New method for GC/FID and GC-C-IRMS analysis of plasma free fatty acid concentration and isotopic enrichment

A simple, direct and accurate method for the determination of concentration and enrichment of free fatty acids (FFAs) in human plasma was developed. The validation and comparison to a conventional method are reported. Three amide derivatives, dimethyl, diethyl and pyrrolidide, were investigated in order to achieve optimal resolution of the individual fatty acids. This method involves the use of dimethylamine/Deoxo-Fluor to derivatize plasma free fatty acids to their dimethylamides. This derivatization method is very mild and efficient, and is selective only towards FFAs so that no separation from a total lipid extract is required. The direct method gave lower concentrations for palmitic acid and stearic acid and increased concentrations for oleic acid and linoleic acid in plasma as compared to methyl ester derivative after thin-layer chromatography. The [(13)C]palmitate isotope enrichment measured using direct method was significantly higher than that observed with the BF(3)/MeOH-TLC method. The present method provided accurate and precise measures of concentration as well as enrichment when analyzed with gas chromatography combustion-isotope ratio-mass spectrometry.     

Enhancing the biocontrol efficacy of Pseudomonas fluorescens F113 by altering the regulation and production of 2,4-diacetylphloroglucinol

Pseudomonas fluorescens F113 is an effective biocontrol agent against Pythium ultimum, the causative agent of damping-off of sugarbeet seedlings. Biocontrol is mediated via the production of the anti-fungal metabolite 2,4-diacetylphloroglucinol (Phl). A genetic approach was used to further enhance the biocontrol ability of F113. Two genetically modified (GM) strains, P. fluorescens F113Rif (pCU8.3) and P. fluorescens F113Rif (pCUP9), were developed for enhanced Phl production and assessed for biocontrol efficacy and impact on sugarbeet in microcosm experiments. The multicopy plasmid pCU8.3 contains the biosynthetic genes (phlA, C, B and D) and the putative permease gene (phlE) of F113 cloned into the rhizosphere stable plasmid pME6010, independent of external promoters. The plasmid pCUP9 consists of the Phl biosynthetic genes cloned downstream of the constitutive Plac promoter in pBBR1MCS. Introduction of pCU8.3 and pCUP9 into P. fluorescens F113 significantly altered the kinetics of Phl biosynthesis when grown in SA medium. A significant and substantial increase in Phl production by the GM strains was observed in the early logarithmic phase and stationary phase of growth compared with the wild-type strain. In microcosm, the two Phl overproducing strains proved to be as effective at controlling damping-off disease as the proprietary fungicide treatment, indicating the potential of genetic modification for plant disease control.     

Host inflammatory response governs fitness in an avian ectoparasite, the northern fowl mite (Ornithonyssus sylviarum)

Vertebrate immune responses to ectoparasites influence pathogen transmission and host fitness costs. Few studies have characterized natural immune responses to ectoparasites and resultant fitness effects on the ectoparasite. These are critical gaps in understanding vertebrate-ectoparasite interaction, disease ecology and host-parasite co-adaptation. This study focused on an ectoparasite of birds—the northern fowl mite (NFM) (Ornithonyssus sylviarum). Based on prior evidence that chickens develop resistance to NFM, these experiments tested two hypotheses: (i) skin inflammation blocks mite access to blood, impairing development, reproduction and survival; and (ii) host immunogenetic variation influences the inflammatory response and subsequent effects on the ectoparasite. On infested hosts, histology of skin inflammation revealed increased epidermal cell number and size, immigration of leukocytes and deposition of serous exudates on the skin surface. Survival of adult mites and their offspring decreased as the area of skin inflammation increased during an infestation. Inflammation increased the distance to blood vessels beyond the length of mite mouthparts (100-160 μm) and prevented protonymphs and adults from reaching a blood source. Consequently, protonymphs could not complete development, evidenced by a significant inverse relationship between inflammation and protonymph feeding success, as well as an increasing protonymph/adult ratio. Adult females were unable to feed and reproduce, indicated by an inverse relationship between inflammation and egg production, and decreasing female/juvenile ratio. These combined impacts of host inflammation reversed NFM population growth. Intensity of inflammation was influenced by the genotype of the major histocompatibility complex (MHC), supporting previous research that linked these immunological loci with NFM resistance. Overall, these data provide a model for a mechanism of avian resistance to an ectoparasitic arthropod and the fitness costs to the parasite of that host defense.     

Defining the Sequence Elements and Candidate Genes for the Coloboma Mutation

The chicken coloboma mutation exhibits features similar to human congenital developmental malformations such as ocular coloboma, cleft-palate, dwarfism, and polydactyly. The coloboma-associated region and encoded genes were investigated using advanced genomic, genetic, and gene expression technologies. Initially, the mutation was linked to a 990 kb region encoding 11 genes; the application of the genetic and genomic tools led to a reduction of the linked region to 176 kb and the elimination of 7 genes. Furthermore, bioinformatics analyses of capture array-next generation sequence data identified genetic elements including SNPs, insertions, deletions, gaps, chromosomal rearrangements, and miRNA binding sites within the introgressed causative region relative to the reference genome sequence. Coloboma-specific variants within exons, UTRs, and splice sites were studied for their contribution to the mutant phenotype. Our compiled results suggest three genes for future studies. The three candidate genes, SLC30A5 (a zinc transporter), CENPH (a centromere protein), and CDK7 (a cyclin-dependent kinase), are differentially expressed (compared to normal embryos) at stages and in tissues affected by the coloboma mutation. Of these genes, two (SLC30A5 and CENPH) are considered high-priority candidate based upon studies in other vertebrate model systems.     

Cysteine Transport through Excitatory Amino Acid Transporter 3 (EAAT3)

Excitatory amino acid transporters (EAATs) limit glutamatergic signaling and maintain extracellular glutamate concentrations below neurotoxic levels. Of the five known EAAT isoforms (EAATs 1–5), only the neuronal isoform, EAAT3 (EAAC1), can efficiently transport the uncharged amino acid L-cysteine. EAAT3-mediated cysteine transport has been proposed to be a primary mechanism used by neurons to obtain cysteine for the synthesis of glutathione, a key molecule in preventing oxidative stress and neuronal toxicity. The molecular mechanisms underlying the selective transport of cysteine by EAAT3 have not been elucidated. Here we propose that the transport of cysteine through EAAT3 requires formation of the thiolate form of cysteine in the binding site. Using Xenopus oocytes and HEK293 cells expressing EAAT2 and EAAT3, we assessed the transport kinetics of different substrates and measured transporter-associated currents electrophysiologically. Our results show that L-selenocysteine, a cysteine analog that forms a negatively-charged selenolate ion at physiological pH, is efficiently transported by EAATs 1–3 and has a much higher apparent affinity for transport when compared to cysteine. Using a membrane tethered GFP variant to monitor intracellular pH changes associated with transport activity, we observed that transport of either L-glutamate or L-selenocysteine by EAAT3 decreased intracellular pH, whereas transport of cysteine resulted in cytoplasmic alkalinization. No change in pH was observed when cysteine was applied to cells expressing EAAT2, which displays negligible transport of cysteine. Under conditions that favor release of intracellular substrates through EAAT3 we observed release of labeled intracellular glutamate but did not detect cysteine release. Our results support a model whereby cysteine transport through EAAT3 is facilitated through cysteine de-protonation and that once inside, the thiolate is rapidly re-protonated. Moreover, these findings suggest that cysteine transport is predominantly unidirectional and that reverse transport does not contribute to depletion of intracellular cysteine pools.