011; 170(3):541-9. DOI:10.1016/j.ygcen.2010.11.013 · 2.47 Impact Factor