The role of very long chain fatty acids in Arabidopsis growth and development

Very long chain fatty acids (VLCFAs) are essential to Arabidopsis growth and development. VLCFAs are found in sphingolipids, glycerophospholipids, triacylglycerols, suberin and cuticular waxes. VLCFAs are synthesized by the addition of 2 carbons from malonyl-CoA to pre-existing acyl-CoAs to produce chain lengths of greater than 18 carbon atoms. VLCFA synthesis involves four consecutive reactions that are catalysed by the microsomal Fatty Acid Elongase. In Arabidopsis the first reaction is catalysed by one of 21 different Keto-CoA Synthases (KCS) with diverse levels of expression and overlapping tissue specificities. The other three enzymes are ubiquitously expressed throughout the plant, and form the core components of the elongase. Lipidomic profiling has been performed on roots and shoots of plants with reduced levels of VLCFAs. Mutants of the core components of the elongase were analysed along with herbicides that inhibit a number of KCS enzymes, this allowed the whole elongase complex to be analysed. Differences were seen in the lipidomic profiles of the different elongase mutants and between the roots and shoots of the same mutants. This has revealed correlations between phenotypic differences and lipidomic changes giving insight into which lipid classes might be responsible for the various phenotypes. A forward genetic screen has been conducted in the Arabidopsis cer10-1 mutant to identify novel genes involved in VLCFA metabolism. CER10 encodes for the fourth component of the elongase complex. One suppressor mutant that has been identified has flower buds and fertility comparable to wild type plants but still displays the reduced size of the cer10-1 mutant. The second suppressor mutant identified showed restored size of aerial organs but the flower buds remained fused. Whole genome sequencing allowed localisation of these suppressor mutations on Chromosome 3. Partial biochemical characterisation of these mutants revealed interesting changes in their acyl-CoA and cuticular lipid profiles

The role of very long chain fatty acids in Arabidopsis growth and development

Very long chain fatty acids (VLCFAs) are essential to Arabidopsis growth and development. VLCFAs are found in sphingolipids, glycerophospholipids, triacylglycerols, suberin and cuticular waxes. VLCFAs are synthesized by the addition of 2 carbons from malonyl-CoA to pre-existing acyl-CoAs to produce chain lengths of greater than 18 carbon atoms. VLCFA synthesis involves four consecutive reactions that are catalysed by the microsomal Fatty Acid Elongase. In Arabidopsis the first reaction is catalysed by one of 21 different Keto-CoA Synthases (KCS) with diverse levels of expression and overlapping tissue specificities. The other three enzymes are ubiquitously expressed throughout the plant, and form the core components of the elongase. Lipidomic profiling has been performed on roots and shoots of plants with reduced levels of VLCFAs. Mutants of the core components of the elongase were analysed along with herbicides that inhibit a number of KCS enzymes, this allowed the whole elongase complex to be analysed. Differences were seen in the lipidomic profiles of the different elongase mutants and between the roots and shoots of the same mutants. This has revealed correlations between phenotypic differences and lipidomic changes giving insight into which lipid classes might be responsible for the various phenotypes. A forward genetic screen has been conducted in the Arabidopsis cer10-1 mutant to identify novel genes involved in VLCFA metabolism. CER10 encodes for the fourth component of the elongase complex. One suppressor mutant that has been identified has flower buds and fertility comparable to wild type plants but still displays the reduced size of the cer10-1 mutant. The second suppressor mutant identified showed restored size of aerial organs but the flower buds remained fused. Whole genome sequencing allowed localisation of these suppressor mutations on Chromosome 3. Partial biochemical characterisation of these mutants revealed interesting changes in their acyl-CoA and cuticular lipid profiles

Phase 1 Safety and Immunogenicity Evaluation of ADMVA, a Multigenic, Modified Vaccinia Ankara-HIV-1 B'/C Candidate Vaccine

in a modified vaccinia Ankara viral vector. Sequences were derived from a prevalent circulating HIV-1 recombinant form in Yunnan, China, an area of high HIV incidence. The objective was to evaluate the safety and immunogenicity of ADMVA in human volunteers.. Two volunteers mounted antibodies that were able to neutralize clade-matched viruses.ADMVA was well-tolerated and elicited durable humoral and cellular immune responses

Phase 1 Safety and Immunogenicity Evaluation of ADVAX, a Multigenic, DNA-Based Clade C/B' HIV-1 Candidate Vaccine

BACKGROUND: We conducted a Phase I dose escalation trial of ADVAX, a DNA-based candidate HIV-1 vaccine expressing Clade C/B' env, gag, pol, nef, and tat genes. Sequences were derived from a prevalent circulating recombinant form in Yunnan, China, an area of high HIV-1 incidence. The objective was to evaluate the safety and immunogenicity of ADVAX in human volunteers. METHODOLOGY/PRINCIPAL FINDINGS: ADVAX or placebo was administered intramuscularly at months 0, 1 and 3 to 45 healthy volunteers not at high risk for HIV-1. Three dosage levels [0.2 mg (low), 1.0 mg (mid), and 4.0 mg (high)] were tested. Twelve volunteers in each dosage group were assigned to receive ADVAX and three to receive placebo in a double-blind design. Subjects were followed for local and systemic reactogenicity, adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA. Cellular immunogenicity was assessed by a validated IFNgamma ELISpot assay and intracellular cytokine staining. ADVAX was safe and well-tolerated, with no vaccine-related serious adverse events. Local and systemic reactogenicity events were reported by 64% and 42% of vaccine recipients, respectively. The majority of events were mild. The IFNgamma ELISpot response rates to any HIV antigen were 0/9 (0%) in the placebo group, 3/12 (25%) in the low-dosage group, 4/12 (33%) in the mid-dosage group, and 2/12 (17%) in the high-dosage group. Overall, responses were generally transient and occurred to each gene product, although volunteers responded to single antigens only. Binding antibodies to gp120 were not detected in any volunteers, and HIV seroconversion did not occur. CONCLUSIONS/SIGNIFICANCE: ADVAX delivered intramuscularly is safe, well-tolerated, and elicits modest but transient cellular immune responses. TRIAL REGISTRATION: Clinicaltrials.gov NCT00249106.published_or_final_versio

Involvement of Arabidopsis acyl-coenzyme A desaturase-like2 (At2g31360) in the biosynthesis of the very-long-chain monounsaturated fatty acid components of membrane lipids

The Arabidopsis (Arabidopsis thaliana) acyl-coenzyme A (CoA) desaturase-like (ADS) gene family contains nine genes encoding fatty acid desaturase-like proteins. The biological function of only one member of the family, fatty acid desaturase5 (AtADS3/ FAD5, At3g15850), is known, and this gene encodes the plastidic palmitoyl-monogalactosyldiacylglycerol D7 desaturase. We cloned seven members of the gene family that are predicted not to have a chloroplast transit peptide and expressed them in the yeast Saccharomyces cerevisiae. All seven have previously undescribed desaturase activity on very-long-chain fatty acid (VLCFA) substrates and exhibit diverse regiospeci\ufb01city, catalyzing introduction of double bonds relative to the methyl end of the molecule (n-x) at n-6 (AtADS4, At1g06350), n-7 (AtADS1.3, At1g06100 and AtADS4.2, At1g06360), n-9 (AtADS1, At1g06080 and AtADS2, At2g31360) or D9 (relative to the carboxyl end of the molecule) positions (AtADS1.2, At1g06090 and AtADS1.4, At1g06120). Through forward and reverse genetics it was shown that AtADS2 is involved in the synthesis of the 24:1(n-9) and 26:1(n-9) components (X:Y, where X is chain length and Y is number of double bonds) of seed lipids, sphingolipids, and the membrane phospholipids phosphatidylserine, and phosphatidylethanolamine. Plants de\ufb01cient in AtADS2 expression showed no obvious phenotype when grown under normal growing conditions, but showed an almost complete loss of phosphatidylethanolamine (42:4), phosphatidylserine(42:4), dihydroxy-monohexosylceramide(42:2)-2, trihydroxy-monohexosylceramide(42:2)-3, and trihydroxy-glycosylinositolphosphoceramide(42:2)-3, lipid species that contain the VLCFA 24:1(n-9), and trihydroxyglycosylinositolphosphoceramide(44:2)-3, a lipid containing 26:1(n-9). Acyl-CoA pro\ufb01ling of these plants revealed a major reduction in 24:1-CoA and a small reduction in 26:1-CoA. Overexpression of AtADS2 resulted in a substantial increase in the percentage of glycerolipid and sphingolipids species containing 24:1 and a dramatic increase in the percentage of very-long-chain monounsaturated fatty acids in the acyl-CoA pool. Plants de\ufb01cient in AtADS1 expression had reduced levels of 26:1(n-9) in seed lipids, but no signi\ufb01cant changes in leaf phospholipids or sphingolipids were observed. These \ufb01ndings indicate that the 24-carbon and 26-carbon monounsaturated VLCFAs of Arabidopsis result primarily from VLCFA desaturation, rather than by elongation of long chain monounsaturated fatty acids.Peer reviewed: YesNRC publication: Ye

Concordant Proficiency in Measurement of T-Cell Immunity in Human Immunodeficiency Virus Vaccine Clinical Trials by Peripheral Blood Mononuclear Cell and Enzyme-Linked Immunospot Assays in Laboratories from Three Continents ▿

The gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In order to compare candidates and pool data from multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter- and intralaboratory performance, as well as assay performance, over time. Seven International AIDS Vaccine Initiative-sponsored trial sites participated in the proficiency panels described in this study. At each laboratory, two operators independently processed identical sample sets consisting of frozen peripheral blood mononuclear cell (PBMC) samples from different donors by using four blind stimuli. PBMC recovery and viability after overnight resting and the IFN-γ ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting, with a median recovery of 78% and median viability of 95%. The laboratories were able to detect similar antigen-specific T-cell responses, ranging from 50 to >3,000 spot-forming cells per million PBMC. An approximate range of a half log in results from operators within or across sites was seen in comparisons of antigen-specific responses. Consistently low background responses were seen in all laboratories. The results of these proficiency panels demonstrate the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN-γ ELISPOT responses. These findings also illustrate the ability to standardize the IFN-γ ELISPOT assay across multiple laboratories when common training methods, reagents such as fetal calf serum, and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV vaccines and other vaccines