Three-phase equilibria of hydrates from computer simulation. I. Finite-size effects in the methane hydrate.

Clathrate hydrates are vital in energy research and environmental applications. Understanding their stability is crucial for harnessing their potential. In this work, we employ direct coexistence simulations to study finite-size effects in the determination of the three-phase equilibrium temperature (T3) for methane hydrates. Two popular water models, TIP4P/Ice and TIP4P/2005, are employed, exploring various system sizes by varying the number of molecules in the hydrate, liquid, and gas phases. The results reveal that finite-size effects play a crucial role in determining T3. The study includes nine configurations with varying system sizes, demonstrating that smaller systems, particularly those leading to stoichiometric conditions and bubble formation, may yield inaccurate T3 values. The emergence of methane bubbles within the liquid phase, observed in smaller configurations, significantly influences the behavior of the system and can lead to erroneous temperature estimations. Our findings reveal finite-size effects on the calculation of T3 by direct coexistence simulations and clarify the system size convergence for both models, shedding light on discrepancies found in the literature. The results contribute to a deeper understanding of the phase equilibrium of gas hydrates and offer valuable information for future research in this field.

Three-phase equilibria of hydrates from computer simulation. II. Finite-size effects in the carbon dioxide hydrate.

In this work, the effects of finite size on the determination of the three-phase coexistence temperature (T3) of the carbon dioxide (CO2) hydrate have been studied by molecular dynamic simulations and using the direct coexistence technique. According to this technique, the three phases involved (hydrate-aqueous solution-liquid CO2) are placed together in the same simulation box. By varying the number of molecules of each phase, it is possible to analyze the effect of simulation size and stoichiometry on the T3 determination. In this work, we have determined the T3 value at 8 different pressures (from 100 to 6000 bar) and using 6 different simulation boxes with different numbers of molecules and sizes. In two of these configurations, the ratio of the number of water and CO2 molecules in the aqueous solution and the liquid CO2 phase is the same as in the hydrate (stoichiometric configuration). In both stoichiometric configurations, the formation of a liquid drop of CO2 in the aqueous phase is observed. This drop, which has a cylindrical geometry, increases the amount of CO2 available in the aqueous solution and can in some cases lead to the crystallization of the hydrate at temperatures above T3, overestimating the T3 value obtained from direct coexistence simulations. The simulation results obtained for the CO2 hydrate confirm the sensitivity of T3 depending on the size and composition of the system, explaining the discrepancies observed in the original work by Míguez et al. [J. Chem Phys. 142, 124505 (2015)]. Non-stoichiometric configurations with larger unit cells show a convergence of T3 values, suggesting that finite-size effects for these system sizes, regardless of drop formation, can be safely neglected. The results obtained in this work highlight that the choice of a correct initial configuration is essential to accurately estimate the three-phase coexistence temperature of hydrates by direct coexistence simulations.

Three-phase equilibria of hydrates from computer simulation. III. Effect of dispersive interactions in the methane and carbon dioxide hydrates.

In this work, the effect of the range of dispersive interactions in determining the three-phase coexistence line of the CO2 and CH4 hydrates has been studied. In particular, the temperature (T3) at which solid hydrate, water, and liquid CO2/gas CH4 coexist has been determined through molecular dynamics simulations using different cutoff values (from 0.9 to 1.6 nm) for dispersive interactions. The T3 of both hydrates has been determined using the direct coexistence simulation technique. Following this method, the three phases in equilibrium are put together in the same simulation box, the pressure is fixed, and simulations are performed at different temperatures T. If the hydrate melts, then T > T3. Conversely, if the hydrate grows, then T < T3. The effect of the cutoff distance on the dissociation temperature has been analyzed at three different pressures for CO2 hydrate: 100, 400, and 1000 bar. Then, we have changed the guest and studied the effect of the cutoff distance on the dissociation temperature of the CH4 hydrate at 400 bar. Moreover, the effect of long-range corrections for dispersive interactions has been analyzed by running simulations with homo- and inhomogeneous corrections and a cutoff value of 0.9 nm. The results obtained in this work highlight that the cutoff distance for the dispersive interactions affects the stability conditions of these hydrates. This effect is enhanced when the pressure is decreased, displacing the T3 about 2-4 K depending on the system and the pressure.

Homogeneous nucleation rate of methane hydrate formation under experimental conditions from seeding simulations.

In this work, we shall estimate via computer simulations the homogeneous nucleation rate for the methane hydrate at 400 bars for a supercooling of about 35 K. The TIP4P/ICE model and a Lennard-Jones center were used for water and methane, respectively. To estimate the nucleation rate, the seeding technique was employed. Clusters of the methane hydrate of different sizes were inserted into the aqueous phase of a two-phase gas-liquid equilibrium system at 260 K and 400 bars. Using these systems, we determined the size at which the cluster of the hydrate is critical (i.e., it has 50% probability of either growing or melting). Since nucleation rates estimated from the seeding technique are sensitive to the choice of the order parameter used to determine the size of the cluster of the solid, we considered several possibilities. We performed brute force simulations of an aqueous solution of methane in water in which the concentration of methane was several times higher than the equilibrium concentration (i.e., the solution was supersaturated). From brute force runs, we infer the value of the nucleation rate for this system rigorously. Subsequently, seeding runs were carried out for this system, and it was found that only two of the considered order parameters were able to reproduce the value of the nucleation rate obtained from brute force simulations. By using these two order parameters, we estimated the nucleation rate under experimental conditions (400 bars and 260 K) to be of the order of log10 (J/(m3 s)) = -7(5).

Intestinal variation of serotonin, melatonin, and digestive enzymes activities along food passage time through GIT in Salmo salar fed with supplemented diets with tryptophan and melatonin.

In teleosts, peripheral serotonin (5-HT) and melatonin (MEL) are synthesised in the gastrointestinal tract (GIT) and regulate secretion and motility processes. Their production is regulated by diet and the passage of food through the GIT. This study aimed to evaluate how intestinal 5-HT, melatonin, and the activity of digestive enzymes varied with food passage time through GIT in Atlantic salmon (Salmo salar). We fed fish diets supplemented with tryptophan and melatonin (L-Trp 2.5% and MEL 0.01%) and measured the activity of digestive enzymes (amylase, lipase, and total protease) in the pyloric caeca, midgut, and hindgut at different times after feeding. 5-HT levels increased in all GIT portions and diets at 120 min post-intake and were highest in the pyloric caeca. Intestinal enzymatic activity was varied with diet, post-intake time and in different intestinal portions. In conclusion, food passage time directly affects GIT 5-HT secretion and digestive enzyme activity in S. salar, and diet composition regulates S. salar GIT function.

[Argentinean consensus guidelines on the identification and clinical care of secondary progressive multiple sclerosis]. / Consenso sobre la identificación y seguimiento de la esclerosis múltiple secundaria progresiva en Argentina.

INTRODUCTION: The identification, diagnosis, follow-up, and treatment of patients with secondary progressive multiple sclerosis (SPMS) show significant differences between health care professionals in Argentina. AIM: To provide consensus recommendations on the management of patients with SPMS in Argentina to optimize patient care. DEVELOPMENT: A panel of expert neurologists from Argentina dedicated to the diagnosis and care of multiple sclerosis patients gathered during 2019 and 2020 to carry out a consensus recommendation on the diagnosis and treatment of SPMS patients in Argentina. To achieve consensus, the methodology of 'formal consensus-RAND/UCLA method' was used. Recommendations were established based on published evidence and the expert opinion. Recommendations focused on how to define SPMS and how to follow SPMS patients. CONCLUSION: The recommendations of this consensus guidelines attempt to optimize the care of SPMS patients in Argentina.

TITLE: Consenso sobre la identificación y seguimiento de la esclerosis múltiple secundaria progresiva en Argentina.Introducción. Existen diferencias significativas en el diagnóstico, la identificación y el seguimiento de pacientes con esclerosis múltiple secundaria progresiva (EMSP) entre los profesionales de la salud a cargo de su tratamiento. Objetivo. Proveer recomendaciones sobre el tratamiento de los pacientes con EMSP en Argentina con el fin de optimizar su cuidado. Desarrollo. Un grupo de neurólogos expertos en esclerosis múltiple de Argentina elaboró un consenso para el tratamiento de pacientes con EMSP en la región mediante metodología de ronda de encuestas a distancia y reuniones presenciales. Se establecieron 33 recomendaciones basadas en la evidencia publicada y en el criterio de los expertos que participaron. Las recomendaciones se enfocaron en el diagnóstico y el seguimiento de los pacientes con EMSP. Conclusión. Las recomendaciones establecidas en el presente consenso permitirían optimizar el cuidado y el seguimiento de los pacientes con EMSP en Argentina.

Brain volume loss and no evidence of disease activity over 3 years in multiple sclerosis patients under interferon beta 1a subcutaneous treatment.

The objective of our study was to evaluate the relationship of percentage of annualized brain volume loss (aBVL) and no evidence of disease activity (NEDA) in multiple sclerosis (MS) patients under interferon beta 1-a subcutaneous treatment (IFN-beta) during 3 years of follow up. Relapsing remitting MS (RRMS) patients, with less than three years from disease onset, expanded disability status scale (EDSS) ≤3 and in which IFN beta 1-a 44 mcg was indicated, were included. Demographic, clinical and structural parameters from the magnetic resonance (MR) during the 3 years of follow up were analyzed and compared between patients with and without NEDA (defined as the absence of: (a) three-month confirmed disability progression defined as an increase in EDSS score of 1.0; (b) confirmed relapses; and (c) new or enlarged T2 lesions and/or Gd+ lesions). A total of 87 patients, mean age 33 ±â€¯6 years were included. NEDA was reached by 39 patients at year 3. Percentage of aBVL from baseline to months 24 in NEDA patients was -1.1% vs. -1.54% in patients without NEDA (p < 0.001) and from baseline to month 36 it was -1.43% vs. -2.1% (p < 0.001) in patients with and without NEDA, respectively. At 3 years follow up, patients who received IFN beta 1a and were disease-free had lower percentage of aBVL compared to patients with disease activity.

Glucosensing capacity of rainbow trout telencephalon.

To assess the hypothesis of glucosensing systems present in fish telencephalon, we first demonstrated in rainbow trout, by in situ hybridisation, the presence of glucokinase (GK). Then, we assessed the response of glucosensing markers in rainbow trout telencephalon 6 hours after i.c.v. treatment with glucose or 2-deoxyglucose (inducing glucoprivation). We evaluated the response of parameters related to the mechanisms dependent on GK, liver X receptor (LXR), mitochondrial activity, sweet taste receptor and sodium-glucose linked transporter 1 (SGLT-1). We also assessed mRNA abundance of neuropeptides involved in the metabolic control of food intake (agouti-related protein, neuropeptide Y, pro-opiomelanocortin, and cocaine- and amphetamine-related transcript), as well as the abundance and phosphorylation status of proteins possibly involved in linking glucosensing with neuropeptide expression, such as protein kinase B (AkT), AMP-activated protein kinase (AMPK), mechanistic target of rapamycin and cAMP response element-binding protein (CREB). The responses obtained support the presence in the telencephalon of a glucosensing mechanism based on GK and maybe one based on LXR, although they do not support the presence of mechanisms dependent on mitochondrial activity and SGLT-1. The mechanism based on sweet taste receptor responded to glucose but in a converse way to that characterised previously in the hypothalamus. In general, systems responded only to glucose but not to glucoprivation. Neuropeptides did not respond to glucose or glucoprivation. By contrast, the presence of glucose activates Akt and inhibits AMPK, CREB and forkhead box01. This is the first study in any vertebrate species in which the response to glucose of putative glucosensing mechanisms is demonstrated in the telencephalon. Their role might relate to processes other than homeostatic control of food intake, such as the hedonic and reward system.

Brain structural changes in patients in the early stages of multiple sclerosis with depression.

Some studies suggest an inflammatory mechanism associated with the presence of depression in multiple sclerosis (MS); however, there is little data concerning these findings. The purpose of this study was to investigate the presence of brain structural changes in patients with MS and depression and to compare them with patients suffering from MS without depression and healthy controls. METHODS: A case-control study that included patients with relapsing-remitting MS (RRMS) defined by validated criteria, over 18 years of age, with less than three years from disease onset, EDSS ≤ 3, with no history of previous depression and under immunomodulatory treatment with interferon beta, if any. A control group paired by age and gender was also included. Patients were clinically assessed to determine the presence of depression. Demographic clinical and structural aspects of parameters from the scan, such as lesion volume, total brain volume (TBV), white matter volume (WMV), neocortical gray matter volume (NGMV), and fractional anisotropy (FA) were analyzed. RESULTS: Sixty-five individuals were enrolled: 20 healthy controls, 22 patients with MS without depression, and 23 patients with MS with depression. Patients with MS and depression showed a lower TBV (P = 0.01), NGMV (0.01) together with an increase in lesion burden in T2 (P < 0.01) but not in T1 (P = 0.09) and no differences in global FA among groups (P = 0.23) and in WMV (P = 0.12). CONCLUSION: Patients with RRMS and depression had a reduced total brain volume and a significantly increased lesion burden at T2 MR than patients with RRMS without depression.

Computational study of the interplay between intermolecular interactions and CO2 orientations in type I hydrates.

Carbon dioxide (CO2) molecules show a rich orientation landscape when they are enclathrated in type I hydrates. Previous studies have described experimentally their preferential orientations, and some theoretical works have explained, but only partially, these experimental results. In the present paper, we use classical molecular dynamics and electronic density functional theory to advance in the theoretical description of CO2 orientations within type I hydrates. Our results are fully compatible with those previously reported, both theoretical and experimental, the geometric shape of the cavities in hydrate being, and therefore, the steric constraints, responsible for some (but not all) preferential angles. In addition, our calculations also show that guest-guest interactions in neighbouring cages are a key factor to explain the remaining experimental angles. Besides the implication concerning equation of state hydrate modeling approximations, the conclusion is that these guest-guest interactions should not be neglected, contrary to the usual practice.

Gender ratio trends over time in multiple sclerosis patients from Argentina.

Several studies in multiple sclerosis (MS) suggest a trend of increasing disease frequency in women during the last decades. A direct comparison of gender ratio trends among MS populations from Argentina remains to be carried out. The objective of the study was to compare gender ratio trends, over a 50-year span in MS populations from Argentina. METHODS: multicenter study that included patients from 14 MS Centers of Argentina. Patients with definite MS with birth years ranging from 1940 to 1989 were included. Gender ratios were calculated by five decades based on year of birth and were adjusted for the F/M born-alive ratio derived from the Argentinean national registry of births. The F/M ratios were calculated using a multivariate logistic regression per five decades by the year of birth approach. Analyses were performed using Stata 10.1. RESULTS: 1069 patients were included. Gender ratios showed a significant increase from the first to the last decade in the whole MS sample (from 1.8 to 2.7; p value for trend=0.023). The Gender ratio did not show differences considering MS subtype. CONCLUSION: our study showed a modest increase of the F/M ratio (from 1.8 to 2.7) over time among patients affected by MS in Argentina.

Increasing prevalence of multiple sclerosis in Buenos Aires, Argentina.

UNLABELLED: In 1996, the prevalence of multiple sclerosis (MS) for the metropolitan area of Buenos Aires using the capture-recapture method was estimated to be between 14 and 19.8 cases per 100,000 inhabitants. The aim of this study was to update the prevalence to 2014 following the same methodology. METHODS: Gran Buenos Aires is the denomination that refers to the megalopolis comprised by the autonomous city of Buenos Aires and the surrounding conurbation of the province of Buenos Aires. The study was carried out taking December 2014 as the prevalence month. We used the capture-recapture method to estimate the prevalence of MS cross-matching registries from 6 MS Centers from the metropolitan area of Buenos Aires. Log-linear model Poisson regression was used to estimate the number of affected MS patients not detected by any of the 6 sources considered. RESULTS: 1035 registries were obtained from the 6 lists from 910 different patients detected. The population of the area based on 2010 census was 12,806,866, the number of MS cases estimated amongst source interactions were 4901. The estimated prevalence was 38.2 per 100,000 inhabitants (95% CI 36.1-41.2). CONCLUSION: The study is an update almost 20 years after the first one in the area showing a significant increase in the previous reported prevalence. Our findings are in line with previous studies performed in other regions of the world.

Increasing incidence of multiple sclerosis among women in Buenos Aires: a 22 year health maintenance organization based study.

Studies in multiple sclerosis (MS) suggest a trend of increasing disease prevalence and incidence, and especially, a disproportional increase in the incidence of multiple sclerosis in women. The objective of this study was to evaluate the incidence of MS over 22 years and to determine the ratio in incidence of men to women in a health maintenance organization from Buenos Aires, Argentina. The population was made up of all members of a hospital-based HMO affiliated between January 1992 and December 2013. Each person was followed contributing time at risk. Cases with definite diagnosis of MS were included. Incidence density was calculated with 95 % confidence intervals and compared between women and men. 165,456 subjects were followed for a total of 1,488,575 person-years, of whom 42 developed MS. Incidence density was 3/100,000 person-years (95 % CI 2.1-3.5/100,000 person-years). During this period (1992-2013), the incidence rate in women increased from 1/100,000 (95 % CI 0.8-1.6) to 4.9/100,000 (95 % CI 4.1-5.4) (p < 0.001), while in men the incidence ranged from 1.4/100,000 (95 % CI 1-1.7) to 1.8 (1.3-2.1) (p = 0.16). Incidence density during the study period increased significantly in women but not in men. This is the first report of this phenomenon in Latin America region.

Disease onset in familial and sporadic multiple sclerosis in Argentina.

UNLABELLED: The present study was carried out to assess if there is an anticipation of age at onset in younger generations of familial multiple sclerosis (FMS) vs. sporadic MS (SMS) in Argentina. METHODS: multicenter study that included patients from 14 MS Centers of Argentina. Patients were considered as FMS if they had in their family at least one relative of first or second degree diagnosed with MS; otherwise, patients were considered to have SMS. We compared the age at onset between familial and sporadic cases as well as the age at onset between relatives from different generations in FMS vs. SMS. RESULTS: 1333 patients were included, 97 of them were FMS (7.3%). A lower age at onset in the younger generations of FMS cases was found compared with older generations of FMS as well as. SMS cases (24.1±3.7 years vs. 30.3±5.7 years, and 32.4±9.4 respectively; p<0.001). No differences were observed between older generations of FMS vs. SMS cases (p=0.12). CONCLUSION: we observed an anticipation of age at onset of MS in younger generations of patients with FMS vs. older generations of FMS and SMS.

On interfacial properties of tetrahydrofuran: Atomistic and coarse-grained models from molecular dynamics simulation.

We have determined the interfacial properties of tetrahydrofuran (THF) from direct simulation of the vapor-liquid interface. The molecules are modeled using six different molecular models, three of them based on the united-atom approach and the other three based on a coarse-grained (CG) approach. In the first case, THF is modeled using the transferable parameters potential functions approach proposed by Chandrasekhar and Jorgensen [J. Chem. Phys. 77, 5073 (1982)] and a new parametrization of the TraPPE force fields for cyclic alkanes and ethers [S. J. Keasler et al., J. Phys. Chem. B 115, 11234 (2012)]. In both cases, dispersive and coulombic intermolecular interactions are explicitly taken into account. In the second case, THF is modeled as a single sphere, a diatomic molecule, and a ring formed from three Mie monomers according to the SAFT-γ Mie top-down approach [V. Papaioannou et al., J. Chem. Phys. 140, 054107 (2014)]. Simulations were performed in the molecular dynamics canonical ensemble and the vapor-liquid surface tension is evaluated from the normal and tangential components of the pressure tensor along the simulation box. In addition to the surface tension, we have also obtained density profiles, coexistence densities, critical temperature, density, and pressure, and interfacial thickness as functions of temperature, paying special attention to the comparison between the estimations obtained from different models and literature experimental data. The simulation results obtained from the three CG models as described by the SAFT-γ Mie approach are able to predict accurately the vapor-liquid phase envelope of THF, in excellent agreement with estimations obtained from TraPPE model and experimental data in the whole range of coexistence. However, Chandrasekhar and Jorgensen model presents significant deviations from experimental results. We also compare the predictions for surface tension as obtained from simulation results for all the models with experimental data. The three CG models predict reasonably well (but only qualitatively) the surface tension of THF, as a function of temperature, from the triple point to the critical temperature. On the other hand, only the TraPPE united-atoms models are able to predict accurately the experimental surface tension of the system in the whole temperature range.

Understanding the Phase Behavior of Tetrahydrofuran + Carbon Dioxide, + Methane, and + Water Binary Mixtures from the SAFT-VR Approach.

The high-pressure phase diagrams of the tetrahydrofuran(1) + carbon dioxide(2), + methane(2), and + water(2) mixtures are examined using the SAFT-VR approach. Carbon dioxide molecule is modeled as two spherical segments tangentially bonded, water is modeled as a spherical segment with four associating sites to represent the hydrogen bonding, methane is represented as an isolated sphere, and tetrahydrofuran is represented as a chain of m tangentially bonded spherical segments. Dispersive interactions are modeled using the square-well intermolecular potential. In addition, two different molecular model mixtures are developed to take into account the subtle balance between water-tetrahydrofuran hydrogen-bonding interactions. The polar and quadrupolar interactions present in water, tetrahydrofuran, and carbon dioxide are treated in an effective way via square-well potentials of variable range. The optimized intermolecular parameters are taken from the works of Giner et al. (Fluid Phase Equil. 2007, 255, 200), Galindo and Blas (J. Phys. Chem. B 2002, 106, 4503), Patel et al. (Ind. Eng. Chem. Res. 2003, 42, 3809), and Clark et al. (Mol. Phys. 2006, 104, 3561) for tetrahydrofuran, carbon dioxide, methane, and water, respectively. The phase diagrams of the binary mixtures exhibit different types of phase behavior according to the classification of van Konynenburg and Scott, ranging from types I, III, and VI phase behavior for the tetrahydrofuran(1) + carbon dioxide(2), + methane(2), and + water(2) binary mixtures, respectively. This last type is characterized by the presence of a Bancroft point, positive azeotropy, and the so-called closed-loop curves that represent regions of liquid-liquid immiscibility in the phase diagram. The system exhibits lower critical solution temperatures (LCSTs), which denote the lower limit of immiscibility together with upper critical solution temperatures (UCSTs). This behavior is explained in terms of competition between the incompatibility with the alkyl parts of the tetrahydrofuran ring chain and the hydrogen bonding between water and the ether group. A minimum number of unlike interaction parameters are fitted to give the optimal representation of the most representative features of the binary phase diagrams. In the particular case of tetrahydrofuran(1) + water(2), two sets of intermolecular potential model parameters are proposed to describe accurately either the hypercritical point associated with the closed-loop liquid-liquid immiscibility region or the location of the mixture lower- and upper-critical end-points. The theory is not only able to predict the type of phase behavior of each mixture, but also provides a reasonably good description of the global phase behavior whenever experimental data are available.

Effects of insulin treatment on the response to oleate and octanoate of food intake and fatty acid-sensing systems in rainbow trout.

We hypothesized that food intake and the response of fatty acid (FA)-sensing systems in hypothalamus, liver, and Brockmann bodies of rainbow trout to raised levels of oleate (OL) or octanoate (OCT) is modified by insulin treatment. To assess this hypothesis, 15 fish per group received intraperitoneally 10-mL/kg injection of saline solution alone (control), or containing insulin (2-mg bovine insulin/kg body mass), OL (300 µg/kg), OCT (300 µg/kg), insulin + OL, or insulin + OCT to be sampled 6 h later to assess parameters related to FA sensing. Our results suggest that the modulatory role of insulin on the responses of hypothalamic FA-sensing systems to changes in circulating levels of OL or OCT was of minor importance in contrast to the mammalian model. However, this is in contrast with the effects observed in another experiment assessing changes in food intake after similar treatments because insulin treatment enhanced the anorectic effects of FA alone, and the effect was especially relevant (P < 0.001) for OCT, in contrast with the mammalian model where this FA is not inducing an anorectic response. In liver and Brockmann bodies, insulin treatment enhanced the responses to OL or OCT treatment in parameters related to FA sensing. Therefore, we provide for the first time in fish, and in a non-mammalian vertebrate, evidence for the modulation of FA-sensing systems by insulin.

Molecular dynamics simulation of CO2 hydrates: Prediction of three phase coexistence line.

The three phase equilibrium line (hydrate-liquid water-liquid carbon dioxide) has been estimated for the water + carbon dioxide binary mixture using molecular dynamics simulation and the direct coexistence technique. Both molecules have been represented using rigid nonpolarizable models. TIP4P/2005 and TIP4P/Ice were used for the case of water, while carbon dioxide was considered as a three center linear molecule with the parameterizations of MSM, EPM2, TraPPE, and ZD. The influence of the initial guest occupancy fraction on the hydrate stability has been analyzed first in order to determine the optimal starting configuration for the simulations, paying attention to the influence of the two different cells existing in the sI hydrate structure. The three phase coexistence temperature was then determined for a pressure range from 2 to 500 MPa. The qualitative shape of the equilibrium curve estimated is correct, including the high pressure temperature maximum that determines the hydrate re-entrant behaviour. However, in order to obtain quantitative agreement with experimental results, a positive deviation from the classical Lorentz-Berthelot combining rules must be considered.

Gradation of the stress response in rainbow trout exposed to stressors of different severity: the role of brain serotonergic and dopaminergic systems.

After an intense acute stressor, fish develop a metabolic and behavioural response that usually lasts for several hours. Brain monoaminergic systems, particularly the serotonergic system, appear 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 hypothesise that serotonergic system could have a direct role in the integration of sensory information during stressor exposure and in the organisation 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 a 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 analysed (hypothalamus, telencephalon and medulla oblongata), independently of the duration of the handling disturbance, whereas the effects on the dopaminergic system were minor and brain region-dependent. These data suggest that the brain serotonergic system, although 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.

Oral administration of melatonin counteracts several of the effects of chronic stress in rainbow trout.

To assess a possible antistress role of melatonin in fish, we orally administered melatonin to rainbow trout for 10 d and then kept the fish under normal or high stocking density conditions during the last 4 d. Food intake; biochemical parameters in plasma (cortisol, glucose, and lactate concentrations); liver (glucose and glycogen concentrations, and glycogen synthase activity); enzyme activities of amylase, lipase, and protease in foregut and midgut; and content of the hypothalamic neurotransmitters dopamine and serotonin, as well as their oxidized metabolites, 3,4-dihydroxyphenylacetic acid and 5-hydroxy-3-indoleacetic acid, were evaluated under those conditions. High stocking density conditions alone induced changes indicative of stress conditions in plasma cortisol concentrations, liver glycogenolytic potential, the activities of some digestive enzymes, and the 3,4-dihydroxyphenylacetic acid-to-dopamine and 5-hydroxy-3-indoleacetic acid-to-serotonin ratios in the hypothalamus. Melatonin treatment in nonstressed fish induced an increase in liver glycogenolytic potential, increased the activity of some digestive enzymes, and enhanced serotoninergic and dopaminergic metabolism in hypothalamus. The presence of melatonin in stressed fish resulted in a significant interaction with cortisol concentrations in plasma, glycogen content, and glycogen synthase activity in liver and dopaminergic and serotoninergic metabolism in the hypothalamus. In general, the presence of melatonin mitigated several of the effects induced by stress, supporting an antistress role for melatonin in rainbow trout.