Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase

Citation: Bansal, S., & Durrett, T. P. (2016). Defining the extreme substrate specificity of Euonymus alatus diacylglycerol acetyltransferase, an unusual membrane-bound O-acyltransferase. Bioscience Reports, 36, 9. doi:10.1042/bsr20160277Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes the unusually structured 3-acetyl-1,2-diacylglycerols (acetyl-TAG) found in the seeds of a few plant species. A member of the membrane-bound O-acyltransferase (MBOAT) family, EaDAcT transfers the acetyl group from acetyl-CoA to sn-1,2-diacylglycerol (DAG) to produce acetyl-TAG. In vitro assays demonstrated that the enzyme is also able to utilize butyryl-CoA and hexanoyl-CoA as acyl donors, though with much less efficiency compared with acetyl-CoA. Acyl-CoAs longer than eight carbons were not used by EaDAcT. This extreme substrate specificity of EaDAcT distinguishes it from all other MBOATs which typically catalyze the transfer of much longer acyl groups. In vitro selectivity experiments revealed that EaDAcT preferentially acetylated DAG molecules containing more double bonds over those with less. However, the enzyme was also able to acetylate saturated DAG containing medium chain fatty acids, albeit with less efficiency. Interestingly, EaDAcT could only acetylate the free hydroxyl group of sn-1,2-DAG but not the available hydroxyl groups in sn-1,3-DAG or in monoacylglycerols (MAG). Consistent with its similarity to the jojoba wax synthase, EaDAcT could acetylate fatty alcohols in vitro to produce alkyl acetates. Likewise, when coexpressed in yeast with a fatty acyl-CoA reductase capable of producing fatty alcohols, EaDAcT synthesized alkyl acetates although the efficiency of production was low. This improved understanding of EaDAcT specificity confirms that the enzyme preferentially utilizes acetyl-CoA to acetylate sn-1,2-DAGs and will be helpful in engineering the production of acetyl-TAG with improved functionality in transgenic plants

Defining the substrate specificity of an unusual acyltransferase: a step towards the production of an advanced biofuel

Doctor of PhilosophyBiochemistry and Molecular Biophysics Interdepartmental ProgramTimothy P. DurrettThe direct use of vegetable oils as a biofuel suffers from problems such as high viscosity, low volatility and poor cold temperature properties. 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) have lower viscosity and freezing temperature than regular vegetable oils. However, by modifying their fatty acid composition, further improvement in their fuel properties is possible. Our goal was to develop plants that synthesize seed oils with further improved fuel properties. Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) synthesizes acetyl-TAGs by the acetyl-CoA dependent acylation of diacylglycerol (DAG). Knowledge of the substrate specificity of EaDAcT for its acetyl-CoA donor and DAG acceptor substrates is important to generate the required acetyl-TAG composition in seed oil. A rapid method to quantify acetyl-TAGs was developed based on electrospray ionization mass spectrometry to gain information about the substrate specificity of EaDAcT. This method is as accurate and more rapid than the traditional radiolabeled substrate based assay and additionally provides information on acetyl-TAG molecular species present. Using this assay, EaDAcT specificity for different chain length acyl-CoA and DAGs was tested. It was found that although EaDAcT can use other short chain length acyl-CoAs as acyl donors, it has high preference for acetyl-CoA. Further, EaDAcT can acetylate a variety of DAGs with short, medium and long chain length fatty acids with high preference for DAGs containing unsaturated fatty acids. To generate acetyl-TAGs with lower molecular mass, EaDAcT was transformed into transgenic Camelina sativa lines producing high amounts of medium chain fatty acids (MCFAs). EaDAcT expression was also combined with the knockdown of DGAT1 and PDAT enzymes, which compete with EaDAcT for their common DAG substrate. High acetyl-TAG yielding homozygous T3 transgenic lines were generated but the incorporation of MCFAs into acetyl-TAGs was inefficient. A small increase in the viscosity of acetyl-TAGs from these lines was observed compared to acetyl-TAGs produced in wild type Camelina plant. The combined effect of insufficient lowering of molecular mass and increased fatty acid saturation levels of acetyl-TAGs might be responsible for this increased viscosity. Overall, it was concluded that the molecular mass and the saturation levels of fatty acids of acetyl-TAGs need to be considered at the same time in future attempts to further decrease their viscosity

Smoking Pattern in Family Members of Smokers in Slums of Surat City, Western India

Background: The relationship between becoming a smoker and having smoker parents, siblings, and relatives is still uncovered in India. The influences of a smoking role model in a family on smoking habits of individuals are yet to be revealed. This study aimed to understand the relationship of smoking abuse of a person with smoking of their family members. Methods: This community-based cross-sectional study was conducted in the slums of 20 urban health centers (UHCs) of Surat city (India). A pretested semi-structured questionnaire was used to collect data. The data was analyzed using Epi-Info software. Findings: Among 281 smoker participants, 168 (59.8%), 55 (19.6%), 95 (33.8%), and 50 (17.8%) had smoking fathers, grandparents, siblings, and other relatives, respectively. While 131 participants (44.6%) had correct information about the law of banned smoking, 249 participants (88.6%) were in favor of this law. The association of smoking abuse in fathers with smoking abuse in siblings came out to be odds ratio (OR) = 3.75 (95% CI: 2.11-6.63) and grandparents to be odds ratio 16.43 (95% CI: 4.98-54.17), respectively. The association between education and following the law of banned smoking was statistically significant OR = 2.98 (95% CI: 1.43-6.00).Conclusion: Substance abuse in parents, siblings, and other relatives is likely to influence the behavior of the person towards it. Persons living in the same vicinity may also greatly influence the behavior of an individual.Keywords: Smoking, Smoker, India

Definitive chemoradiation in non metastatic squamous cell carcinoma anal canal: a single institution experience

Background: To analyze the oncological outcomes in anal canal squamous cell carcinoma treated with concurrent chemoradiotherapy. Materials and Methods: A single centre retrospective hospital based study with sample size of 51 patients of anal canal Squamous cell carcinoma treated with concurrent chemoradiotherapy with mitomycin @10mg/m2 and 5FU based. Disease free survival (DFS), Colostomy free survival (CFS) and Overall survival (OS) rates were calculated by Kaplan-Meier method. Results: Among 51 eligible patients, after a median follow up of 46 months (range 10-68 months). The 3year Disease free survival (DFS) was 73.9%. 3patients developed locoregional recurrence while 1patient developed distant metastasis. At 3 years Overall survival (OS) rate was 77%. Out of 44 patients 6 patients lost to follow up while 2 patients died due to progressive disease and 2 due to non cancer causes. 3year Colostomy free survival (CFS) rate was 59%. Total 18 out of 44 patients underwent colostomy. No grade 3 or 4 late toxicities occurred after completion of treatment. Conclusion: This study concluded that definitive chemoradiotherapy achieves good local control, overall survival and colostomy free survival with acceptable toxicity and can be recommended as standard treatment in patients with carcinoma anal canal

Evaluation of peripheral arterial occlusive disease by computed tomography angiography

Background: Characterization of peripheral arterial occlusive disease (PAOD) can be performed with non-invasive angiography using computed tomography (CT). The present study was conducted to evaluate the CT angiographic spectrum of aortoiliac and the lower limb arterial disease in symptomatic patients of peripheral arterial occlusive disease (PAOD) and to classify the lesions according to the Trans-Atlantic Inter-Society Consensus II (TASC II).Methods: The study was carried out in the department of radio-diagnosis in collaboration with departments of cardiothoracic surgery, cardiology and surgery, Safdarjung Hospital and Vardhman Mahavir Medical College, New Delhi. 50 patients aged above 40 years presenting with symptoms and (or) signs of lower limb peripheral arterial disease were recruited into the study after evaluating the renal function. Patient’s clinical history, ABI index and categories of PAOD according to the classification of Fontaine was noted. CT angiography of aortoiliac and lower limb arteries was performed with Philips Brilliance 40 CT unit. The findings in each CT angiography were analysed in respect to site, number, nature and distribution of the lesions and classified individually according to the TASC II.Results: The patients included in the study were all more than 40 years of age. The age range in the study group was 42 years to 75 years. The majority (86%) were male patients. Smoking and dyslipidemia were found to be the main risk factors in our patients. 24% of patients had documentary evidence of ischemic heart disease. On grading with ABI majority of patients (58%) presented in the end stage of the disease (stage IV). On CT angiography, number of lesions detected was 157. 97.4% of lesions were either stenotic or occlusive and 2.54% are with aneurysm. Maximum number of patients had femoropopliteal lesions followed by aortailiac lesions. 14 Winslow pathways were found in 10 patients. Maximum numbers of femoropopliteal lesions (47.29%) belong to type D, type B lesions account for 50% of total aortoiliac lesions based on TASC II classification. Out of 50, 40 were made follow up. Among them 8 were managed with conservative treatment and remaining 32 managed with treatment based TASC II classification.Conclusions: CT angiography is a reliable noninvasive imaging method for the comprehensive and multi parameter evaluation of patients with PAOD. CT angiographic findings are a highly accurate basis for treatment decisions and planning

Towards the synthetic design of camelina oil enriched in tailored acetyl-triacylglycerols with medium-chain fatty acids

The ability to manipulate expression of key biosynthetic enzymes has allowed the development of genetically modified plants that synthesise unusual lipids that are useful for biofuel and industrial applications. By taking advantage of the unique activities of enzymes from different species, tailored lipids with a targeted structure can be conceived. In this study we demonstrate the successful implementation of such an approach by metabolically engineering the oilseed crop Camelina sativa to produce 3-acetyl-1,2-diacyl-sn-glycerols (acetyl-TAGs) with medium-chain fatty acids (MCFAs). Different transgenic camelina lines that had been genetically modified to produce MCFAs through the expression of MCFA-specific thioesterases and acyltransferases were retransformed with the Euonymus alatus gene for diacylglycerol acetyltransferase (EaDAcT) that synthesises acetyl-TAGs. Concomitant RNAi suppression of acyl-CoA:diacylglycerol acyltransferase increased the levels of acetyl-TAG, with up to 77 mole percent in the best lines. However, the total oil content was reduced. Analysis of the composition of the acetyl-TAG molecular species using electrospray ionisation mass spectrometry demonstrated the successful synthesis of acetyl-TAG containing MCFAs. Field growth of high-yielding plants generated enough oil for quantification of viscosity. As part of an ongoing design–test–learn cycle, these results, which include not only the synthesis of ‘designer’ lipids but also their functional analysis, will lead to the future production of such molecules tailored for specific applications

Community Based Assessment of Biochemical Risk Factors for Cardiovascular Diseases in Rural and Tribal Area of Himalayan Region, India

Context. Evident change in nutrition and lifestyle among individuals of urban and rural areas raises suspicion for similar change in tribal area population of India. Aim. To study the biochemical risk factor for CVDs in rural and tribal population of Sub-Himalayan state of India. Settings and Design. Cross-sectional study in rural (low altitude) and tribal (high altitude) area of Himachal Pradesh, India. Methodology. Blood lipid profile using standard laboratory methods. Statistical Analysis. Chi-square test and multiple linear regression analysis. Results. Total of 900 individuals were studied in both areas. As per Asian criteria, obesity (BMI 27.5–30.0 kg/m2) was observed to be significantly high (P=0.00) as 13.7% in tribal area as compared to 5.5% in rural area. Normal level of TC (<200 mg/dL) and LDL (<130 mg/dL) was observed in the majority of the population of both areas, whereas, at risk level of HDL (<40 mg/dL) was present in half of the population of both rural and tribal areas. The prevalence of borderline to high level of TGs was observed to be 60.2% and 55.2% in rural and tribal (P=0.10) area, respectively. Conclusion. Prevalent abnormal lipid profile in tribal area demands establishment of an effective surveillance system for development of chronic diseases

Molecular tools enabling pennycress (\u3ci\u3eThlaspi arvense\u3c/i\u3e) as a model plant and oilseed cash cover crop

Thlapsi arvense L. (pennycress) is being developed as a profitable oilseed cover crop for the winter fallow period throughout the temperate regions of the world, controlling soil erosion and nutrients run-off on otherwise barren farmland. We demonstrate that pennycress can serve as a user-friendly model system akin to Arabidopsis that is well-suited for both laboratory and field experimentation. We sequenced the diploid genome of the spring-type Spring 32-10 inbred line (1C DNA content of 539 Mb; 2n = 14), identifying variation that may explain phenotypic differences with winter-type pennycress, as well as predominantly a one-to-one correspondence with Arabidopsis genes, which makes translational research straightforward. We developed an Agrobacterium-mediated floral dip transformation method (0.5% transformation efficiency) and introduced CRISPR-Cas9 constructs to produce indel mutations in the putative FATTY ACID ELONGATION1 (FAE1) gene, thereby abolishing erucic acid production and creating an edible seed oil comparable to that of canola. We also stably transformed pennycress with the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene, producing low-viscosity acetyltriacylglycerol- containing seed oil suitable as a diesel-engine drop-in fuel. Adoption of pennycress as a model system will accelerate oilseed-crop translational research and facilitate pennycress’ rapid domestication to meet the growing sustainable food and fuel demands