Unsaturated fatty acids play an essential role in the biophysical characteristics of cell membranes and determine the proper function of membrane-attached proteins. Thus, the ability of cells to alter the degree of unsaturation in their membranes is an important factor in cellular acclimatization to environmental conditions. Many eukaryotic organisms can synthesize dienoic fatty acids, but Saccharomyces cerevisiae can introduce only a single double bond at the ⌬ 9 position. We expressed two sunflower (Helianthus annuus) oleate ⌬ 12 desaturases encoded by FAD2-1 and FAD2-3 in yeast cells of the wild-type W303-1A strain (trp1) and analyzed their effects on growth and stress tolerance. Production of the heterologous desaturases increased the content of dienoic fatty acids, especially 18:2⌬ 9,12 , the unsaturation index, and the fluidity of the yeast membrane. The total fatty acid content remained constant, and the level of monounsaturated fatty acids decreased. Growth at 15°C was reduced in the FAD2 strains, probably due to tryptophan auxotrophy, since the trp1 (TRP1) transformants that produced the sunflower desaturases grew as well as the control strain did. Our results suggest that changes in the fluidity of the lipid bilayer affect tryptophan uptake and/or the correct targeting of tryptophan transporters. The expression of the sunflower desaturases, in either Trp ؉ or Trp ؊ strains, increased NaCl tolerance. Production of dienoic fatty acids increased the tolerance to freezing of wild-type cells preincubated at 30°C or 15°C. Thus, membrane fluidity is an essential determinant of stress resistance in S. cerevisiae, and engineering of membrane lipids has the potential to be a useful tool of increasing the tolerance to freezing in industrial strains.Tolerance to freezing is an essential trait influencing the viability and leavening capacity of baker's yeast in frozen dough (4, 42). The so-called frozen-dough technology has been widely accepted by consumers and bakers due to several advantages, which include supplying oven-fresh bakery products and improving labor conditions. However, no appropriate industrial strain with a high tolerance to freezing is available, and it is unlikely that the classical breeding program could significantly improve this trait. Freezing is a complex and multifaceted stress, in which different stressors and stress responses appear to play important roles. Cells exposed to subzero temperatures are injured by the formation of ice crystals and crystal growth during frozen storage (33). At a slow freezing rate, cells are exposed to hyperosmotic solutions and equilibrate by movement of water across the membranes (65). Finally, during the thawing process, cells can suffer biochemical damage by oxidative stress (16). It is not surprising, therefore, that tolerance to freezing involves several mechanisms working in concert.Biological membranes are the first barrier that separates cells from their environment and are a primary target for damage during environmental stress. Sudden changes in envi...

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Oleoyl-phosphatidylcholine desaturase (FAD2) is a key enzyme involved in fatty acid desaturation in oilseeds, which is affected by environmental temperature. The results of this study show that FAD2 is regulated in vivo via temperature-dependent endogenous oxygen concentrations in developing sunflower (Helianthus annuus L.) seeds. By combining in vivo oxygen profiling, in situ hybridization of FAD2 genes, an assay of energy status, fatty acid analysis, and an in vitro FAD2 enzyme activity assay, it is shown that: (i) the oil-storing embryo is characterized by a very low oxygen level that is developmentally regulated. Oxygen supply is mainly limited by the thin seed coat. (ii) Elevations of external oxygen supply raised the energy status of seed and produced a dramatic increase of the FAD2 enzyme activity as well as the linoleic acid content. (iii) A clear negative correlation exists between temperature and internal oxygen concentration. The changes occurred almost instantly and the effect was fully reversible. The results indicate that the internal oxygen level acts as a key regulator for the activity of the FAD2 enzyme. It is concluded that a major mechanism by which temperature modifies the unsaturation degree of the sunflower oil is through its effect on dissolved oxygen levels in the developing seed.

Coenzyme Q (CoQ), a redox-active lipid, is comprised of a quinone group and a polyisoprenoid tail. It is an electron carrier in the mitochondrial respiratory chain, a cofactor of other mitochondrial dehydrogenases, and an essential antioxidant. CoQ requires a large set of enzymes for its biosynthesis; mutations in genes encoding these proteins cause primary CoQ deficiency, a clinically and genetically heterogeneous group of diseases. Patients with CoQ deficiency often respond to oral CoQ10 supplementation. Treatment is however problematic because of the low bioavailability of CoQ10 and the poor tissue delivery. In recent years, bypass therapy using analogues of the precursor of the aromatic ring of CoQ has been proposed as a promising alternative. We have previously shown using a yeast model that vanillic acid (VA) can bypass mutations of COQ6, a monooxygenase required for the hydroxylation of the C5 carbon of the ring. In this work, we have generated a human cell line lacking functional COQ6 using CRISPR/Cas9 technology. We show that these cells cannot synthesize CoQ and display severe ATP deficiency. Treatment with VA can recover CoQ biosynthesis and ATP production. Moreover, these cells display increased ROS production, which is only partially corrected by exogenous CoQ, while VA restores ROS to normal levels. Furthermore, we show that these cells accumulate 3-decaprenyl-1,4-benzoquinone, suggesting that in mammals, the decarboxylation and C1 hydroxylation reactions occur before or independently of the C5 hydroxylation. Finally, we show that COQ6 isoform c (transcript NM_182480) does not encode an active enzyme. VA can be produced in the liver by the oxidation of vanillin, a nontoxic compound commonly used as a food additive, and crosses the blood-brain barrier. These characteristics make it a promising compound for the treatment of patients with CoQ deficiency due to COQ6 mutations.

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The temperature and oxygen regulation of the microsomal oleate desaturase (FAD2, EC 1.3.1.35) activity has been studied in developing sunflower (Helianthus annuus L.) seeds. In plants cultivated in growth chambers, the linoleic acid content in the seed lipids increased along the 25/15°C (day/night) cycle, except during the first hours of the warm period, where it decreased significantly. In contrast, FAD2 activity decreased notably at the beginning of the warm period, showing a small and continuous increase during the rest of the cycle. The temperature effect on the linoleic acid content and the FAD2 activity was also investigated using peeled seeds and detached achenes subjected to temperature changes. In peeled seeds, a change of temperature from 10 to 30°C brought about a significant decrease of FAD2 activity. On the contrary, when the temperature shifted from 30 to 10°C, FAD2 activity only increased slightly. Unlike peeled seeds, detached achenes showed a fast and dramatic increase or decrease in the level of FAD2 activity in response to a temperature change from 30 to 10°C, or from 10 to 30°C, respectively. The in vivo and in vitro thermal properties of the FAD2 enzyme were also studied. Optimal temperature and heat‐resistance profile showed similar patterns in both conditions. All these data support the hypothesis that temperature regulates FAD2 activity by two different and independent mechanisms: a direct effect, and an indirect effect affecting oxygen availability. Furthermore, these results suggest that the low thermal stability of the enzyme is the main factor responsible for the direct temperature effect on FAD2 activity.

Intraocular lens (IOL) opacification is an infrequent complication of cataract sur-gery. Surface analysis has demonstrated that the opacification of IOLs is related to calcium or phosphate precipitation on or within the lenses, but the associated mechanisms are unknown, and the scientific literature is heterogeneous and limited to case series and retrospective studies. The purpose of this systematic review was to analyse the most frequent conditions associated with opacification of IOLs reported by studies. A search was carried out using the PubMed MEDLINE, Web of Science and Scopus databases. The quality of the studies selected was evaluated using the Pierson tool. The search provided a total of 811 articles, of which 39 were selected following the inclusion and exclusion criteria. The most common opacified lenses were hydrophilic IOLs according to our analysis. The mean time of appearance of lens opacification was 14.93 ± 17.82 months. The most frequent conditions associated with opacification of the IOLs were Descemet Stripping with Automated Endothelial Keratoplasty (DSAEK/DSEK) and diabetes mellitus (DM), followed by pars plana vitrectomy (PPV), blood hypertension (HT), and glaucoma.Concerning the quality analysis, the mean score was 7.00 ± 1.43 (scoring range from 0 to 10), indicating an acceptable quality of the case reports and retrospective studies. In conclusion, DSAEK/DSEK, DM, PPV, glaucoma and hypertension are conditions with potential risk of IOL opacification after cataract surgery, especially when implanting hydrophilic acrylic IOLs. K E Y W O R D Sacrylic IOL, cataract surgery, intraocular lens, IOL explantation, IOL opacification