Trauma pain management in the emergency department: use of methoxyflurane as a patient-administered analgesic.

Evidence suggests that pain is highly prevalent among patients with traumatic injuries who attend emergency departments (EDs), yet accurate assessment and management of patients with acute pain can be challenging in this setting. Effective and rapid pain management is beneficial for patients and can support timely discharge from the ED, which is particularly important in the context of the coronavirus disease 2019 (COVID-19) pandemic. This article describes a service development project that introduced the use of a patient-administered analgesic, methoxyflurane, for patients with traumatic injuries with moderate to severe pain in one ED. The author outlines the benefits and rationale for using methoxyflurane as a first-line analgesic in this patient group and describes the main elements of training sessions for emergency nurses and other ED clinicians in the administration and supervision of patient-administered methoxyflurane.

Is there a role for bariatric surgery in patients with severe obesity and type 1 diabetes?

BACKGROUND: The prevalence of obesity in type 1 diabetes has been increasing over the past decades. Multiple studies have demonstrated suboptimal outcomes with dietary control and medical management for obesity and type 2 diabetes. This study's objective was to evaluate insulin and diabetic medication requirements in patients with type 1 diabetes 2 years after bariatric surgery. METHODS: This was a retrospective medical-record review study from 2002 to 2019 at Geisinger Health System. Of 4549 total bariatric surgeries, 38 bariatric surgery patients were confirmed to have type 1 diabetes. Type 1 diabetes was confirmed by medical-record review and/or the presence of C-peptide <5 ng/mL. RESULTS: The patient cohort had a mean age of 41 years, with 87% being female. The mean body mass index was 43.0 kg/m2, with a mean HbA1C of 8.4% before surgery. During follow-up, the insulin requirements improved from 114 units preoperatively to 60 units at 1 year postoperatively (SD = 54.5, P = .0018) and 60 units at 2 years postoperatively (SD = 60.3, P = .0033). Though not significant, the number of patients on more than 1 diabetic medication decreased from 66% preoperatively to 53% 1 year postoperatively (P = .343) and 52% at 2 years (P = .149). CONCLUSION: This study demonstrated significant improvement in the insulin and total number of diabetic medication requirements after bariatric surgery, suggesting that bariatric surgery may be a viable treatment for patients with type 1 diabetes.

Aqueous Metabolite Trends for the Progression of Nonalcoholic Fatty Liver Disease in Female Bariatric Surgery Patients by Targeted 1H-NMR Metabolomics.

Determining biomarkers and better characterizing the biochemical progression of nonalcoholic fatty liver disease (NAFLD) remains a clinical challenge. A targeted 1H-NMR study of serum, combined with clinical variables, detected and localized biomarkers to stages of NAFLD in morbidly obese females. Pre-surgery serum samples from 100 middle-aged, morbidly obese female subjects, grouped on gold-standard liver wedge biopsies (non-NAFLD; steatosis; and fibrosis) were collected, extracted, and analyzed in aqueous (D2O) buffer (1H, 600 MHz). Profiled concentrations were subjected to exploratory statistical analysis. Metabolites varying significantly between the non-NAFLD and steatosis groups included the ketone bodies 3-hydroxybutyrate (↓; p = 0.035) and acetone (↓; p = 0.012), and also alanine (↑; p = 0.004) and a putative pyruvate signal (↑; p = 0.003). In contrast, the steatosis and fibrosis groups were characterized by 2-hydroxyisovalerate (↑; p = 0.023), betaine (↓; p = 0.008), hypoxanthine (↓; p = 0.003), taurine (↓; p = 0.001), 2-hydroxybutyrate (↑; p = 0.045), 3-hydroxyisobutyrate (↑; p = 0.046), and increasing medium chain fatty acids. Exploratory classification models with and without clinical variables exhibited overall success rates ca. 75-85%. In the study conditions, inhibition of fatty acid oxidation and disruption of the hepatic urea cycle are supported as early features of NAFLD that continue in fibrosis. In fibrosis, markers support inflammation, hepatocyte damage, and decreased liver function. Complementarity of NMR concentrations and clinical information in classification models is shown. A broader hypothesis that standard-of-care sera can yield metabolomic information is supported.

A Vernalization Response in a Winter Safflower (Carthamus tinctorius) Involves the Upregulation of Homologs of FT, FUL, and MAF.

Safflower (Carthamus tinctorius) is a member of the Asteraceae family that is grown in temperate climates as an oil seed crop. Most commercially grown safflower varieties can be sown in late winter or early spring and flower rapidly in the absence of overwintering. There are winter-hardy safflower accessions that can be sown in autumn and survive over-wintering. Here, we show that a winter-hardy safflower possesses a vernalization response, whereby flowering is accelerated by exposing germinating seeds to prolonged cold. The impact of vernalization was quantitative, such that increasing the duration of cold treatment accelerated flowering to a greater extent, until the response was saturated after 2 weeks exposure to low-temperatures. To investigate the molecular-basis of the vernalization-response in safflower, transcriptome activity was compared and contrasted between vernalized versus non-vernalized plants, in both 'winter hardy' and 'spring' cultivars. These genome-wide expression analyses identified a small set of transcripts that are both differentially expressed following vernalization and that also have different expression levels in the spring versus winter safflowers. Four of these transcripts were quantitatively induced by vernalization in a winter hardy safflower but show high basal levels in spring safflower. Phylogenetic analyses confidently assigned that the nucleotide sequences of the four differentially expressed transcripts are related to FLOWERING LOCUS T (FT), FRUITFUL (FUL), and two genes within the MADS-like clade genes. Gene models were built for each of these sequences by assembling an improved safflower reference genome using PacBio-based long-read sequencing, covering 85% of the genome, with N50 at 594,000 bp in 3000 contigs. Possible evolutionary relationships between the vernalization response of safflower and those of other plants are discussed.

A Synthetic Biology Workflow Reveals Variation in Processing and Solubility of Nitrogenase Proteins Targeted to Plant Mitochondria, and Differing Tolerance of Targeting Sequences in a Bacterial Nitrogenase Assay.

While industrial nitrogen fertilizer is intrinsic to modern agriculture, it is expensive and environmentally harmful. One approach to reduce fertilizer usage is to engineer the bacterial nitrogenase enzyme complex within plant mitochondria, a location that may support enzyme function. Our current strategy involves fusing a mitochondrial targeting peptide (MTP) to nitrogenase (Nif) proteins, enabling their import to the mitochondrial matrix. However, the process of import modifies the N-terminus of each Nif protein and may impact nitrogenase assembly and function. Here we present our workflow assessing the mitochondrial processing, solubility and relative abundance of 16 Klebsiella oxytoca Nif proteins targeted to the mitochondrial matrix in Nicotiana benthamiana leaf. We found that processing and abundance of MTP::Nif proteins varied considerably, despite using the same constitutive promoter and MTP across all Nif proteins tested. Assessment of the solubility for all MTP::Nif proteins when targeted to plant mitochondria found NifF, M, N, S, U, W, X, Y, and Z were soluble, while NifB, E, H, J, K, Q, and V were mostly insoluble. The functional consequence of the N-terminal modifications required for mitochondrial targeting of Nif proteins was tested using a bacterial nitrogenase assay. With the exception of NifM, the Nif proteins generally tolerated the N-terminal extension. Proteomic analysis of Nif proteins expressed in bacteria found that the relative abundance of NifM with an N-terminal extension was increased ~50-fold, while that of the other Nif proteins was not influenced by the N-terminal extension. Based on the solubility, processing and functional assessments, our workflow identified that K. oxytoca NifF, N, S, U, W, Y, and Z successfully met these criteria. For the remaining Nif proteins, their limitations will need to be addressed before proceeding towards assembly of a complete set of plant-ready Nif proteins for reconstituting nitrogenase in plant mitochondria.

Plant expression of NifD protein variants resistant to mitochondrial degradation.

To engineer Mo-dependent nitrogenase function in plants, expression of the structural proteins NifD and NifK will be an absolute requirement. Although mitochondria have been established as a suitable eukaryotic environment for biosynthesis of oxygen-sensitive enzymes such as NifH, expression of NifD in this organelle has proven difficult due to cryptic NifD degradation. Here, we describe a solution to this problem. Using molecular and proteomic methods, we found NifD degradation to be a consequence of mitochondrial endoprotease activity at a specific motif within NifD. Focusing on this functionally sensitive region, we designed NifD variants comprising between one and three amino acid substitutions and distinguished several that were resistant to degradation when expressed in both plant and yeast mitochondria. Nitrogenase activity assays of these resistant variants in Escherichia coli identified a subset that retained function, including a single amino acid variant (Y100Q). We found that other naturally occurring NifD proteins containing alternate amino acids at the Y100 position were also less susceptible to degradation. The Y100Q variant also enabled expression of a NifD(Y100Q)-linker-NifK translational polyprotein in plant mitochondria, confirmed by identification of the polyprotein in the soluble fraction of plant extracts. The NifD(Y100Q)-linker-NifK retained function in bacterial nitrogenase assays, demonstrating that this polyprotein permits expression of NifD and NifK in a defined stoichiometry supportive of activity. Our results exemplify how protein design can overcome impediments encountered when expressing synthetic proteins in novel environments. Specifically, these findings outline our progress toward the assembly of the catalytic unit of nitrogenase within mitochondria.

Insights into Nitrogenase Bioelectrocatalysis for Green Ammonia Production.

There is a growing interest in using ammonia as a liquid carrier of hydrogen for energy applications. Currently, ammonia is produced industrially by the Haber-Bosch process, which requires high temperature and high pressure. In contrast, bacteria have naturally evolved an enzyme known as nitrogenase, that is capable of producing ammonia and hydrogen at ambient temperature and pressure. Therefore, nitrogenases are attractive as a potentially more efficient means to produce ammonia via harnessing the unique properties of this enzyme. In recent years, exciting progress has been made in bioelectrocatalysis using nitrogenases to produce ammonia. Here, the prospects for developing biological ammonia production are outlined, key advances in bioelectrocatalysis by nitrogenases are highlighted, and possible solutions to the obstacles faced in realising this goal are discussed.

Producing Cyclopropane Fatty Acid in Plant Leafy Biomass via Expression of Bacterial and Plant Cyclopropane Fatty Acid Synthases.

Saturated mid-chain branched fatty acids (SMCBFAs) are widely used in the petrochemical industry for their high oxidative stability and low melting temperature. Dihydrosterculic acid (DHSA) is a cyclopropane fatty acid (CPA) that can be converted to SMCBFA via hydrogenation, and therefore oils rich in DHSA are a potential feedstock for SMCBFA. Recent attempts to produce DHSA in seed oil by recombinant expression of cyclopropane fatty acid synthases (CPFASes) resulted in decreased oil content and poor germination or low DHSA accumulation. Here we explored the potential for plant vegetative tissue to produce DHSA by transiently expressing CPFAS enzymes in leaf. When CPFASes from plant and bacterial origin were transiently expressed in Nicotiana benthamiana leaf, it accumulated up to 1 and 3.7% DHSA in total fatty acid methyl ester (FAME), respectively, which increased up to 4.8 and 11.8%, respectively, when the N. benthamiana endogenous oleoyl desaturase was silenced using RNA interference (RNAi). Bacterial CPFAS expression produced a novel fatty acid with a cyclopropane ring and two carbon-carbon double bonds, which was not seen with plant CPFAS expression. We also observed a small but significant additive effect on DHSA accumulation when both plant and bacterial CPFASes were co-expressed, possibly due to activity upon different oleoyl substrates within the plant cell. Lipidomics analyses found that CPFAS expression increased triacylglycerol (TAG) accumulation relative to controls and that DHSA was distributed across a range of lipid species, including diacylglycerol and galactolipids. DHSA and the novel CPA were present in phosphatidylethanolamine when bacterial CPFAS was expressed in leaf. Finally, when plant diacylglycerol acyltransferase was coexpressed with the CPFASes DHSA accumulated up to 15% in TAG. This study shows that leaves can readily produce and accumulate DHSA in leaf oil. Our findings are discussed in line with current knowledge in leaf oil production for a possible route to DHSA production in vegetative tissue.

M3 muscarinic receptor activation reduces hepatocyte lipid accumulation via CaMKKß/AMPK pathway.

Previously, we reported that hepatic muscarinic receptors modulate both acute and chronic liver injury, however, the role of muscarinic receptors in fatty liver disease is unclear. We observed in patients who underwent weight loss surgery, a decrease in hepatic expression of M3 muscarinic receptors (M3R). We also observed that fat loading of hepatocytes, increased M3R expression. Based on these observations, we tested the hypothesis that M3R regulate hepatocyte lipid accumulation. Incubation of AML12 hepatocytes with 1 mM oleic acid resulted in lipid accumulation that was significantly reduced by co-treatment with a muscarinic agonist (pilocarpine or carbachol), an effect blocked by atropine (a muscarinic antagonist). Similar treatment of Hepa 1-6 cells, a mouse hepatoblastoma cell line, showed comparable results. In both, control and fat-loaded AML12 cells, pilocarpine induced time-dependent AMPKα phosphorylation and significantly up-regulated lipolytic genes (ACOX1, CPT1, and PPARα). Compound C, a selective and reversible AMPK inhibitor, significantly blunted pilocarpine-mediated reduction of lipid accumulation and pilocarpine-mediated up-regulation of lipolytic genes. BAPTA-AM, a calcium chelator, and STO-609, a calcium/calmodulin-dependent protein kinase kinase inhibitor, attenuated agonist-induced AMPKα phosphorylation. Finally, M3R siRNA attenuated agonist-induced AMPKα phosphorylation as well as agonist-mediated reduction of hepatocyte steatosis. In conclusion, this proof-of-concept study demonstrates that M3R has protective effects against hepatocyte lipid accumulation by activating AMPK pathway and is a potential therapeutic target for non-alcoholic fatty liver disease.

Is age a real or perceived discriminator for bariatric surgery? A long-term analysis of bariatric surgery in the elderly.

BACKGROUND: Bariatric surgery is the most effective treatment of obesity. There are few studies evaluating long-term outcomes in elderly patients. OBJECTIVES: Our study was designed to evaluate the safety and long-term outcomes of bariatric surgery in the elderly compared with a contemporary medically managed cohort. SETTING: University hospital. METHODS: Three hundred thirty-seven patients age ≥60 who underwent a sleeve gastrectomy or Roux-en-Y gastric bypass between January 2007 and April 2017 were identified (ElderSurg) and compared with a matched cohort of medically managed elderly patients with obesity (ElderNonSurg). RESULTS: Thirty-two patients underwent laparoscopic sleeve gastrectomy, 190 underwent laparoscopic Roux-en-Y gastric bypass, and 115 underwent open Roux-en-Y gastric bypass. The cohort was a mean of 64.4-years old, 75.4% female, mean preoperative body mass index was 46.9, and 62.6% had type 2 diabetes. During a median follow-up period of 56.2 months (confidence interval 49.5-62.9), mean percent excess weight loss (EWL) at nadir was 72.1 ± 24.7% and EWL at 36 months or beyond was 60.9 ± 27.6%. On regression analysis, diabetes, body mass index, and laparoscopic sleeve gastrectomy were negatively associated with EWL at all time periods (P < .05). Mean %EWL was greater for Roux-en-Y gastric bypass compared with laparoscopic sleeve gastrectomy (61.7 versus 41.2; P = .039). Diabetes remission rate was 45.8%. There was a statistically significant decrease in the risk of death in ElderSurg (hazard ratio .584, 95% confidence interval .362-.941) compared with ElderNonSurg. CONCLUSIONS: Our study supports that bariatric surgery is safe in elderly patients with effective long-term control of obesity, diabetes, and with improved overall survival.

Identification of Genes Involved in Lipid Biosynthesis through de novo Transcriptome Assembly from Cocos nucifera Developing Endosperm.

Cocos nucifera (coconut), a member of the Arecaceae family, is an economically important woody palm that is widely grown in tropical and subtropical regions. The coconut palm is well known for its ability to accumulate large amounts of oil, approximately 63% of the seed weight. Coconut oil varies significantly from other vegetable oils as it contains a high proportion of medium-chain fatty acids (MCFA; 85%). The unique composition of coconut oil raises interest in understanding how the coconut palm produces oil of a high saturated MCFA content, and if such an oil profile could be replicated via biotechnology interventions. Although some gene discovery work has been performed there is still a significant gap in the knowledge associated with coconut's oil production pathways. In this study, a de novo transcriptome was assembled for developing coconut endosperm to identify genes involved in the synthesis of lipids, particularly triacylglycerol. Of particular interest were thioesterases, acyltransferases and oleosins because of their involvement in the processes of releasing fatty acids for assembly, esterification of fatty acids into glycerolipids and protecting oils from degradation, respectively. It is hypothesized that some of these genes may exhibit a strong substrate preference for MCFA and hence may assist the future development of vegetable oils with an enriched MCFA composition. In this study, we identified and confirmed functionality of five candidate genes from the gene families of interest. This study will benefit future work in areas of increasing vegetable oil production and the tailoring of oil fatty acid compositions.

Seed-specific RNAi in safflower generates a superhigh oleic oil with extended oxidative stability.

Vegetable oils extracted from oilseeds are an important component of foods, but are also used in a range of high value oleochemical applications. Despite being biodegradable, nontoxic and renewable current plant oils suffer from the presence of residual polyunsaturated fatty acids that are prone to free radical formation that limit their oxidative stability, and consequently shelf life and functionality. Many decades of plant breeding have been successful in raising the oleic content to ~90%, but have come at the expense of overall field performance, including poor yields. Here, we engineer superhigh oleic (SHO) safflower producing a seed oil with 93% oleic generated from seed produced in multisite field trials spanning five generations. SHO safflower oil is the result of seed-specific hairpin-based RNA interference of two safflower lipid biosynthetic genes, FAD2.2 and FATB, producing seed oil containing less than 1.5% polyunsaturates and only 4% saturates but with no impact on lipid profiles of leaves and roots. Transgenic SHO events were compared to non-GM safflower in multisite trial plots with a wide range of growing season conditions, which showed no evidence of impact on seed yield. The oxidative stability of the field-grown SHO oil produced from various sites was 50 h at 110°C compared to 13 h for conventional ~80% oleic safflower oils. SHO safflower produces a uniquely stable vegetable oil across different field conditions that can provide the scale of production that is required for meeting the global demands for high stability oils in food and the oleochemical industry.

Obesity as a risk factor for developing functional limitation among older adults: A conditional inference tree analysis.

OBJECTIVE: To examine the risk factors of developing functional decline and make probabilistic predictions by using a tree-based method that allows higher order polynomials and interactions of the risk factors. METHODS: The conditional inference tree analysis, a data mining approach, was used to construct a risk stratification algorithm for developing functional limitation based on BMI and other potential risk factors for disability in 1,951 older adults without functional limitations at baseline (baseline age 73.1 ± 4.2 y). We also analyzed the data with multivariate stepwise logistic regression and compared the two approaches (e.g., cross-validation). Over a mean of 9.2 ± 1.7 years of follow-up, 221 individuals developed functional limitation. RESULTS: Higher BMI, age, and comorbidity were consistently identified as significant risk factors for functional decline using both methods. Based on these factors, individuals were stratified into four risk groups via the conditional inference tree analysis. Compared to the low-risk group, all other groups had a significantly higher risk of developing functional limitation. The odds ratio comparing two extreme categories was 9.09 (95% confidence interval: 4.68, 17.6). CONCLUSIONS: Higher BMI, age, and comorbid disease were consistently identified as significant risk factors for functional decline among older individuals across all approaches and analyses.

Expression of 16 Nitrogenase Proteins within the Plant Mitochondrial Matrix.

The industrial production and use of nitrogenous fertilizer involves significant environmental and economic costs. Strategies to reduce fertilizer dependency are required to address the world's increasing demand for sustainable food, fibers, and biofuels. Biological nitrogen fixation, a process unique to diazatrophic bacteria, is catalyzed by the nitrogenase complex, and reconstituting this function in plant cells is an ambitious biotechnological strategy to reduce fertilizer use. Here we establish that the full array of biosynthetic and catalytic nitrogenase (Nif) proteins from the diazotroph Klebsiella pneumoniae can be individually expressed as mitochondrial targeting peptide (MTP)-Nif fusions in Nicotiana benthamiana. We show that these are correctly targeted to the plant mitochondrial matrix, a subcellular location with biochemical and genetic characteristics potentially supportive of nitrogenase function. Although Nif proteins B, D, E, F, H, J, K, M, N, Q, S, U, V, X, Y, and Z were all detectable by Western blot analysis, the NifD catalytic component was the least abundant. To address this problem, a translational fusion between NifD and NifK was designed based on the crystal structure of the nitrogenase MoFe protein heterodimer. This fusion protein enabled equimolar NifD:NifK stoichiometry and improved NifD expression levels in plants. Finally, four MTP-Nif fusion proteins (B, S, H, Y) were successfully co-expressed, demonstrating that multiple components of nitrogenase can be targeted to plant mitochondria. These results establish the feasibility of reconstituting the complete componentry for nitrogenase in plant cells, within an intracellular environment that could support the conversion of nitrogen gas into ammonia.

A reconfigured Kennedy pathway which promotes efficient accumulation of medium-chain fatty acids in leaf oils.

Medium-chain fatty acids (MCFA, C6-14 fatty acids) are an ideal feedstock for biodiesel and broader oleochemicals. In recent decades, several studies have used transgenic engineering to produce MCFA in seeds oils, although these modifications result in unbalance membrane lipid profiles that impair oil yields and agronomic performance. Given the ability to engineer nonseed organs to produce oils, we have previously demonstrated that MCFA profiles can be produced in leaves, but this also results in unbalanced membrane lipid profiles and undesirable chlorosis and cell death. Here we demonstrate that the introduction of a diacylglycerol acyltransferase from oil palm, EgDGAT1, was necessary to channel nascent MCFA directly into leaf oils and therefore bypassing MCFA residing in membrane lipids. This pathway resulted in increased flux towards MCFA rich leaf oils, reduced MCFA in leaf membrane lipids and, crucially, the alleviation of chlorosis. Deep sequencing of African oil palm (Elaeis guineensis) and coconut palm (Cocos nucifera) generated candidate genes of interest, which were then tested for their ability to improve oil accumulation. Thioesterases were explored for the production of lauric acid (C12:0) and myristic (C14:0). The thioesterases from Umbellularia californica and Cinnamomum camphora produced a total of 52% C12:0 and 40% C14:0, respectively, in transient leaf assays. This study demonstrated that the introduction of a complete acyl-CoA-dependent pathway for the synthesis of MFCA-rich oils avoided disturbing membrane homoeostasis and cell death phenotypes. This study outlines a transgenic strategy for the engineering of biomass crops with high levels of MCFA rich leaf oils.

Metabolic Health Status and the Obesity Paradox in Older Adults.

The explanation for reduced mortality among older persons with overweight or class I obesity compared to those of desirable weight remains unclear. Our objective was to investigate the joint effects of body mass index (BMI) and metabolic health status on all-cause mortality in a cohort of advanced age. Adults aged 74 ± 4.7 (mean ± SD) years at baseline (n = 4551) were categorized according to BMI (18.5-24.9, 25.0-29.9, 30.0-34.9, and ≥35.0 kg/m(2)) and the presence or absence of a metabolically healthy phenotype (i.e., 0 or 1 risk factors based on a modified Adult Treatment Panel III). Metabolically unhealthy was ≥2 risk factors. There were 2294 deaths over a mean 10.9 years of follow up. Relative to metabolically healthy desirable weight, metabolically healthy overweight or class I obesity was not associated with a greater mortality risk (HR 0.90; 95 CI% 0.73-1.13 and HR 0.58; 95 CI% 0.42-0.80, respectively) (P-interaction <0.001). Results remained consistent in rigorous sensitivity analyses. The "obesity paradox" may be partially explained by the inclusion of metabolically healthy overweight and obese older persons, who do not have elevated mortality risk, in population studies of BMI and mortality.

Body mass index and all-cause mortality among older adults.

OBJECTIVE: To examine the association between baseline body mass index (BMI, kg/m(2) ) and all-cause mortality in a well-characterized cohort of older persons. METHODS: The association between BMI (both as a categorical and continuous variable) and all-cause mortality was investigated using 4,565 Geisinger Rural Aging Study participants with baseline age 74.0 ± 4.7 years (mean ± SD) and BMI 29.5 ± 5.3 kg/m(2) over a mean of 10.9 ± 3.8 years of follow-up. RESULTS: The relationship between BMI (as a continuous variable) and all-cause mortality was found to be U-shaped (P nonlinearity <0.001). Controlling for age, sex, smoking, alcohol, laboratory values, medications, and comorbidity status, underweight (BMI <18.5 kg/m(2) ) individuals had significantly greater adjusted risk of all-cause mortality than persons of BMI 18.5 to 24.9 kg/m(2) (reference range). Participants with overweight (BMI 25.0-29.9 kg/m(2) ) and class I obesity (BMI 30.0-34.9 kg/m(2) ) had significantly lower adjusted-risk of all-cause mortality. Those with classes II/III obesity (BMI ≥ 35.0 kg/m(2) ) did not have significantly greater adjusted-risk of all-cause mortality. Findings were consistent using propensity score weights and among never-smokers with 2- and 5-year lag analysis and among those with no identified chronic disease. CONCLUSIONS: A U-shaped association was observed between BMI and all-cause mortality with lower risk among older persons with overweight and class I obesity in comparison with those with BMI 18.5 to 24.9 kg/m(2) .

Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway.

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.

A case study on the genetic origin of the high oleic acid trait through FAD2-1 DNA sequence variation in safflower (Carthamus tinctorius L.).

The safflower (Carthamus tinctorius L.) is considered a strongly domesticated species with a long history of cultivation. The hybridization of safflower with its wild relatives has played an important role in the evolution of cultivars and is of particular interest with regards to their production of high quality edible oils. Original safflower varieties were all rich in linoleic acid, while varieties rich in oleic acid have risen to prominence in recent decades. The high oleic acid trait is controlled by a partially recessive allele ol at a single locus OL. The ol allele was found to be a defective microsomal oleate desaturase FAD2-1. Here we present DNA sequence data and Southern blot analysis suggesting that there has been an ancient hybridization and introgression of the FAD2-1 gene into C. tinctorius from its wild relative C. palaestinus. It is from this gene that FAD2-1Δ was derived more recently. Identification and characterization of the genetic origin and diversity of FAD2-1 could aid safflower breeders in reducing population size and generations required for the development of new high oleic acid varieties by using perfect molecular marker-assisted selection.

Metabolic engineering of medium-chain fatty acid biosynthesis in Nicotiana benthamiana plant leaf lipids.

Various research groups are investigating the production of oil in non-seed biomass such as leaves. Recently, high levels of oil accumulation have been achieved in plant biomass using a combination of biotechnological approaches which also resulted in significant changes to the fatty acid composition of the leaf oil. In this study, we were interested to determine whether medium-chain fatty acids (MCFA) could be accumulated in leaf oil. MCFA are an ideal feedstock for biodiesel and a range of oleochemical products including lubricants, coatings, and detergents. In this study, we explore the synthesis, accumulation, and glycerolipid head-group distribution of MCFA in leaves of Nicotiana benthamiana after transient transgenic expression of C12:0-, C14:0-, and C16:0-ACP thioesterase genes. We demonstrate that the production of these MCFA in leaf is increased by the co-expression of the WRINKLED1 (WRI1) transcription factor, with the lysophosphatidic acid acyltransferase (LPAAT) from Cocos nucifera being required for the assembly of tri-MCFA TAG species. We also demonstrate that the newly-produced MCFA are incorporated into the triacylglycerol of leaves in which WRI1 + diacylglycerol acyltransferase1 (DGAT1) genes are co-expressed for increased oil accumulation.