Diego López Alonso

Rapid simultaneous lipid extraction and transesterification for fatty acid analyses

An improved adaptation of the direct transesterification method of Lepage and Roy (J. Lipid Res. 25, 1391–96, 1984) for the preparation of fatty acid methyl esters allows notable saving of time and reagents. The material being analysed is heated for 10 minutes with methanol, acetyl chloride and hexane.

Acyl lipid composition variation related to culture age and nitrogen concentration in continuous culture of the microalga Phaeodactylum tricornutum

The influence of culture age and nitrogen concentration on the distribution of fatty acids among the different acyl lipid classes has been studied in continuous cultures of the microalga Phaeodactylum tricornutum. The culture age was tested in the range of 1.15-7 days, controlled by adjusting the dilution rate of fresh medium supplied. The effect of nitrogen concentration was tested from saturating conditions to starvation by modifying nitrate concentration in the fresh medium. Culture age had almost no influence on the fatty acid content; 16:0, 16:3 and 20:5 increased moderately wherein the level of 16:1 decreased when the culture age decreased. Culture age had no effect on the total fatty acid content that remained around 11% of dry weight. Conversely, culture age had a greater impact on lipid classes, producing changes in amounts of triacylglycerols (TAG) which ranged between 43% and 69%, and galactolipids (GLs) that oscillated between 20% and 40%. In general, the content of polar lipids of the biomass decreased with culture age. The other factor assayed, nitrogen content, affected the fatty acid profile. Saturated and monounsaturated fatty acids accumulated when the nitrogen concentration was decreased. The experiments regarding the effect of nitrogen concentration on lipid species were carried out with cells of an average age of 3.5 days. A decrease of the nitrogen concentration caused the GL fraction to decrease from 21 to 12%. Conversely, both neutral lipids (NLs) and phospolipids (PLs) increased from about 73 to 79% and from 6 to 8%, respectively. In these experiments, TAG was the lipid class with the highest increase, from 69 to 75%.

Cloning and molecular characterization of the Δ6-desaturase from two Echium plant species: Production of GLA by heterologous expression in yeast and tobacco

The synthesis of GLA (Δ6, 9, 12-18:3) is carried out in a number of plant taxa by introducing a double bond at the Δ6 position of its precursor, linoleic acid (Δ9, 12-18:2), through a reaction catalyzed by a Δ6-desaturase enzyme. We have cloned genes encoding the Δ6-desaturase (D6DES) from two different Macaronesian Echium species, E. pitardii and E. gentianoides (Boraginaceae), which are characterized by the accumulation of high amounts of GLA in their seeds. The Echium D6DES genes encode proteins of 438 amino acids bearing the prototypical cytochrome b5 domain at the N-terminus. Cladistic analysis of desaturases from higher plants groups the Echium D6DES proteins together with other Δ6-desaturases in a different cluster from that of the highly related Δ8-desaturases. Expression analysis carried out in E. pitardii shows a positive correlation between the D6DES transcript level and GLA accumulation in different tissues of the plant. Although a ubiquitous expression in all organs is observed, the transcript is particularly abundant in developing fruits, whereas a much lower level is present in mature leaves. Functional characterization of the D6DES gene from E. gentianoides has been achieved by heterologous expression in tobacco plants and in the yeast Saccharomyces cerevisiae. In both cases, overexpression of the gene led to the synthesis of GLA. Biotechnological application of these results can be envisaged as an initial step toward the generation of transgenic oleaginous plants producing GLA.

Acyl lipids of three microalgae

Abstract Acyl-lipid composition of Isochrysis galbana, Porphyridium cruentum and Phaeodactylum tricornutum from stationary-phase cultures has been analyzed by TLC and GC. Additionally, P. tricornutum from an outdoor tubular photobioreactor was also studied. Neutral (NLs) and glyco (GLs) lipids were found in similar amounts of ca 40–45% each, with phospholipids (PLs) representing ca 10–20%. The major lipid classes were triacylglycerol (TAG), monogalactosylaylglycerols (MGD), and galactosylacylglycerols (DGD), usually in that order. The P. tricornutum biomass taken from the bioreactor showed a distinctive acyl-lipid composition. GL content was nearly twice (56%) that of the indoor culture (31%) and NL content (31%) was nearly half that of the indoor culture (54%). These changes mainly involved TAG and MGD. The fatty acid composition of lipids in the outdoor culture generally remained unaffected, except for MGD, DGD, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), in which eicosapentaenoic acid (EPA) content was greatly increased. TAG, the main class of lipids, always contained a high proportion of EPA, 16:0 and 16:1. In the typical (SQD) composition, 14:0, 16:0 and 16:1 accounted for around 70% of fatty acids, with small amounts of polunsaturated fatty acids (PUFAs). The lipid classes which typically had the highest PUFA content in the microalgae were MGD and DGD. In P. tricornutum , these lipids characteristically contained a large proportion of 16:1, 16:2, 16:3 and 16:4. PC, which was composed of the main C 16 fatty acids and the major PUFAs, was usually the most abundant phospholipid. Phosphatidylgycerol showed an accumulation of 16:1 (probably a mixture of 16:1n7 and 16:1n3 trans ) as a distinctive feature. Phosphatidylinositol was characterized by 16:0 and 16:1 (and 14:0 in I. galbana ), which together accounted for over 50% of fatty acids, and significant presence of the main PUFAs. There was no consistent fatty acid pattern for PE in the three microalgae studied. PE was exceptional in I. galbana , containing 61% docosahexaenoic acid.

Acquisition and generalization of action effects

Three experiments studied the acquisition of action-contingent events (action effects). In a first, acquisition phase participants performed free-choice reactions with each keypress leading to the presentation of either a particular category word (e.g., animal or furniture) or an exemplar word (e.g., dog or chair). In the test phase, choice responses were made to category or exemplar words by using a word-key mapping that was either compatible or incompatible with the key-word mapping during acquisition. Compatible mapping produced better performance than incompatible mapping if the words in the practice and the test phase were the same (e.g., animal M animal), if they had a subordinate-superordinate relationship (e.g., dog M animal), belonged to the same category (e.g., dog M cat), or referred to visually related concepts (e.g., orange M circle). The findings support the assumption that action effects are acquired and integrated with the accompanying action automatically, so that perceiving the effect leads to the priming of the associated response. And, most importantly, they demonstrate that effect acquisition generalizes to other, feature-overlapping events.

Δ6-Desaturase sequence evidence for explosive Pliocene radiations within the adaptive radiation of Macaronesian Echium (Boraginaceae).

The oceanic islands of Macaronesia provide an ideal temporal and spatial context to test hypotheses of plant evolution using a novel set of phylogenetic markers, Delta(6)-desaturase sequences. In contrast to the limited resolution of standard molecular markers (nrDNA and plastid sequences), the Delta(6)-desaturase sequence phylogeny of Echium unequivocally reconstructs its active colonization across islands and archipelagos (Madeira, the Canary Islands, and Cape Verde), as well as its subsequent geographical and ecological speciation. Molecular-clock estimates using penalized likelihood and Bayesian inference reveal two radiation processes coincident with two dramatic climatic changes recorded in the region: the advent of the cold Canarian sea current (ca. 4 Ma) and the establishment of a strong seasonality in the Pleistocene (1.8 Ma). Though Echium had available all the diversity of present-day Macaronesian environments (xeric and mesic scrub, laurisilva, pine forest, and subalpine habitats) in the Miocene, evolutionary divergence appears to have been triggered by an extension of fluctuating xeric and mesic habitats with the advent of Pliocene conditions. These Echium radiations not only fulfill traditional predictions of adaptive radiation (i.e., common ancestry, rapid speciation, and phenotype-environment correlation), but also, uniquely among Macaronesian species, trait utility of woodiness. A Pliocene transition from annuality to a bush or tree-like condition occurred in early Echium lineages. Maintenance of woodiness in major lineages, and reversal to an herbaceous condition by three independent events, is reported for the first time in plants of oceanic islands.

Irrational decisions: attending to numbers rather than ratios

When judging the probability of a low probability event, many people judge it as less likely when it is expressed as a ratio of small numbers (e.g. 1:10) than of large numbers (e.g. 10:100). This is known as the ratio-bias (RB) phenomenon. Besides confirming the phenomenon, in this experiment participants made irrational decisions selecting probabilities of 10% in preference to 20 and 30%, in spite of the great discrepancy of probabilities between options (greater than in previous studies). These results support an interpretation in terms of the principles of cognitive-experiential self-theory (CEST). Relation between tendency to present the RB phenomenon and degree of rationality (as measured by the Need for Cognition Scale) was also examined. Scores in rationality obtained by participants who chose the nonoptimal response were lower than scores of participants who chose the optimal response.

FIRST INSIGHTS INTO IMPROVEMENT OF EICOSAPENTAENOIC ACID CONTENT IN PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) BY INDUCED MUTAGENESIS1

A strain improvement program was initiated based on mutagenesis with the goal of commercial production of eicosapentaenoic acid (EPA)from EPA‐overproducing microalgal strains. Two rounds of mutation and selection were conducted using Phaeodactylum tricornutum Bohlin UTEX #640 as the parent strain. After the first round of mutagenesis, a putative mutant (provisionally labeled 114) was obtained. The EPA content (% of dry weight) of this mutant strain was 37% higher than that of the wild type. 114 was further mutated and another putative mutant (provisionally called II242) was isolated, the EPA content of which was 44% higher than that of the wild type. When cultured with aeration in 1‐L flasks, EPA content of the wild type and putative mutants 114 and II242 was, 17.3 mg · g−1, 31.5mg · g−1, and 38.6 mg · g−1 dry biomass, respectively. EPA productivity was 3.48 mg · L−1· d−1 4.01 mg · L−1· d−1, and 4.98 mg · L−1· d−1 respectively. These figures compare favorably with many other promising EPA‐producing microorganisms and suggest that the use of a single methodology such as mutation and selection is a way to improve the polyunsaturated fatty acid content of microalgae and other microorganisms.

Enhancing challenged students' recognition of mathematical relations through differential outcomes training

Previous studies have reported that the differential outcomes procedure enhances learning and memory in special populations with cognitive deficits (see Goeters, Blakely, & Poling, 1992, for a review). In the present study we extend these findings to healthy adults who were asked to discriminate between the symbols “ > ” and “ < ” in mathematical statements. In Experiment 1, the performance of participants who showed difficulties in discriminating between these symbols was better (shorter response times) for the differential outcomes condition than for the nondifferential outcomes condition. In Experiment 2, the difficulty of the task was increased by using signed decimal numbers. Similar to Experiment 1, participants who initially had difficulties in discriminating between the symbols showed better performance (higher accuracy) for the differential outcomes condition than for the nondifferential outcomes condition, but only when both numbers were negative. These findings suggest that the differential outcomes procedure can be used to improve performance of challenged healthy adults on discrimination tasks with mathematical symbols and relations.

Overcoming illusory inferences in a probabilistic counterintuitive problem: the role of explicit representations.

In the context of conditional probabilities, a good example of the marked discrepancy between intuition and formal reasoning is the Monty Hall dilemma (MHD). We used the MHD to study the effects of practicing the game, making explicit the underlying structure, or enhancing the representation of the different possibilities, on reaching and stating the correct answer. The results of the experiments showed that accumulated experience with the MHD increased the proportion of switching responses but did not change erroneous intuitions (Experiment 1). However, when the dilemma was presented in the form of an adversary game that made the underlying structure more explicit, more participants formed complete mental representations that enabled them to reason correctly (Experiment 2). This result was observed even without any practice with the game if the participants were encouraged to represent possibilities (Experiment 3). Therefore, in this context, correct reasoning seems to depend more on the ability to consider different possibilities than on extensive practice with the game.