Genetically modified plants are an alternative to oily fish for providing n-3 polyunsaturated fatty acids in the human diet: A summary of the findings of a Biotechnology and Biological Sciences Research Council funded project.

The n-3 polyunsaturated fatty acids (PUFA) present primarily in oily fish, namely eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are important components of cell membranes and that are needed for normal development and cell function. Humans have very limited capacity for EPA and DHA synthesis from α-linolenic acid and so they must be obtained pre-formed from the diet. However, perceived unpalatability of oily fish and fish oil concerns about contamination with environmental pollutants, dietary choices that exclude fish and animal products, and price limit the effectiveness of recommendations for EPA and DHA intakes. Moreover, marine sources of EPA and DHA are diminishing in the face of increasing demands. Therefore, an alternative source of EPA and DHA is needed that is broadly acceptable, can be upscaled and is sustainable. This review discusses these challenges and, using findings from recent nutritional trials, explains how they may be overcome by seed oils from transgenic plants engineered to produce EPA and DHA. Trials in healthy men and women assessed the acute uptake and appearance in blood over 8 hours of EPA and DHA from transgenic Camelina sativa compared to fish oil, and the incorporation of these PUFA into blood lipids after dietary supplementation. The findings showed that postprandial EPA and DHA incorporation into blood lipids and accumulation in plasma lipids after dietary supplementation was as good as that achieved with fish oil. The oil derived from this transgenic plant was well tolerated. This review also discusses the implications for human nutrition, marine ecology and agriculture.

A service evaluation of an occupational health Covid-19 referral pathway.

BACKGROUND: A rapid management referral pathway was established by a private UK occupational health (OH) provider to offer assessments and advice on managing individual risk relating to Covid-19 in the workplace. AIMS: The aim of this service evaluation was to assess the utilization and effectiveness of the pathway in supporting referrers during a pandemic. METHODS: Referrals between March-August 2020 were analysed by date and industry to assess service utilization. A survey was sent to a convenience sample of referrers throughout this period, requesting feedback on whether the report led to a change in how the worker was managed, and whether it increased referrer confidence in managing the worker. RESULTS: Five hundred and seventy referrals were made, predominantly from wholesale and retail; professional, scientific and technical; and food and drink production. There was a small peak of referrals from manufacturing in April and a larger peak in July-August from wholesale and retail, and food and drink production. Of 166 surveys sent, 58 were completed (35% response rate). In 71% of cases, referrers indicated that the report led to change in how the worker was managed, and in 86% of cases, referrers reported being more confident in managing the worker. CONCLUSIONS: The pathway was well-utilized. OH assessments and advice have an important role to play in a pandemic, with useful impact on how workers are managed and how confident managers feel in managing workers.

Oil from transgenic Camelina sativa as a source of EPA and DHA in feed for European sea bass (Dicentrarchus labrax L.).

Aquaculture, the fastest growing food production sector cannot continue to rely on finite stocks of marine fish as the primary source of the omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA), eicosapentaenoic acid (EPA; 20:5n3) and docosahexaenoic acid (DHA; 22:6n-3), for feeds. A four-month feeding trial was conducted to investigate the impact of a de novo oil, with high levels of EPA and DHA, obtained from transgenic Camelina sativa on growth performance, tissue fatty acid profiles, and expression of lipid metabolism genes when used as a replacement for fish oil in feed for European seabass (Dicentrachus labrax). Triplicate groups of 50 juvenile fish (initial weight 16.7 ± 0.92 g) per tank were fed for 4 months with one of three isolipidic and isoproteic experimental diets consisting of a standard diet containing a commercial blend of fish oil and rapeseed oil (CFO), a diet containing transgenic Camelina oil (TCO), or a blend of fish oil and rapeseed oil with enhanced levels of EPA and DHA (EFO) formulated to match the n-3 LC-PUFA profile of the TCO feed. Final weight of fish fed the GM-derived oil was not different to fish fed either CFO or EFO. Slight lower growth performance of fish fed TCO at the beginning of the trial was related to transient reduced feed intake, possibly caused by glucosinolates in the raw Camelina sativa oil. The GM-derived oil improved the nutritional quality of the fish fillet by enhancing total n-3 PUFA levels compared to the fish fed the other two feeds, and maintained flesh EPA and DHA at the same levels as in fish fed the diets containing fish oil. The metabolic response in liver and intestine was generally relatively mild although diets TCO and EFO seemed to trigger a metabolic response consisting of an up-regulation of both ß-oxidation (cpt1a) and fatty acid transport (fabp1), possibly reflecting higher levels of LC-PUFA. Overall, the present study indicated that an oil of terrestrial origin, Camelina sativa, when engineered to contain high levels of EPA and DHA can replace fish oil in feeds for European seabass with no detrimental impact on growth or feed efficiency, while also maintaining or increasing tissue n-3 LC-PUFA contents.

Job strain and retirement decisions in UK general practice.

BACKGROUND: Falling retention in UK general practice is a well-described problem but there has been little previous research into its underlying causes. Poor psychosocial work conditions may help explain falling workforce morale and early retirement from the profession. AIMS: To explore the impact upon morale and retirement decisions of changes in psychosocial aspects of UK general practice over the course of a career. METHODS: Biographical narrative interviewing method (BNIM) was used to obtain and analyse career narratives of 12 London general practitioners (GPs), aged 55-65, half of whom had retired. Findings were theorized using the Job Demands-Control-Support (JDCS) model. RESULTS: A spontaneous, consistent theme was evident across all 12 interviews: changes in the psychosocial work environment had contributed to a steady decline in morale. Sequential, multilayered reductions in autonomy were the most commonly cited causes for reduced enthusiasm. Increasing demands in the form of both a rising workload as well as a complaints culture drained energy and morale. The GPs described increasingly fragmented teams and therefore reduced social support for the role. Nonetheless, retirement decisions were not straightforward, provoking complex emotions. CONCLUSIONS: The combination of increasing demands with reduced autonomy puts practitioners under intense strain, diminishing the satisfaction they derive from their work and affecting retirement decisions. The Job Demands-Control-Support (JDCS) model is an empirically tested model that could be used to inform improved work design in general practice.

Action on diabetic macular oedema: achieving optimal patient management in treating visual impairment due to diabetic eye disease.

This paper identifies best practice recommendations for managing diabetes and sight-threatening diabetic eye disease. The authors provide an update for ophthalmologists and allied healthcare professionals on key aspects of diabetes management, supported by a review of the pertinent literature, and recommend practice principles for optimal patient management in treating visual impairment due to diabetic eye disease. In people with diabetes, early optimal glycaemic control reduces the long-term risk of both microvascular and macrovascular complications. The authors propose more can and should be done to maximise metabolic control, promote appropriate behavioural modifications and encourage timely treatment intensification when indicated to ameliorate diabetes-related complications. All people with diabetes should be screened for sight-threatening diabetic retinopathy promptly and regularly. It is shown that attitudes towards treatment adherence in diabetic macular oedema appear to mirror patients' views and health behaviours towards the management of their own diabetes. Awareness of diabetic macular oedema remains low among people with diabetes, who need access to education early in their disease about how to manage their diabetes to delay progression and possibly avoid eye-related complications. Ophthalmologists and allied healthcare professionals play a vital role in multidisciplinary diabetes management and establishment of dedicated diabetic macular oedema clinics is proposed. A broader understanding of the role of the diabetes specialist nurse may strengthen the case for comprehensive integrated care in ophthalmic practice. The recommendations are based on round table presentations and discussions held in London, UK, September 2016.

Replacement of Marine Fish Oil with de novo Omega-3 Oils from Transgenic Camelina sativa in Feeds for Gilthead Sea Bream (Sparus aurata L.).

Omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) are essential components of the diet of all vertebrates. The major dietary source of n-3 LC-PUFA for humans has been fish and seafood but, paradoxically, farmed fish are also reliant on marine fisheries for fish meal and fish oil (FO), traditionally major ingredients of aquafeeds. Currently, the only sustainable alternatives to FO are vegetable oils, which are rich in C18 PUFA, but devoid of the eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) abundant in FO. Two new n-3 LC-PUFA sources obtained from genetically modified (GM) Camelina sativa containing either EPA alone (ECO) or EPA and DHA (DCO) were compared to FO and wild-type camelina oil (WCO) in juvenile sea bream. Neither ECO nor DCO had any detrimental effects on fish performance, although final weight of ECO-fed fish (117 g) was slightly lower than that of FO- and DCO-fed fish (130 and 127 g, respectively). Inclusion of the GM-derived oils enhanced the n-3 LC-PUFA content in fish tissues compared to WCO, although limited biosynthesis was observed indicating accumulation of dietary fatty acids. The expression of genes involved in several lipid metabolic processes, as well as fish health and immune response, in both liver and anterior intestine were altered in fish fed the GM-derived oils. This showed a similar pattern to that observed in WCO-fed fish reflecting the hybrid fatty acid profile of the new oils. Overall the data indicated that the GM-derived oils could be suitable alternatives to dietary FO in sea bream.

Evaluation of a high-EPA oil from transgenic Camelina sativa in feeds for Atlantic salmon (Salmo salar L.): Effects on tissue fatty acid composition, histology and gene expression.

Currently, one alternative for dietary fish oil (FO) in aquafeeds is vegetable oils (VO) that are devoid of omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFAs). Entirely new sources of n-3 LC-PUFA such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids through de novo production are a potential solution to fill the gap between supply and demand of these important nutrients. Camelina sativa was metabolically engineered to produce a seed oil (ECO) with > 20% EPA and its potential to substitute for FO in Atlantic salmon feeds was tested. Fish were fed with one of the three experimental diets containing FO, wild-type camelina oil (WCO) or ECO as the sole lipid sources for 7 weeks. Inclusion of ECO did not affect any of the performance parameters studied and enhanced apparent digestibility of individual n-6 and n-3 PUFA compared to dietary WCO. High levels of EPA were maintained in brain, liver and intestine (pyloric caeca), and levels of DPA and DHA were increased in liver and intestine of fish fed ECO compared to fish fed WCO likely due to increased LC-PUFA biosynthesis based on up-regulation of the genes. Fish fed ECO showed slight lipid accumulation within hepatocytes similar to that with WCO, although not significantly different to fish fed FO. The regulation of a small number of genes could be attributed to the specific effect of ECO (311 features) with metabolism being the most affected category. The EPA oil from transgenic Camelina (ECO) could be used as a substitute for FO, however it is a hybrid oil containing both FO (EPA) and VO (18:2n-6) fatty acid signatures that resulted in similarly mixed metabolic and physiological responses.

A nutritionally-enhanced oil from transgenic Camelina sativa effectively replaces fish oil as a source of eicosapentaenoic acid for fish.

For humans a daily intake of up to 500 mg omega-3 (n-3) long-chain polyunsaturated fatty acids (LC-PUFA) is recommended, amounting to an annual requirement of 1.25 million metric tonnes (mt) for a population of 7 billion people. The annual global supply of n-3 LC-PUFA cannot meet this level of requirement and so there is a large gap between supply and demand. The dietary source of n-3 LC-PUFA, fish and seafood, is increasingly provided by aquaculture but using fish oil in feeds to supply n-3 LC-PUFA is unsustainable. Therefore, new sources of n-3 LC-PUFA are required to supply the demand from aquaculture and direct human consumption. One approach is metabolically engineering oilseed crops to synthesize n-3 LC-PUFA in seeds. Transgenic Camelina sativa expressing algal genes was used to produce an oil containing n-3 LC-PUFA to replace fish oil in salmon feeds. The oil had no detrimental effects on fish performance, metabolic responses or the nutritional quality of the fillets of the farmed fish.

Expression of epitope-tagged LMW glutenin subunits in the starchy endosperm of transgenic wheat and their incorporation into glutenin polymers.

The low-molecular-weight (LMW) glutenin subunits are components of the highly cross-linked glutenin polymers that confer viscoelastic properties to gluten and dough. They have both quantitative and qualitative effects on dough quality that may relate to differences in their ability to form the inter-chain disulphide bonds that stabilise the polymers. In order to determine the relationship between dough quality and the amounts and properties of the LMW subunits, we have transformed the pasta wheat cultivars Svevo and Ofanto with three genes encoding proteins, which differ in their numbers or positions of cysteine residues. The transgenes were delivered under control of the high-molecular-weight (HMW) subunit 1Dx5 gene promoter and terminator regions, and the encoded proteins were C-terminally tagged by the introduction of the c-myc epitope. Stable transformants were obtained with both cultivars, and the use of a specific antibody to the c-myc epitope tag allowed the transgene products to be readily detected in the complex mixture of LMW subunits. A range of transgene expression levels was observed. The addition of the epitope tag did not compromise the correct folding of the trangenic subunits and their incorporation into the glutenin polymers. Our results demonstrate that the ability to specifically epitope-tag LMW glutenin transgenes can greatly assist in the elucidation of their individual contributions to the functionality of the complex gluten system.

Expression of the gamma-zein protein of maize in seeds of transgenic barley: effects on grain composition and properties.

A cDNA clone encoding the gamma-zein protein of maize was expressed in developing grain of barley using the starchy endosperm cell-specific promoter from the wheat Glu-1D-1 (HMW subunit 1Dx5) gene. Seven transgenic lines were recovered from 226 bombarded immature embryos, of which two were sterile and four tetraploid, while five were shown to express the gamma-zein protein based on western blotting. Southern blot analysis showed the presence of between about three and twelve transgene insertions. Detailed comparative studies of five null and five homozygous transformed sub-lines from transgenic line A showed that gamma-zein accounted for over 4% of the total prolamin fraction, corresponding to about 1.9% of the total grain N. Comparison of the proteins present in the gel protein fraction demonstrated that the gamma-zein was incorporated into polymers, as in maize. However, there was no effect on grain hardness measured using the Perten Single Kernel Characterisation System or on the vitreousness measured by visual inspection. This contrasts with the situation in maize where a clear association with vitreousness has been reported.

Physiological characterization of 'stay green' mutants in durum wheat.

Four mutants with delayed leaf senescence were selected from seed of durum wheat mutagenized with ethylmethane sulphonate. Changes in net photosynthetic rate, efficiency of photosystem II and chlorophyll concentration during the maturation and senescence of the flag leaves of both mutant and parental plants were determined under glasshouse conditions. The four mutant lines maintained photosynthetic competence for longer than the parental line and are therefore functionally 'stay green'. The mutant lines also had higher seed weights and grain yields per plant than the parental line.

Plant stress signalling: understanding and exploiting plant-plant interactions.

When plants are attacked by insects, volatile chemical signals can be released, not only from the damaged parts, but also systemically from other parts of the plant and this continues after cessation of feeding by the insect. These signals are perceived by olfactory sensory mechanisms in both the herbivorous insects and their parasites. Molecular structures involved can be characterized by means of electrophysiological assays, using the insect sensory system linked to chemical analysis. Evidence is mounting that such signals can also affect neighbouring intact plants, which initiate defence by the induction of further signalling systems, such as those that increase parasitoid foraging. Furthermore, insect electrophysiology can be used in the identification of plant compounds having effects on the plants themselves. It has been found recently that certain plants can release stress signals even when undamaged, and that these can cause defence responses in intact plants. These discoveries provide the basis for new crop protection strategies, that are either delivered by genetic modification of plants or by conventionally produced plants to which the signal is externally applied. Delivery can also be made by means of mixed seed strategies in which the provoking and recipient plants are grown together. Related signalling discoveries within the rhizosphere seem set to extend these approaches into new ways of controlling weeds, by exploiting the elusive potential of allelopathy, but through signalling rather than by direct physiological effects.

Isolation and characterization of a cDNA encoding a Delta8 sphingolipid desaturase from Aquilegia vulgaris.

We have isolated a cDNA encoding the Delta(8) sphingolipid desaturase from the plant Aquilegia vulgaris L. via a PCR-based strategy using primers designed to target the conserved histidine box regions of microsomal desaturases. The function of the cDNA was confirmed by expression in the yeast, Saccharomyces cerevisiae. Analysis of the long-chain sphingoid bases as their dinitrophenyl derivatives by reverse-phase HPLC demonstrated the accumulation of cis - and trans -desaturated sphingoid bases which were not present in the wild-type yeast cells. The Delta(8) desaturated products co-eluted with known Delta(8)-desaturated phytosphingenine and the molecular mass of these products was confirmed by liquid chromatography-MS. The Delta(8) long-chain base desaturase was also able to desaturate dihydrosphingosine substrates. This is the first report of the functional characterization of an A. vulgaris gene product.

Genomic and functional characterization of polyunsaturated fatty acid biosynthesis in Caenorhabditis elegans.

The biosynthetic pathway for polyunsaturated fatty acids in the model animal Caenorhabditis elegans was examined in the context of the completed genome sequence. The genomic organization and location of seven desaturase genes and one elongase activity, all previously identified by functional characterization, were elucidated. A pathway for the biosynthesis of polyunsaturated fatty acids in C. elegans was proposed based on these genes. The role of gene duplication in enzyme evolution and proliferation is discussed.

HFE mutations, iron deficiency and overload in 10,500 blood donors.

People with genetic haemochromatosis (GH) accumulate iron from excessive dietary absorption. In populations of northern European origin, over 90% of patients are homozygous for the C282Y mutation of the HFE gene. While about 1 in 200 people in the general population have this genotype the proportion who develop clinical haemochromatosis is not known. The influence of HFE genotype on iron status was investigated in 10 556 blood donors. The allele frequencies of the C282Y and H63D mutations were 8.23% and 15.3% respectively. Heterozygosity for C282Y occurred in 1 in 7.9 donors, for H63D in 1 in 4.2 donors, and 1 in 42 were compound heterozygotes. Homozygosity for H63D occurred in 1 in 42 donors and 1 in 147 (72) were homozygous for C282Y. Mean values increased for transferrin saturation (TS) and serum ferritin (sFn), and decreased for unsaturated iron binding capacity (UIBC) in the order: donors lacking the mutations, H63D heterozygotes, C282Y heterozygotes, H63D homozygotes, compound heterozygotes and C282Y homozygotes, but serum ferritin (sFn) concentrations were no higher in H63D heterozygotes and C282Y heterozygous women than in donors lacking mutations. The percentage of donors failing the screening test for anaemia or of those with sFn < 15 microg/l did not differ among the genotype groups. C282Y and H63D heterozygotes and donors homozygous for H63D were at no greater risk of iron accumulation than donors lacking mutations, of whom 1 in 1200 had both a raised TS and sFn. The risk was higher for compound heterozygotes (1 in 80, P = 0.003) and for C282Y homozygotes (1 in 5, P < 0.0001). There was no correlation between sFn and either age or donation frequency in C282Y homozygotes. None of the 63 C282Y homozygous donors interviewed showed physical signs of overload or were aware of relatives with haemochromatosis. The Welsh Blood Service collects blood from about 140 000 people each year including an estimated 950 who are homozygous for HFE C282Y. They are probably healthy and unaware of any family history of iron overload.

Mutagenesis and heterologous expression in yeast of a plant Delta6-fatty acid desaturase.

Membrane-bound microsomal fatty acid desaturases are known to have three conserved histidine boxes, comprising a total of up to eight histidine residues. Recently, a number of deviations from this consensus have been reported, with the substitution of a glutamine for the first histidine residue of the third histidine box being present in the so called 'front end' desaturases. These enzymes are also characterized by the presence of a cytochrome b5 domain at the protein N-terminus. Site-directed mutagenesis has been used to probe the functional importance of a number of amino acid residues which comprise the third histidine box of a 'front end' desaturase, the borage Delta6-fatty acid desaturase. This showed that the variant glutamine in the third histidine box is essential for enzyme activity and that histidine is not able to substitute for this residue.

Functional identification of a delta8-sphingolipid desaturase from Borago officinalis.

The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids. The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-terminal cytochrome b5 domain. Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves.