The effects of ocean acidification on fish are only partially understood. Studies on olfaction are mostly limited to behavioral alterations of coral reef fish; studies on temperate species and/or with economic importance are scarce. The current study evaluated the effects of short- and medium-term exposure to ocean acidification on the olfactory system of gilthead seabream ( Sparus aurata ), and attempted to explain observed differences in sensitivity by changes in the protonation state of amino acid odorants. Short-term exposure to elevated P CO 2 decreased olfactory sensitivity to some odorants, such as L-serine, L-leucine, L-arginine, L-glutamate, and conspecific intestinal fluid, but not to others, such as L-glutamine and conspecific bile fluid. Seabream were unable to compensate for high P CO 2 levels in the medium term; after 4 weeks exposure to high P CO 2 , the olfactory sensitivity remained lower in elevated P CO 2 water. The decrease in olfactory sensitivity in high P CO 2 water could be partly attributed to changes in the protonation state of the odorants and/or their receptor(s); we illustrate how protonation due to reduced pH causes changes in the charge distribution of odorant molecules, an essential component for ligand-receptor interaction. However, there are other mechanisms involved. At a histological level, the olfactory epithelium contained higher densities of mucus cells in fish kept in high CO 2 water, and a shift in pH of the mucus they produced to more neutral. These differences suggest a physiological response of the olfactory epithelium to lower pH and/or high CO 2 levels, but an inability to fully counteract the effects of acidification on olfactory sensitivity. Therefore, the current study provides evidence for a direct, medium term, global effect of ocean acidification on olfactory sensitivity in fish, and possibly other marine organisms, and suggests a partial explanatory mechanism.

Amyloid-beta protein (Ab) and the scrapie isoform of prion protein (PrP Ss ) have a central role in the pathogenesis of Alzheimer's disease (AD) and prion-related encephalopathies (PRE), respectively. In both disorders, the deposition of these misfolded proteins is accompanied by apoptotic neuronal loss. However, the pathogenesis and molecular basis of Ab-and PrP Sc -neurotoxic effects are not completely understood. The Ca 2+ / calmodulin-dependent phosphatase calcineurin (CaN), through the dephosphorylation of the proapoptotic protein BAD, may be the link between Ca 2+ homeostasis deregulation and apoptotic neuronal death. In this study we used primary cultures of rat brain cortical neurons in order to investigate whether Ab and PrP affect CaN activity. We observed that synthetic peptides of Ab (Ab 25-35 and Ab 1-40 ) and PrP (PrP 106-126 ) increased CaN activity, but did not affect the levels of this protein phosphatase. Moreover, we found that these peptides reduced the levels of BAD phosphorylated at serine residue 112, and this effect was prevented by the CaN inhibitor FK506. Since dephosphorylated BAD translocates to mitochondria, where it triggers cytochrome c release, we determined the levels of BAD in mitochondrial and cytosolic fractions. The data obtained showed that Ab-and PrP-treated neurons had higher levels of BAD in mitochondria than control neurons. This increase in mitochondrial BAD levels was matched by a decrease in cytochrome c. FK506 prevented the alterations of mitochondrial BAD and cytochrome c levels induced by Ab and PrP peptides. Taken together the data suggest that Ab and PrP increased CaN activity, inducing BAD dephosphorylation and translocation to mitochondria and, subsequently, cytochrome c release that may trigger an apoptotic cascade. Therefore, therapeutic strategies targeting CaN might be valuable for these neurodegenerative disorders. #

Cynara cardunculus (Cc) is a multipurpose species; beyond its use in southwestern European cuisine, it is also used for the production of solid biofuel, seed oil, biodiesel, paper pulp and cheese, as well as animal feed. In addition, Cc has a long tradition of use in folk medicine as a diuretic and liver protector. The value of this species as a source of bioactive compounds is known; however, pharmacological use would further increase its cultivation. The main goal of the current work was to evaluate the potential of Cc as source of anti-carcinogenic phytochemicals. Different methanolic extracts obtained from wild and cultivated plants were tested for antioxidant activity and effect on breast tumor cell viability. The most effective extract, both as antioxidant and inhibition of tumor cell viability, was tested for effects on angiogenesis and tumor cell migration capacity. All the extracts tested had high antioxidant activity; however, only green leaves and dry head extracts exhibit anti-proliferative activity. Green cultivated leaves (GCL) were the most effective extract both as antioxidant and inhibiting the proliferation of tumor cells; it is equally active inhibiting tumor cell migration and in vivo angiogenesis. GCL extract is an effective inhibitor of several key points in tumor development and thus a promising source of anti-carcinogenic phytochemicals

Olfactory sensitivity to bile salts is wide-spread in teleosts; however, which bile salts are released in suYcient quantities to be detected is unclear. The current study identiWed bile salts in the intestinal and bile Xuids of Solea senegalensis by mass spectrometry-liquid chromatography and assessed their olfactory potency by the electro-olfactogram. The main bile salts identiWed in the bile were taurocholic acid (342 mM) and taurolithocholic acid (271 mM) plus a third, unidentiWed, bile salt of 532.3 Da. These three were also present in the intestinal Xuid (taurocholic acid, 4.13 mM; taurolithocholic acid, 0.4 mM). In sole-conditioned water, only taurocholic acid (0.31 M) was released in suYcient quantities to be measured (release rate: 24 nmol kg ¡1 min ¡1). Sole had high olfactory sensitivity to taurocholic acid but not to taurolithocholic acid. Furthermore, olfactory sensitivity was higher in the upper (right) olfactory epithelium than the lower (left). These two bile acids contribute about 40% of the olfactory potency of intestinal Xuid and account for the diVerence in potency at the two epithelia. Taurocholic acid (but not taurolithocholic acid), and possibly other types of bile acid not tested, could be used as chemical signals and the upper olfactory epithelium is specialised for their detection.

An electrical method to measure extracellular bioelectrical activity in vitro is presented. This method exploits the Helmholtz capacitive double-layer established at the electrode surface. Small extracellular voltage variations in the order of μVs induce through the double-layer capacitor a displacement current that is measured. This current is then enhanced by a gain factor proportional to the electrode capacitance. In addition, when measurements are carried out at low frequencies in current mode the electrode contribution to the noise can be minimized. The performance of the electrodes and the method is demonstrated using zebrafish hearts and glioma cell cultures. We propose that this electrical method is an ideal tool to measure in vitro slow and temporally synchronized events that are often involved in long range intracellular signaling

The two olfactory epithelia of flatfish of the family Soleidae are essentially in contact with two distinct environments; the upper (right) side samples open water while the lower (left) side samples interstitial water. This study assessed whether there are differences in the responsiveness of the two epithelia by use of the electro-olfactogram in the Senegalese sole (Solea senegalensis). The upper epithelium was significantly more responsive to the basic amino acids (L-lysine and L-arginine), glycine, and L-threonine than the lower epithelium. The lower epithelium was significantly more responsive to aromatic amino acids (L-tryptophan, L-tyrosine, L-DOPA, and L-phenylalanine), L-leucine, and L-asparagine than the upper. Both epithelia had similar responsiveness to the sulphur-containing amino acids (L-cysteine and L-methionine), L-alanine, L-serine, and L-glutamine. Neither side was responsive to the acidic amino acids (L-aspartate and L-glutamate) or the D-isomers of any amino acid tested. The upper olfactory organ was much more responsive to conspecific-derived stimuli (bile and intestinal fluid) than the lower organ. We suggest that these differences in responsiveness may be related to different functional roles of the upper and lower epithelia in feeding and chemical communication.

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In the past decade, many studies have investigated the effects of low pH/high CO2 as a proxy for ocean acidification on olfactory-mediated behaviours of marine organisms. The effects of ocean acidification on the behaviour of fish vary from very large to none at all, and most of the maladaptive behaviours observed have been attributed to changes in acid–base regulation, leading to changes in ion distribution over neural membranes, and consequently affecting the functioning of gamma-aminobutyric acid-mediated (GABAergic) neurotransmission. Here, we highlight a possible additional mechanism by which ocean acidification might directly affect olfaction in marine fish and invertebrates. We propose that a decrease in pH can directly affect the protonation, and thereby, 3D conformation and charge distribution of odorants and/or their receptors in the olfactory organs of aquatic animals. This can sometimes enhance signalling, but most of the time the affinity of odorants for their receptors is reduced in high CO2/low pH; therefore, the activity of olfactory receptor neurons decreases as measured using electrophysiology. The reduced signal reception would translate into reduced activation of the olfactory bulb neurons, which are responsible for processing olfactory information in the brain. Over longer exposures of days to weeks, changes in gene expression in the olfactory receptors and olfactory bulb neurons cause these neurons to become less active, exacerbating the problem. A change in olfactory system functioning leads to inappropriate behavioural responses to odorants. We discuss gaps in the literature and suggest some changes to experimental design in order to improve our understanding of the underlying mechanisms and their effects on the associated behaviours to resolve some current controversy in the field regarding the extent of the effects of ocean acidification on marine fish.