Abundant synthesis of long-chain polyunsaturated fatty acids in Eutreptiella sp. (Euglenozoa) revealed by chromatographic and transcriptomic analyses.

Algal lipids are important molecules to store energy in algae and transfer energy in the marine food chain, and are potential materials for high value nutraceuticals (e.g., omega-3 fatty acids) or biofuel production. However, how lipid biosynthesis is regulated is not well understood in many species including Eutreptiella from the phylum of Euglenozoa. Here, we characterized the fatty acid (FA) profile of an Eutreptiella species isolated from Long Island Sound, USA, using gas chromatography-tandem mass spectrometry (GC/MS/MS) and investigated their biosynthesis pathways by transcriptome sequencing. We discovered 24 types of FAs including a relatively high proportion of long-chain unsaturated FAs. The abundances of C16, C18, and saturated FAs decreased when phosphate in the culture medium was depleted. Among the 24 FAs, docosahexaenoic acid (C22:6∆4,7,10,13,16,19 ) was most abundant, suggesting that Eutreptiella sp. preferentially invests in the synthesis of long-chain polyunsaturated fatty acids (LC-PFAs). Further transcriptomic analysis revealed that Eutreptiella sp. likely synthesizes LC-PFAs via ∆8 pathway and uses type I and II fatty acid synthases. Using RT-qPCR, we found that some of the lipid synthesis genes, such as ß-ketoacyl-ACP reductase, fatty acid desaturase, acetyl-CoA carboxylase, acyl carrier protein, ∆8 desaturase, and Acyl-ACP thioesterase, were more actively expressed during light period, and two carbon fixation genes were up-regulated in the high-lipid illuminated cultures, suggesting a linkage between photosynthesis and lipid production. The lipid profile renders Eutreptiella sp. a nutritional prey and valuable source for nutraceuticals, and the biosynthesis pathway documented here will be useful for future research and applications.

Rapid quick, easy, cheap, effective, rugged, and safe extraction with novel phospholipid cleanup: A streamlined ultra high performance liquid chromatography with ultraviolet detection approach for screening polycyclic aromatic hydrocarbons in avian blood cells and plasma.

A streamlined method has been developed for the isolation and analysis of polycyclic aromatic hydrocarbons in avian blood cells and plasma utilizing quick, easy, cheap, effective, rugged, and safe extraction in combination with novel phospholipid cleanup technology. A variety of traditional extraction and cleanup techniques have been employed in the preparation and analysis of polycyclic aromatic hydrocarbonsin a variety of matrices; liquid-liquid partitioning, solid-phase extractions, gel permeation chromatography, and column chromatography are all effective techniques, however they are laborious and time consuming processes that require large amounts of solvent. Using quick, easy, cheap, effective, rugged, and safe extraction coupled with phospholipid cleanup, samples can be quickly screened while maintaining high throughput and sensitivity. With a liquid chromatography approach, analysis times may be kept short at 16 min while maintaining high analyte recovery. Recoveries in quality control samples ranged from 70 to 109%, with average surrogate recoveries of 80.6 ± 1.10%. The result of using a quick, easy, cheap, effective, rugged, and safe extraction approach in conjunction with phospholipid cleanup is a methodology that significantly reduces sample preparation time and solvent use while maintaining high sensitivity and reproducibility.

The feasibility of converting Cannabis sativa L. oil into biodiesel.

Cannabis sativa Linn, known as industrial hemp, was utilized for biodiesel production in this study. Oil from hemp seed was converted to biodiesel through base-catalyzed transesterification. The conversion is greater than 99.5% while the product yield is 97%. Several ASTM tests for biodiesel quality were implemented on the biodiesel product, including acid number, sulfur content, flash point, kinematic viscosity, and free and total glycerin content. In addition, the biodiesel has a low cloud point (-5 degrees C) and kinematic viscosity (3.48mm(2)/s). This may be attributed to the high content of poly-unsaturated fatty acid of hemp seed oil and its unique 3:1 ratio of linoleic to alpha-linolenic acid.

Oxidation of aminonitrotoluenes by 2,4-DNT dioxygenase of Burkholderia sp. strain DNT.

Aminonitrotoluenes form rapidly from the reduction of dinitrotoluenes (DNTs) which are priority pollutants and animal carcinogens. For example, 4-amino-2-nitrotoluene (4A2NT) and 2A4NT accumulate from the reduction of 2,4-DNT during its aerobic biodegradation. Here, we show that 2,4-DNT dioxygenase (DDO) from Burkholderia sp. strain DNT oxidizes the aminonitrotoluenes 2A3NT, 2A6NT, 4A3NT, and 5A2NT to 2-amino-3-nitrobenzylalcohol, 2-amino-4-nitro-m-cresol and 3-amino-5-nitro-p-cresol, 4-amino-3-nitrobenzylalcohol and aminonitrocresol, and 2-amino-5-nitro-o-cresol, respectively. 2A5NT and 3A4NT are oxidized to aminonitrocresols and/or aminonitrobenzylalcohols, and 4A2NT is oxidized to aminonitrocresol. Only 2A4NT, a reduced compound derived from 2,4-DNT, was not oxidized by DDO or its three variants. The alpha subunit mutation I204Y resulted in two to fourfold faster oxidization of the aminonitrotoluenes. Though these enzymes are dioxygenases, they acted like monooxygenases by adding a single hydroxyl group, which did not result in the release of nitrite.

Analyses of phenolic endocrine disrupting chemicals in marine samples by both gas and liquid chromatography-mass spectrometry.

With the renovation of Boston Harbor's Deer Island wastewater treatment plant and the extension of its diffuser pipes 15 km further into Massachusetts Bay, there arose the question whether the increased load of its secondary-treated wastewater contained significant amounts of phenolic endocrine disrupting chemicals (EDCs). Sampling from an oceanographic research vessel during the summers of 2003 and 2004 allowed for a unique opportunity to obtain clam, zooplankton, and bottom sediment samples. The samples were prepared by enhanced organic-solvent microwave digestion, followed by solid-phase extraction (SPE), derivatization and then analyzed by gas chromatography-mass spectrometry (GC-MS) or left un-derivatized and analyzed by LC-UV and liquid chromatography-mass spectrometry (LC-MS). The marine samples, especially parts of the clams, zooplankton and certain bottom sediments were found to contain primarily bisphenol A (BPA) at concentrations of 1-30 ng/g.

Methyl farnesoate controls adult male morphogenesis in the crayfish, Procambarus clarkii.

Adult male crayfish Procambarus clarkii exist in two morphotypes. They continue to molt as adults, switching between Form Is and Form IIs. Form Is are primary reproductive types, with large chelae and spines on the ischiopodites of the third and fourth pair of walking legs. Form IIs are non-reproductive types with smaller chelae and no spines on the ischiopodites. We investigated the hormonal control of these transitions in two ways, by eyestalk ablation and by methyl farnesoate (MF) treatments. Eyestalk ablation accelerates molting and increases MF levels in the blood. MF is a hormone that regulates both reproduction and morphogenesis. MF concentrations were determined in two ways. The hemolymph samples were extracted first, then purified, using normal phase HPLC. The fractions containing MF were collected and analyzed for MF concentration, utilizing both internal and external standards by GC/MS. The other hemolymph samples were analyzed from individual animals by HPLC. The concentrations of ecdysteroids were determined by radioimmunoassay. In the control animals, 4 out of 4 untreated Form I males molted into Form II, while 6 out of 7 Form IIs molted into Form Is. Eight of 8 ablated Form Is molted into Form IIs as expected, while 5 of 5 ablated Form IIs molted into Form IIs, instead of Form Is. MF treatment of intact animals resulted in 6 of 7 Form Is becoming Form IIs and 5 of 6 Form IIs becoming Form IIs. These results were highly significant in comparison of Form I and IIs in each treatment (eyestalk intact, eyestalk ablated and eyestalk intact with MF) by a chi square analysis, P = 0.006, P < 0.0005, and P = 0.013, respectively. MF premolt blood levels suggested that Form IIs were produced in the presence of 1.3 ng/ml MF, while Form Is result from MF levels less than 0.5 ng/ml. Since both eyestalk ablation and MF treatment resulted in the failure of Form IIs becoming Form Is, it was concluded that the control of morphogenesis of primary reproductives (Form Is) depends on a low level of MF prior to the molt, while Form IIs are formed in the presence of increased levels of MF.

Ontogenic variations of ascorbic acid and phenethyl isothiocyanate concentrations in watercress (Nasturtium officinale R.Br.) leaves.

Watercress (Nasturtium officinale R.Br.) is the richest source of glucosinolate nasturtiin, which on hydrolysis produces phenethyl isothiocyante (PEITC). Interest in growing watercress is stimulated since demonstration of the role of PEITC in protection against cancers associated with tobacco specific carcinogens. Twenty-one days old watercress seedlings were transplanted into growth chambers (16-h days/8-h nights of 25/22 degrees C and photosynthetic photon flux (PPF) of approximately 265 micromol m(-2) s(-)(1)). The study was replicated three times. Leaves were analyzed for PEITC and ascorbic acid concentrations at transplant, and harvested at 10-days intervals until 60 days after transplant. The PEITC and ascorbic acid concentrations were the highest in leaves harvested at 40 days and the lowest at transplant. Leaves harvested at 40 days produced about 150% higher PEITC concentrations compared to the leaves at transplant. Both PEITC and ascorbic acid concentrations of leaves increased linearly with age until 40 days after transplant after which there was no significant increase. Seedlings at transplant had the lowest dry mass and leaf area, while plants harvested at 60 days had the highest dry mass and leaf area.

Oscillating formation of 8-oxoguanine during DNA oxidation.

Oxidation of free guanine and guanine in salmon testes ds-DNA by hydroxyl radicals generated with Fenton reagent resulted in oscillating 8-oxoguanine concentrations. These oscillations were superimposed on a general trend of decreasing ratio of [8-oxoguanine]/{[8-oxoguanine] + [guanine]} with time, suggesting that a steady state 8-oxoguanine concentration would not be achieved. Mass spectrometry detected 8-oxoguanine and 5-guanidinohydantoin as products, suggesting that the latter was the product of oxidation of 8-oxoguanine. Guanidinohydantoin and other possible intermediates and products may be involved in a complex mechanism leading to the observed behavior. Oscillatory fluctuations in 8-oxoguanine may need to be considered in assessing its clinical significance as a biomarker for oxidative DNA damage.

Toxicity screening by electrochemical detection of DNA damage by metabolites generated in situ in ultrathin DNA-enzyme films.

Rapid detection of DNA damage could serve as a basis for in vitro genotoxicity screening for new organic compounds. Ultrathin films (20-40 nm) containing myoglobin or cytochrome P450(cam) and DNA grown layer-by-layer on electrodes were activated by hydrogen peroxide, and the enzyme in the film generated metabolite styrene oxide from styrene. This styrene oxide reacted with double stranded (ds)-DNA in the same film, mimicking metabolism and DNA damage in human liver. DNA damage was detected by square wave voltammetry (SWV) by using catalytic oxidation with Ru(bpy)(3)(2+) (bpy = 2,2'-bipyridine) and by monitoring the binding of Co(bpy)(3)(3+). Damaged DNA reacts more rapidly than intact ds-DNA with Ru(bpy)(3)(3+), giving SWV peaks at approximately 1 V versus SCE that grow larger with reaction time. Co(bpy)(3)(3+) binds more strongly to intact ds-DNA, and its SWV peaks at 0.04 V decreased as DNA was damaged. Little change in SWV signals was found for incubations of DNA/enzyme films with unreactive organic controls or hydrogen peroxide. Capillary electrophoresis and HPLC-MS suggested the formation of styrene oxide adducts of DNA bases under similar reaction conditions in thin films and in solution. The catalytic SWV method was more sensitive than the Co(bpy)(3)(3+) binding assay, providing multiple measurements over a 5 min reaction time.