Co-expressing Eranthis hyemalis lysophosphatidic acid acyltransferase 2 and elongase improves two very long chain polyunsaturated fatty acid production in Brassica carinata.

Docosadienoic acid (DDA, 22:2-13,16) and docosatrienoic acid (DTA, 22:3-13,16,19) are two very long chain polyunsaturated fatty acids (VLCPUFAs) that are recently shown to possess strong anti-inflammatory and antitumor properties. An ELO type elongase (EhELO1) from wild plant Eranthis hyemalis can synthesize the two fatty acids by sequential elongation of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop Brassica carinata produced a considerable amount of DDA and DTA in transgenic seeds. However, these fatty acids were excluded from the sn-2 position of triacylglycerols (TAGs). To improve the production level and nutrition value of the VLCPUFAs in the transgenic oilseed crop, a cytoplasmic lysophosphatidic acid acyltransferase (EhLPAAT2) for the incorporation of the two fatty acids into the sn-2 position of triacylglycerols was identified from E. hyemalis. RT-PCR analysis showed that it was preferentially expressed in developing seeds where EhELO1 was exclusively expressed in E. hyemalis. Seed specific expression of EhLPAAT2 along with EhELO1 in B. carinata resulted in the effective incorporation of DDA and DTA at the sn-2 position of TAGs, thereby increasing the total amount of DDA and DTA in transgenic seeds. To our knowledge, this is the first plant LPAAT that can incorporate VLCPUFAs into TAGs. Improved production of DDA and DTA in the oilseed crop using EhLPAAT2 and EhELO1 provides a real commercial opportunity for high value agriculture products for nutraceutical uses.

An engineered oilseed crop produces oil enriched in two very long chain polyunsaturated fatty acids with potential health-promoting properties.

Very long chain polyunsaturated fatty acids (VLCPUFAs) are well recognized for their health benefits in humans and animals. Here we report that identification and characterization of a gene (EhELO1) encoding the first functional ELO type elongase (3-ketoacyl-CoA synthase) in higher plants that is involved in the biosynthesis of two VLCPUFAs docosadienoic acid (DDA, 22:2n-6) and docosatrienoic acid (DTA, 22:3n-3) that possess potential health-promoting properties. Functional analysis of the gene in yeast indicated that this novel enzyme could elongate a wide range of polyunsaturated fatty acids with 18-22 carbons and effectively catalyze the biosynthesis of DDA and DTA by the sequential elongations of linoleic acid and alpha-linolenic acid, respectively. Seed-specific expression of this gene in oilseed crop Brassica carinata showed that the transgenic plants produced the level of DDA and DTA at approximately 30% of the total fatty acids in seeds, and the amount of the two fatty acids remained stable over four generations. The oilseed crop producing a high and sustained level of DDA and DTA provides an opportunity for high value agricultural products for nutritional and medical uses.

An effective identification and quantification method for Ginkgo biloba flavonol glycosides with targeted evaluation of adulterated products.

BACKGROUND: Ginkgo biloba L. (Ginkgoaceae) leaf extract is one of the most popular herbal products on the market, as it contains flavone glycosides (≥ 24%) and terpene lactones (≥ 6%), which are proposed to have significant physiological effects. Unfortunately, the challenging financial climate has resulted in a natural health product market containing adulterated ginkgo products. PURPOSE: 42 ginkgo samples were analyzed to establish an HPLC profile for authentic ginkgo and common ginkgo adulterants, and to develop a method capable of easily detecting adulteration in ginkgo commercial products. METHOD: In this study an efficient and targeted HPLC analysis method was established that is capable of distinguishing flavonol glycosides and aglycones simultaneously for the evaluation of ginkgo powdered extracts (PEs) and finished products in a single, 13 min run. Thirteen ginkgo leaf samples, fifteen standardized powdered extracts, and fourteen commercially available ginkgo products have been analyzed using this new HPLC method. Chromatograms were compared to six standard reference materials: one flavonol glycoside (rutin), three aglycones (quercetin, kaempferol and isorhamnetin), and two isoflavones (genestin and genistein). The quantitative chromatographic data was interpreted by principal component analysis (PCA), which assisted in the detection of unexpected chromatographic features in various adulterated botanical products. RESULTS: Only three of the commercially available ginkgo finished products tested in this study were determined to be authentic, with flavonol glycoside rutin, and aglycones quercetin, kaempferol, and isorhamnetin found to be common adulterants in the ginkgo powdered extract and finished product samples. CONCLUSION: Despite evidence of adulteration in most of the samples, each of the samples discussed herein met most of the current pharmacopeial standards. It is therefore critical that a preliminary evaluation be utilized to detect adulteration in commercial ginkgo products, prior to the acid hydrolysis procedure utilized in the current testing methods.

An automated, sheathless capillary electrophoresis-mass spectrometry platform for discovery of biomarkers in human serum.

A capillary electrophoresis-mass spectrometry (CE-MS) method has been developed to perform routine, automated analysis of low-molecular-weight peptides in human serum. The method incorporates transient isotachophoresis for in-line preconcentration and a sheathless electrospray interface. To evaluate the performance of the method and demonstrate the utility of the approach, an experiment was designed in which peptides were added to sera from individuals at each of two different concentrations, artificially creating two groups of samples. The CE-MS data from the serum samples were divided into separate training and test sets. A pattern-recognition/feature-selection algorithm based on support vector machines was used to select the mass-to-charge (m/z) values from the training set data that distinguished the two groups of samples from each other. The added peptides were identified correctly as the distinguishing features, and pattern recognition based on these peptides was used to assign each sample in the independent test set to its respective group. A twofold difference in peptide concentration could be detected with statistical significance (p-value < 0.0001). The accuracy of the assignment was 95%, demonstrating the utility of this technique for the discovery of patterns of biomarkers in serum.

Zwitterionic SAMs that Resist Nonspecific Adsorption of Protein from Aqueous Buffer.

This paper describes the use of surface plasmon resonance spectroscopy and self-assembled monolayers (SAMs) of alkanethiols on gold to evaluate the ability of surfaces terminating in different combinations of charged groups to resist the nonspecific adsorption of proteins from aqueous buffer. Mixed SAMs formed from a 1:1 combination of a thiol terminated in a trimethylammonium group and a thiol terminated in a sulfonate group adsorbed less than 1% of a monolayer of two proteins with different characteristics: fibrinogen and lysozyme. Single-component SAMs formed from thiols terminating in groups combining a positively charged moiety and a negatively charged moiety were also capable of resisting the adsorption of proteins. Single-component SAMs presenting single charges adsorbed nearly a full monolayer of protein. The amount of protein that adsorbed to mixed zwitterionic SAMs did not depend on the ionic strength or the pH of the buffer in which the protein was dissolved. The amount of protein that adsorbed to single-component zwitterionic SAMs increased as the ionic strength of the buffer decreased; it also decreased as the pH of the buffer increased (at constant ionic strength). Single-component zwitterionic SAMs composed of thiols terminating in N,N-dimethyl-amino-propane-1-sulfonic acid (-N+(CH3)2CH2CH2CH2SO3-) groups were substantially more effective at resisting adsorption of fibrinogen and lysozyme from buffer at physiological ionic strength and pH than single-component zwitterionic SAMs composed of thiols terminating in phosphoric acid 2-trimethylamino-ethyl ester (-OP(O)2-OCH2CH2N+(CH3)3). Several of these zwitterionic SAMs were comparable to the best known systems for resisting nonspecific adsorption of protein.