Antarctic thraustochytrids: Producers of long-chain omega-3 polyunsaturated fatty acids.

Thraustochytrids have been isolated from different aquatic systems; however, few studies have reported their occurrence in Antarctica. In this study, 13 strains close to strains belonging to the genera Oblongichytrium, Thraustochytrium, and Aurantiochytrium were isolated from seawater samples collected near the Antarctic Base Professor Julio Escudero (S 62°12'57' E 58°57'35″). Docosahexaenoic acid (DHA) was found in the total lipids of all the isolates; DHA content of the biomass (dry weight) varied between 3.3 and 33 mg/g under the growth conditions for isolation. Five of the Antarctic thraustochytrids were able to accumulate lipids at levels higher than 20% w/w. Two strains, RT2316-7 and RT2316-13, were selected to test the effect of the incubation temperature (at 5°C for 14 days and at 15°C for 5 days). Incubation temperature had little effect on the lipid content and biomass yield; however, its effect on the fatty acid composition was significant (p < .05). The low incubation temperature favored the accumulation of eicosapentaenoic acid (EPA), palmitic acid and stearic acid in the total lipids of RT2316-7. Percentage of EPA, DHA and the omega-6 fatty acid dihomo-γ-linolenic acid of total fatty acids of RT2316-13 was higher at the low incubation temperature. RT2316-13 accumulated the highest lipid content (30.0 ± 0.5%) with a carbon to nitrogen mass ratio equal to 16.9. On the contrary, lipid accumulation in RT2316-7 occurred at high concentration of the nitrogen sources (monosodium glutamate or yeast extract). The capability to accumulate lipids with a fatty acid profile that can be tuned through cultivation temperature make the Antarctic thraustochytrid RT2316-13 a candidate for the production of lipids with different uses.

Production of Lipids and Proteome Variation in a Chilean Thraustochytrium striatum Strain Cultured under Different Growth Conditions.

Total lipids and docosahexaenoic acid (DHA) production by a Chilean isolated thraustochytrid were evaluated under different growth conditions in shake flasks. The analyzed strain was identified as Thraustochytrium striatum according to an 18S rRNA gene sequence analysis. The strain (T. striatum AL16) showed negligible growth in media prepared with artificial seawater at concentrations lower than 50% v/v and pH lower than 5. Maltose and starch were better carbon sources for growth than glucose. DHA content of the biomass grown with maltose (60 g L-1) was doubled by increasing the agitation rate from 150 to 250 rpm. The DHA (0.8-6%) and eicosapentaenoic acid (0.2-21%) content in the total lipids varied depending on culture conditions and culture age. Lipid and DHA concentration increased (up to 5 g L-1 and 66 mg L-1, respectively) by regularly feeding the culture with a concentrated starch solution. Carotenoid accumulation was detected in cells grown with maltose or starch. Contrasting conditions of starch and glucose cultures were selected for comparative proteomics. Total protein extracts were separated by two-dimensional gel electrophoresis; 25 spots were identified using ESI-MS/MS. A protein database (143,006 entries) for proteomic interrogation was generated using de novo assembling of Thraustochytrium sp. LLF1b - MMETSP0199_2 transcriptome; 18 proteins differentially expressed were identified. Three ATP synthases were differentially accumulated in cultures with glucose, whereas malate dehydrogenase was more abundant in cells cultured with starch.

Molecular and phenotypic profiling from the base to the crown in maritime pine wood-forming tissue.

Environmental, developmental and genetic factors affect variation in wood properties at the chemical, anatomical and physical levels. Here, the phenotypic variation observed along the tree stem was explored and the hypothesis tested that this variation could be the result of the differential expression of genes/proteins during wood formation. Differentiating xylem samples of maritime pine (Pinus pinaster) were collected from the top (crown wood, CW) to the bottom (base wood, BW) of adult trees. These samples were characterized by Fourier transform infrared spectroscopy (FTIR) and analytical pyrolysis. Two main groups of samples, corresponding to CW and BW, could be distinguished from cell wall chemical composition. A genomic approach, combining large-scale production of expressed sequence tags (ESTs), gene expression profiling and quantitative proteomics analysis, allowed identification of 262 unigenes (out of 3512) and 231 proteins (out of 1372 spots) that were differentially expressed along the stem. A good relationship was found between functional categories from transcriptomic and proteomic data. A good fit between the molecular mechanisms involved in CW-BW formation and these two types of wood phenotypic differences was also observed. This work provides a list of candidate genes for wood properties that will be tested in forward genetics.

Slow and Fast Desiccation of Single-cell Thick Fronds of Filmy Ferns.

Filmy ferns can desiccate and recover after rehydration to resume photosynthesis. Slow and fast desiccation rates were compared in filmy fern fronds to determine whether structural or physiological differences may occur between desiccation rates. Slow desiccation is considered to be more similar to natural conditions experienced by plants that grow under the forest canopy. A fast desiccation rate will help to understand whether slow desiccation is important for recovery and viability.

Increasing the Membrane Permeability of a Fern with DMSO.

Cell membrane prevents the entrance of extra molecules (e.g., transcription and translation inhibitors) into the cell. For studying the physiological effects of transcription and translation inhibitors on Hymenophyllum caudiculatum fronds, we incubate fronds with 0.1% DMSO to test if this increases cell membrane permeability relative to incubation with ultrapure water. The study showed that DMSO could significantly improve the cell membrane permeability of filmy fronds.

Proteomic analysis during ontogenesis of secondary xylem in maritime pine.

Secondary xylem (wood) is formed through an intricate biological process that results in a highly variable final product. Studies have focused on understanding the molecular events for wood formation in conifers. In this process environmental, ontogenic and genetic factors influence variation in wood characteristics, including anatomical, chemical and physical properties. The main objective of this study was to analyse the ageing (ontogenic) effect on protein accumulation in wood-forming tissues along a cambial age (CA) gradient, ranging from juvenile wood (JW) sampled at the top of the tree, to mature wood (MW) sampled at the bottom of the tree. A total of 62 proteins whose accumulation varied by at least 1.5-fold according to CA were selected and identified by ESI-MS/MS; 30 of these were more abundant in MW and 32 were more abundant in JW. Consistent with earlier findings, our results show that JW is a tissue characterized by a high energy demand with the accumulation of gene products involved in energy, protein fate and cellular transport, while proteins identified in MW (heat shock response, oxygen and radical detoxification, and the S-adenosyl methionine cycle) support the idea that this tissue undergoes extended cell-wall thickening and a delay of programmed cell death.

La translocación in vitro citoplasma/núcleo del factor de transcripción embrionario oct-4 en células perivasculares propone a la aorta como un nicho quiescente de células madres adultas / In vitro translocation cytoplasm/nucleus of embryonic transcription factor oct-4 in perivascular cells suggests that aorta as a niche of quiescent adult stem cells

Las células perivasculares tienen un origen común en las células madre embrionarias y en los vasos sanguíneos que proporcionan un nicho para la mantención de su troncalidad. La expresión de marcadores embrionarios y de células indiferenciadas, como también la gran variedad de fenotipos celulares generados desde los pericitos, podrían ser explicados por la capacidad de estas células de ser inducidas a un estado "stemness" cuando son tratadas con factores adecuados. Nuestros resultados describen la expresión de células con OCT-4 citoplasmático en una ubicación anatómica perivascular donde de su nicho se encuentra en la región intima de la aorta en rata. In vitro las células aisladas por el método de explante que promueve el aislamiento de células migratorias desde los tejidos muestran un fenotipo con un citoplasma alargado y que expresan aSMA, PDGFRa y b, siendo estos dos últimos marcadores específicos de pericitos. En estas células se presenta una tranlocación a la variante nuclear de OCT-4 que ha sido descrito como el principal regulador de los procesos de autorrenovación y pluripotencia. La expresión de OCT-4 confirma y amplía aún más las observaciones obtenidas en nuestras investigaciones anteriores y demuestra que células madre se encuentran en los vasos sanguíneos en un microambiente que, probablemente, les permite que sobrevivan y permanezcan en reposo como un tipo de célula troncal quiescente.

Perivascular cells have a common origin from embryonic stem cells and blood vessels provide a niche for the maintenance of their stemness. Embryonic markers expression of undifferentiated cells, as well as, the wide variety of cellular phenotypes generated from pericytes, could be explained by the ability of these cells to be induced to a state of "stemness" when treated with appropriate factors. Our findings describe the expression of cells with cytoplasmic OCT-4 in perivascular anatomical location where their niche region is in the intima of the aorta in rats. In vitro isolated cells by explant method that promotes the isolation of migratory cells from tissues show an elongated cytoplasm phenotype, expressing aSMA, PDGFRa & b where the last two are specific markers of pericytes. These cells present a translocated nuclear variant of OCT-4 that has been described as the master regulator of self-renewal processes and pluripotency. The expression of OCT-4 further confirms and extends the observations obtained in our previous research and proves that stem cells found in the blood vessels in a microenvironment that probably allows them to survive and remain at rest as a type of quiescent stem cell.