Docosahexaenoic Acid Is Naturally Concentrated at the sn-2 Position in Triacylglycerols of the Australian Thraustochytrid Aurantiochytrium sp. Strain TC 20.

The Labyrinthulomycetes or Labyrinthulea are a class of protists that produce a network of filaments that enable the cells to glide along and absorb nutrients. One of the main two Labyrinthulea groups is the thraustochytrids, which are becoming an increasingly recognised and commercially used alternate source of long-chain (LC, ≥C20) omega-3 containing oils. This study demonstrates, to our knowledge for the first time, the regiospecificity of the triacylglycerol (TAG) fraction derived from Australian thraustochytrid Aurantiochytrium sp. strain TC 20 obtained using 13C nuclear magnetic resonance spectroscopy (13C NMR) analysis. The DHA present in the TC 20 TAG fraction was determined to be concentrated in the sn-2 position, with TAG (16:0/22:6/16:0) identified as the main species present. The sn-2 preference is similar to that found in salmon and tuna oil, and differs to seal oil containing largely sn-1,3 LC-PUFA. A higher concentration of sn-2 DHA occurred in the thraustochytrid TC 20 oil compared to that of tuna oil.

Microwave pre-treatment of canola seeds and flaked seeds for increased hot expeller oil yield.

Microwave (MW) pre-treatment of canola seeds or flaked seeds was found to be a superior alternative to the conventional thermal pre-treatment (steam). Flaked seeds were "cooked" (heat-treated) with steam or using microwave treatments in the temperature range of 62-130 °C prior to expeller pressing. Microwave cooking at 100 °C resulted in the highest increase in the pressed oil yield, which is an increase of 3.7% (w/w) on a pressed oil basis or 9.0% (oil in seed basis) compared with steam cooking. Whole canola seeds conditioning was conducted with microwaves or steam, in the temperature range of 40-75 °C, followed by microwave or steam cooking at 100 °C to evaluate the effect of MW treatment during conditioning on the expeller oil yield. The use of a continuous microwave process for combined conditioning of whole seeds at 55 °C and subsequent cooking of flaked seeds at 100 °C resulted in a 4.0% increase in expeller oil yield, compared with steam conditioning and cooking. The influence of dry basis (db %) moisture contents of 5%, 11.5%, and 16.5% on oil yield after steam or MW treatments of seeds and flaked seeds was also studied. The moisture content of 11.5% (db %) yielded the highest net oil yield for both MW and steam at best conditioning and cooking temperatures of 55 °C and 100 °C, respectively. No significant impact of MW cooking was seen on oil quality compared with conventional steam cooking.

Development of a Brassica napus (Canola) Crop Containing Fish Oil-Like Levels of DHA in the Seed Oil.

Plant seeds have long been promoted as a production platform for novel fatty acids such as the ω3 long-chain (≥ C20) polyunsaturated fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) commonly found in fish oil. In this article we describe the creation of a canola (Brassica napus) variety producing fish oil-like levels of DHA in the seed. This was achieved by the introduction of a microalgal/yeast transgenic pathway of seven consecutive enzymatic steps which converted the native substrate oleic acid to α-linolenic acid and, subsequently, to EPA, docosapentaenoic acid (DPA) and DHA. This paper describes construct design and evaluation, plant transformation, event selection, field testing in a wide range of environments, and oil profile stability of the transgenic seed. The stable, high-performing event NS-B50027-4 produced fish oil-like levels of DHA (9-11%) in open field trials of T3 to T7 generation plants in several locations in Australia and Canada. This study also describes the highest seed DHA levels reported thus far and is one of the first examples of a deregulated genetically modified crop with clear health benefits to the consumer.

Fecal occult blood testing as a diagnostic test in symptomatic patients is not useful: a retrospective chart review.

BACKGROUND: The fecal occult blood test (FOBT) is a screening tool designed for the early detection of colorectal cancer in primary care. Although not validated for use in hospitalized patients, it is often used by hospital physicians for reasons other than asymptomatic screening. OBJECTIVE: To profile the in-hospital use of the FOBT and assess its impact on patient care. METHODS: Patient charts were retrospectively reviewed for all FOBTs conducted over a three-month period in 2011 by the central laboratory supporting the three acute care campuses of Hamilton Health Sciences (Hamilton, Ontario). RESULTS: A total of 229 patients underwent 351 tests; 52% were female and the mean age was 49 years (range one to 104 years). A total of 80 (34.9%) patients had at least one positive test. The most common indications for testing were anemia (51.0%) and overt gastrointestinal bleeding (19.2%). Only one patient had testing performed for asymptomatic colorectal cancer screening. In only 20 (8.7%) cases medications were modified before testing and diet was modified in only 21 (9.2%) cases. Most patients (85.2%) were taking one or more medications that could result in a false-positive result. Only 18 (7.9%) patients had a digital rectal examinations documented, of which seven were positive. All patients with a positive digital rectal examination underwent endoscopic procedures that revealed a source of bleeding. Among 44 patients with overt gastrointestinal bleeding, 12 (27.3%) had endoscopic investigations delayed to await results of the FOBT. Four patients were referred despite a negative FOBT due to a high degree of suspicion of gastrointestinal bleeding. CONCLUSIONS: The FOBT is often used inappropriately in the hospital setting. Confounding factors, such as diet and medication use, which may lead to false positives, are often ignored. Use of the FOBT in-hospital may lead to inappropriate management of patients, increased length of stay and increased direct medical costs. Use of the FOBT should be limited to validated indications only.

Characterization of oilseed lipids from "DHA-producing Camelina sativa": a new transformed land plant containing long-chain omega-3 oils.

New and sustainable sources of long-chain (LC, ≥C20) omega-3 oils containing DHA (docosahexaenoic acid, 22:6ω3) are required to meet increasing demands. The lipid content of the oilseed of a novel transgenic, DHA-producing land plant, Camelina sativa, containing microalgal genes able to produce LC omega-3 oils, contained 36% lipid by weight with triacylglycerols (TAG) as the major lipid class in hexane extracts (96% of total lipid). Subsequent chloroform-methanol (CM) extraction recovered further lipid (~50% polar lipid, comprising glycolipids and phospholipids) and residual TAG. The main phospholipid species were phosphatidyl choline and phosphatidyl ethanolamine. The % DHA was: 6.8% (of total fatty acids) in the TAG-rich hexane extract and 4.2% in the polar lipid-rich CM extract. The relative level of ALA (α-linolenic acid, 18:3ω3) in DHA-camelina seed was higher than the control. Major sterols in both DHA- and control camelina seeds were: sitosterol, campesterol, cholesterol, brassicasterol and isofucosterol. C16-C22 fatty alcohols, including iso-branched and odd-chain alcohols were present, including high levels of iso-17:0, 17:0 and 19:0. Other alcohols present were: 16:0, iso-18:0, 18:0 and 18:1 and the proportions varied between the hexane and CM extracts. These iso-branched odd-chain fatty alcohols, to our knowledge, have not been previously reported. These components may be derived from wax esters, or free fatty alcohols.

Metabolic engineering Camelina sativa with fish oil-like levels of DHA.

BACKGROUND: Omega-3 long-chain (≥C20) polyunsaturated fatty acids (ω3 LC-PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are critical for human health and development [corrected].. Numerous studies have indicated that deficiencies in these fatty acids can increase the risk or severity of cardiovascular, inflammatory and other diseases or disorders. EPA and DHA are predominantly sourced from marine fish although the primary producers are microalgae. Much work has been done to engineer a sustainable land-based source of EPA and DHA to reduce pressure on fish stocks in meeting future demand, with previous studies describing the production of fish oil-like levels of DHA in the model plant species, Arabidopsis thaliana. PRINCIPAL FINDINGS: In this study we describe the production of fish oil-like levels (>12%) of DHA in the oilseed crop species Camelina sativa achieving a high ω3/ω6 ratio. The construct previously transformed in Arabidopsis as well as two modified construct versions designed to increase DHA production were used. DHA was found to be stable to at least the T5 generation and the EPA and DHA were found to be predominantly at the sn-1,3 positions of triacylglycerols. Transgenic and parental lines did not have different germination or seedling establishment rates. CONCLUSIONS: DHA can be produced at fish oil-like levels in industrially-relevant oilseed crop species using multi-gene construct designs which are stable over multiple generations. This study has implications for the future of sustainable EPA and DHA production from land-based sources.

The anti-tumour effects of zoledronic acid.

Bone is the most common site for metastasis in patients with solid tumours. Bisphosphonates are an effective treatment for preventing skeletal related events and preserving quality of life in these patients. Zoledronic acid (ZA) is the most potent osteoclast inhibitor and is licensed for the treatment of bone metastases. Clodronate and pamidronate are also licensed for this indication. In addition, ZA has been demonstrated to exhibit antitumour effect. Direct and indirect mechanisms of anti-tumour effect have been postulated and at many times proven. Evidence exists that ZA antitumour effect is mediated through inhibition of tumour cells proliferation, induction of apoptosis, synergistic/additive to inhibitory effect of cytotoxic agents, inhibition of angiogenesis, decrease tumour cells adhesion to bone, decrease tumour cells invasion and migration, disorganization of cell cytoskeleton and activation of specific cellular antitumour immune response. There is also clinical evidence from clinical trials that ZA improved long term survival outcome in cancer patients with and without bone metastases. In this review we highlight the preclinical and clinical studies investigating the antitumour effect of bisphosphonates with particular reference to ZA.

Metabolic engineering of biomass for high energy density: oilseed-like triacylglycerol yields from plant leaves.

High biomass crops have recently attracted significant attention as an alternative platform for the renewable production of high energy storage lipids such as triacylglycerol (TAG). While TAG typically accumulates in seeds as storage compounds fuelling subsequent germination, levels in vegetative tissues are generally low. Here, we report the accumulation of more than 15% TAG (17.7% total lipids) by dry weight in Nicotiana tabacum (tobacco) leaves by the co-expression of three genes involved in different aspects of TAG production without severely impacting plant development. These yields far exceed the levels found in wild-type leaf tissue as well as previously reported engineered TAG yields in vegetative tissues of Arabidopsis thaliana and N. tabacum. When translated to a high biomass crop, the current levels would translate to an oil yield per hectare that exceeds those of most cultivated oilseed crops. Confocal fluorescence microscopy and mass spectrometry imaging confirmed the accumulation of TAG within leaf mesophyll cells. In addition, we explored the applicability of several existing oil-processing methods using fresh leaf tissue. Our results demonstrate the technical feasibility of a vegetative plant oil production platform and provide for a step change in the bioenergy landscape, opening new prospects for sustainable food, high energy forage, biofuel and biomaterial applications.

Phase II study of single agent oral vinorelbine as first-line treatment in patients with HER-2 negative metastatic breast cancer.

PURPOSE: Previous studies indicated that oral chemotherapy is convenient and preferred by many patients. We hereby report the efficacy and safety of oral vinorelbine as first-line chemotherapy for metastatic breast cancer (MBC). METHODS: Thirty-one patients with HER-2 negative MBC were enrolled between January 2007 and December 2010 in a prospective phase II trial. Patients were treated every 3 weeks with oral vinorelbine 60 mg/m² Days 1 and 8 for the 1st cycle and thereafter 80 mg/m² Days 1 and 8 every 3 weeks. Treatment was administered until disease progression or unexpected adverse event or patient refusal to continue. Primary endpoint was objective response rate (ORR); secondary endpoints were time-to-progression (TTP), overall survival (OS) and safety. Follow-up results until October 2012 are reported. RESULTS: Median age was 42 years (range 33-75). 26 (84 %) patients had 2 or more metastatic sites. A median of 6 cycles were administered (range 2-20). ORR was achieved in 9 (29 %) patients including 1 complete and 8 partial responses. 12 (39 %) patients had stable disease, resulting in a disease control rate of 68 %. Median TTP was 5.2 months [95 % CI 2.8-7.5]. Median OS was 16 months [95 % CI 11.3-20.7]. 3 (10 %) patients developed Grade 3-4 neutropenia. No events of febrile neutropenia, cardiac, renal toxicities or alopecia were recorded. Grade 3 thrombocytopenia and nausea-vomiting were reported in 2 (6 %) and 5 (16 %) patients, respectively. CONCLUSION: Results show a good efficacy and tolerance profile of oral vinorelbine as first-line chemotherapy for HER-2 negative MBC patients.

Metabolic engineering plant seeds with fish oil-like levels of DHA.

BACKGROUND: Omega-3 long-chain (≥C(20)) polyunsaturated fatty acids (ω3 LC-PUFA) have critical roles in human health and development with studies indicating that deficiencies in these fatty acids can increase the risk or severity of cardiovascular and inflammatory diseases in particular. These fatty acids are predominantly sourced from fish and algal oils, but it is widely recognised that there is an urgent need for an alternative and sustainable source of EPA and DHA. Since the earliest demonstrations of ω3 LC-PUFA engineering there has been good progress in engineering the C(20) EPA with seed fatty acid levels similar to that observed in bulk fish oil (∼18%), although undesirable ω6 PUFA levels have also remained high. METHODOLOGY/PRINCIPAL FINDINGS: The transgenic seed production of the particularly important C(22) DHA has been problematic with many attempts resulting in the accumulation of EPA/DPA, but only a few percent of DHA. This study describes the production of up to 15% of the C(22) fatty acid DHA in Arabidopsis thaliana seed oil with a high ω3/ω6 ratio. This was achieved using a transgenic pathway to increase the C(18) ALA which was then converted to DHA by a microalgal Δ6-desaturase pathway. CONCLUSIONS/SIGNIFICANCE: The amount of DHA described in this study exceeds the 12% level at which DHA is generally found in bulk fish oil. This is a breakthrough in the development of sustainable alternative sources of DHA as this technology should be applicable in oilseed crops. One hectare of a Brassica napus crop containing 12% DHA in seed oil would produce as much DHA as approximately 10,000 fish.

Recruiting a new substrate for triacylglycerol synthesis in plants: the monoacylglycerol acyltransferase pathway.

BACKGROUND: Monoacylglycerol acyltransferases (MGATs) are predominantly associated with lipid absorption and resynthesis in the animal intestine where they catalyse the first step in the monoacylglycerol (MAG) pathway by acylating MAG to form diacylglycerol (DAG). Typical plant triacylglycerol (TAG) biosynthesis routes such as the Kennedy pathway do not include an MGAT step. Rather, DAG and TAG are synthesised de novo from glycerol-3-phosphate (G-3-P) by a series of three subsequent acylation reactions although a complex interplay with membrane lipids exists. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that heterologous expression of a mouse MGAT acyltransferase in Nicotiana benthamiana significantly increases TAG accumulation in vegetative tissues despite the low levels of endogenous MAG substrate available. In addition, DAG produced by this acyltransferase can serve as a substrate for both native and coexpressed diacylglycerol acyltransferases (DGAT). Finally, we show that the Arabidopsis thaliana GPAT4 acyltransferase can produce MAG in Saccharomyces cerevisiae using oleoyl-CoA as the acyl-donor. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the concept of a new method of increasing oil content in vegetative tissues by using MAG as a substrate for TAG biosynthesis. Based on in vitro yeast assays and expression results in N. benthamiana, we propose that co-expression of a MAG synthesising enzyme such as A. thaliana GPAT4 and a MGAT or bifunctional M/DGAT can result in DAG and TAG synthesis from G-3-P via a route that is independent and complementary to the endogenous Kennedy pathway and other TAG synthesis routes.

Improving serological test ordering patterns for the diagnosis of celiac disease through clinical laboratory audit of practice.

BACKGROUND: Clinical Practice Guidelines (CPG) from both adult medicine and pediatrics recommend tTG to screen for celiac disease (CD). DESIGN AND METHODS: Serological test orders for celiac disease were evaluated against the guidelines. Ordering physicians were categorized as gastroenterologists, immunologists, pediatricians, other hospital physicians and non-hospital physicians. Interventions based on initial audit were implemented, including interacting with physicians, revising test menu and changing test ordering policy. After implementation of interventions, test orders were re-evaluated. RESULTS: After corrective interventions celiac panel (CP) orders were decreased from 48.4% to 3.6% in children, and from 72.3% to 28.1% in adults. Physicians ordered tTG alone for more than 90% of children. In adults the ordering of tTG alone was significantly increased from 7.2% to 61.3% (from 8.9% to 63.9% for gastroenterologists and from 8.1% to 44.4% for other physicians (p<0.05)). CONCLUSIONS: The audit reduced the CPG-practice gap that existed in the screening of CD.

Transgenic production of arachidonic acid in oilseeds.

We describe a transgenic microalgal Δ9-elongase pathway transformed in both Brassica napus and Arabidopsis thaliana seed resulting in the production of arachidonic acid (ARA). This pathway is noteworthy for both the production of ARA in seed tissue and the low levels of intermediate C20 fatty acids that accumulate. We also demonstrate that the arachidonic acid is naturally enriched at the sn2 position in triacylglycerol. This is the first report of ARA production by the Δ9-elongase pathway in an oilseed.

Odd-chain polyunsaturated fatty acids in thraustochytrids.

A series of unusual odd-chain fatty acids (OC-FA) were identified in two thraustochytrid strains, TC 01 and TC 04, isolated from waters off the south east coast of Tasmania, Australia. FA compositions were determined by capillary GC and GC-MS, with confirmation of polyunsaturated fatty acids (PUFA) structure performed by analysis of 4,4-dimethyloxazoline derivatives. PUFA constituted 68-74% of the total FA, with the essential PUFA; eicosapentaenoic acid (20:5ω3, EPA), arachidonic acid (20:4ω6, AA) and docosahexaenoic acid (22:6ω3, DHA), accounting for 42-44% of the total FA. High proportions of the saturated OC-FA 15:0 (7.1% in TC 01) and 17:0 (6.2% in TC 04) were detected. The OC-FA 17:1ω8 was present at 2.8% in TC 01. Of particular interest, the C21 PUFA 21:5ω5 and 21:4ω7 were detected at 3.5% and 4.1%, respectively, in TC 04. A proposed biosynthesis pathway for these OC-PUFA is presented. It is possible that the unsaturated OC-PUFA found previously in a number of marine animals were derived from dietary thraustochytrids and they could be useful biomarkers in environmental and food web studies.

Rapid expression of transgenes driven by seed-specific constructs in leaf tissue: DHA production.

BACKGROUND: Metabolic engineering of seed biosynthetic pathways to diversify and improve crop product quality is a highly active research area. The validation of genes driven by seed-specific promoters is time-consuming since the transformed plants must be grown to maturity before the gene function can be analysed. RESULTS: In this study we demonstrate that genes driven by seed-specific promoters contained within complex constructs can be transiently-expressed in the Nicotiana benthamiana leaf-assay system by co-infiltrating the Arabidopsis thaliana LEAFY COTYLEDON2 (LEC2) gene. A real-world case study is described in which we first assembled an efficient transgenic DHA synthesis pathway using a traditional N. benthamiana Cauliflower Mosaic Virus (CaMV) 35S-driven leaf assay before using the LEC2-extended assay to rapidly validate a complex seed-specific construct containing the same genes before stable transformation in Arabidopsis. CONCLUSIONS: The LEC2-extended N. benthamiana assay allows the transient activation of seed-specific promoters in leaf tissue. In this study we have used the assay as a rapid preliminary screen of a complex seed-specific transgenic construct prior to stable transformation, a feature that will become increasingly useful as genetic engineering moves from the manipulation of single genes to the engineering of complex pathways. We propose that the assay will prove useful for other applications wherein rapid expression of transgenes driven by seed-specific constructs in leaf tissue are sought.

Isolation and characterisation of a high-efficiency desaturase and elongases from microalgae for transgenic LC-PUFA production.

The production of long-chain polyunsaturated fatty acids from precursor molecules linoleic acid (LA; 18:2omega6) and alpha-linolenic acid (ALA; 18:3omega3) is catalysed by sequential desaturase and elongase reactions. We report the isolation of a front-end Delta6-desaturase gene from the microalgae Ostreococcus lucimarinus and two elongase genes, a Delta6-elongase and a Delta5-elongase, from the microalga Pyramimonas cordata. These enzymes efficiently convert their respective substrates when transformed in yeast (39-75% conversion for omega3 substrate fatty acids), and the Delta5-elongase in particular displays higher elongation efficiency (75% for conversion of eicosapentaenoic acid (20:5omega3) to docosapentaenoic acid (22:5omega3)) than previously reported genes. In addition, the Delta6-desaturase is homologous with acyl-CoA desaturases and shows a strong preference for the omega3 substrate ALA.

Metabolic engineering of omega-3 long-chain polyunsaturated fatty acids in plants using an acyl-CoA Delta6-desaturase with omega3-preference from the marine microalga Micromonas pusilla.

Long-chain (> or = C20) polyunsaturated fatty acids (LC-PUFA) EPA and DHA (20:5(Delta5,8,11,14,17) and 22:6(Delta4,7,10,13,16,19)) have well-documented health benefits against coronary heart disease, rheumatoid arthritis and other disorders. Currently, the predominant sources of these fatty acids are marine fish and algal oils, but research is being conducted to ensure that a sustainable, land-based production system can be developed. We here describe the metabolic engineering of an artificial pathway that produces 26% EPA in leaf triacylglycerol using a newly-identified Delta6-desaturase from the marine microalga Micromonas pusilla. We also demonstrate that this enzyme appears to function as an acyl-CoA desaturase that has preference for omega3 substrates both in planta and in yeast. Phylogenetic analysis indicates that this desaturase shares highly conserved motifs with previously described acyl-CoA Delta6-desaturases.