Wound-induced increases in the glucosinolate content of oilseed rape and their effect on subsequent herbivory by a crucifer specialist

Damage to the oilseed rape plant (Brassica napus L.) by the cabbage stem flea beetle, Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) induces systemic changes to the glucosinolate profile, most noticeably an increase in the concentration of indole glucosinolates. When jasmonic acid was applied to the cotyledons of the plant, a similar effect was observed. Feeding tests with artificial substrates compared a glucosinolate fraction from jasmonic acid-treated plants with a similar fraction from untreated plants. In these tests, alterations to the glucosinolate profile increased the feeding of a crucifer-specialist feeder (P. chrysocephala). However, in whole plant tests, P. chrysocephala did not feed more on the jasmonic acid treated plants than on the controls. This implies that other aspects of the damage response are being induced by the jasmonic acid treatment and having a negative effect on subsequent herbivory

Control of Ascorbate Synthesis by Respiration and Its Implications for Stress Responses

We show for the first time that respiration can control ascorbate (AA) synthesis in plants. Evidence for this control is provided by (a) the localization of L-galactono-1,4-lactone dehydrogenase (GalLDH), the terminal enzyme in AA biosynthesis, with mitochondrial complex I, and its regulation by electron transport through this complex, (b) the absolute requirement of the enzyme for oxidized cytochrome c (cyt c(ox)) as substrate, and (c) the coordinated response of respiration and AA synthesis to stress induced by hormone treatment.Instituto de Fisiología VegetalFacultad de Ciencias Agrarias y Forestale

Optimised LAMP allows single copy detection of 35Sp and NOSt in transgenic maize using Bioluminescent Assay in Real Time (BART)

Loop-mediated amplification (LAMP) has been widely used to amplify and hence detect nucleic acid target sequences from various pathogens, viruses and genetic modifications. Two distinct types of primer are required for LAMP; hairpin-forming LAMP and displacement. High specificity arises from this use of multiple primers, but without optimal conditions for LAMP, sensitivity can be poor. We confirm here the importance of LAMP primer design, concentrations and ratios for efficient LAMP amplification. We further show that displacement primers are non-essential to the LAMP reaction at certain concentrations providing accelerating loop primers are present. We investigate various methods to quantify DNA extracts from GM maize certified reference materials to calculate the target copy numbers of template presented to the LAMP reaction, and show that LAMP can amplify transgenic promoter / terminator sequences in DNA extracted from various maize GM events using primers designed to target the 35S promoter (35Sp) or NOS terminator (NOSt) sequences, detection with both bioluminescence in real-time (BART) and fluorescent methods. With prior denaturation and HPLC grade LAMP primers single copy detection was achieved, showing that optimised LAMP conditions can be combined with BART for single copy targets, with simple and cost efficient light detection electronics over fluorescent alternatives

Ascorbate content of wheat leaves is not determined by maximal L-galactono-1,4-lactone dehydrogenase (GalLDH) activity under drought stress

Although ascorbic acid (AA) is a high-abundance metabolite, relatively little is known about the factors controlling its accumulation in leaves. To address this issue, we examined the role of l-galactono-1,4-lactone dehydrogenase (GalLDH), the enzyme which catalyses the last step of this pathway, in the control of AA content under optimal and stress conditions. In a range of species, no clear relationship between AA content and leaf GalLDH protein and activity was found under optimal growth conditions. To explore the effect of drought stress on GalLDH activity and protein content, wheat (Triticum aestivum L.) was selected for detailed analysis, using two cultivars that differ in their constitutive AA level. In well-watered plants, the AA content of cv Buck Chambergo (BCH) was over twice that of cv Cooperativa Maipún (CM) but dehydroascorbic acid content was similar in both cv. In agreement with this, dehydroascorbate reductase and glutathione reductase activities were higher in cv BCH than in cv CM, indicating a higher capacity for AA regeneration. Neither leaf DHA content nor activities of AA regenerating enzymes were modified by drought. Although drought caused a substantial increase in GalLDH protein and activity in the low AA cv CM, this treatment had no effect on these parameters in cv BCH. Notably, leaf AA content was unaffected by drought in either cv. These results suggest that GalLDH protein and activity cannot be used as an indicator for changes in the capacity for ascorbate biosynthesis and that AA biosynthesis is constrained by other factors under stress. This can be explained by the importance of regeneration in maintaining AA levels and possibly also by redox regulation of GalLDH.Instituto de Fisiología Vegeta

Inhaled corticosteroids, blood eosinophils, and FEV1 decline in patients with COPD in a large UK primary health care setting.

Background: Inhaled corticosteroid (ICS)-containing medications slow rate of decline of FEV1. Blood eosinophil (EOS) levels are associated with the degree of exacerbation reduction with ICS. Purpose: We investigated whether FEV1 decline differs between patients with and without ICS, stratified by blood EOS level. Patients and methods: The UK Clinical Practice Research Datalink (primary care records) and Hospital Episode Statistics (hospital records) were used to identify COPD patients aged 35 years or older, who were current or ex-smokers with ≥2 FEV1 measurements ≥6 months apart. Prevalent ICS use and the nearest EOS count to start of follow-up were identified. Patients were classified at baseline as higher stratum EOS (≥150 cell/µL) on ICS; higher stratum EOS not on ICS; lower stratum EOS (<150 cells/µL) on ICS; and lower stratum EOS not on ICS. In addition, an incident ICS cohort was used to investigate the rate of FEV1 change by EOS and incident ICS use. Mixed-effects linear regression was used to compare rates of FEV1 change in mL/year. Results: A total of 26,675 COPD patients met our inclusion criteria (median age 69, 46% female). The median duration of follow up was 4.2 years. The rate of FEV1 change in prevalent ICS users was slower than non-ICS users (-12.6 mL/year vs -21.1 mL/year; P =0.001). The rate of FEV1 change was not significantly different when stratified by EOS level. The rate of FEV1 change in incident ICS users increased (+4.2 mL/year) vs -21.2 mL/year loss in non-ICS users; P<0.001. In patients with high EOS, incident ICS patients showed an increase in FEV1 (+12 mL/year) compared to non-ICS users whose FEV1 decreased (-20.8 mL/year); P<0.001. No statistical difference was seen in low EOS patients. Incident ICS use is associated with an improvement in FEV1 change, however, over time this association is lost. Conclusion: Regardless of blood EOS level, prevalent ICS use is associated with slower rates of FEV1 decline in COPD

Control of Ascorbate Synthesis by Respiration and Its Implications for Stress Responses

We show for the first time that respiration can control ascorbate (AA) synthesis in plants. Evidence for this control is provided by (a) the localization of L-galactono-1,4-lactone dehydrogenase (GalLDH), the terminal enzyme in AA biosynthesis, with mitochondrial complex I, and its regulation by electron transport through this complex, (b) the absolute requirement of the enzyme for oxidized cytochrome c (cyt c(ox)) as substrate, and (c) the coordinated response of respiration and AA synthesis to stress induced by hormone treatment.Instituto de Fisiología VegetalFacultad de Ciencias Agrarias y Forestale

Ascorbate content of wheat leaves is not determined by maximal L-galactono-1,4-lactone dehydrogenase (GalLDH) activity under drought stress

Although ascorbic acid (AA) is a high-abundance metabolite, relatively little is known about the factors controlling its accumulation in leaves. To address this issue, we examined the role of l-galactono-1,4-lactone dehydrogenase (GalLDH), the enzyme which catalyses the last step of this pathway, in the control of AA content under optimal and stress conditions. In a range of species, no clear relationship between AA content and leaf GalLDH protein and activity was found under optimal growth conditions. To explore the effect of drought stress on GalLDH activity and protein content, wheat (Triticum aestivum L.) was selected for detailed analysis, using two cultivars that differ in their constitutive AA level. In well-watered plants, the AA content of cv Buck Chambergo (BCH) was over twice that of cv Cooperativa Maipún (CM) but dehydroascorbic acid content was similar in both cv. In agreement with this, dehydroascorbate reductase and glutathione reductase activities were higher in cv BCH than in cv CM, indicating a higher capacity for AA regeneration. Neither leaf DHA content nor activities of AA regenerating enzymes were modified by drought. Although drought caused a substantial increase in GalLDH protein and activity in the low AA cv CM, this treatment had no effect on these parameters in cv BCH. Notably, leaf AA content was unaffected by drought in either cv. These results suggest that GalLDH protein and activity cannot be used as an indicator for changes in the capacity for ascorbate biosynthesis and that AA biosynthesis is constrained by other factors under stress. This can be explained by the importance of regeneration in maintaining AA levels and possibly also by redox regulation of GalLDH.Instituto de Fisiología Vegeta

Plasma based markers of [11C] PiB-PET brain amyloid burden.

PublishedJournal ArticleResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tChanges in brain amyloid burden have been shown to relate to Alzheimer's disease pathology, and are believed to precede the development of cognitive decline. There is thus a need for inexpensive and non-invasive screening methods that are able to accurately estimate brain amyloid burden as a marker of Alzheimer's disease. One potential method would involve using demographic information and measurements on plasma samples to establish biomarkers of brain amyloid burden; in this study data from the Alzheimer's Disease Neuroimaging Initiative was used to explore this possibility. Sixteen of the analytes on the Rules Based Medicine Human Discovery Multi-Analyte Profile 1.0 panel were found to associate with [(11)C]-PiB PET measurements. Some of these markers of brain amyloid burden were also found to associate with other AD related phenotypes. Thirteen of these markers of brain amyloid burden--c-peptide, fibrinogen, alpha-1-antitrypsin, pancreatic polypeptide, complement C3, vitronectin, cortisol, AXL receptor kinase, interleukin-3, interleukin-13, matrix metalloproteinase-9 total, apolipoprotein E and immunoglobulin E--were used along with co-variates in multiple linear regression, and were shown by cross-validation to explain >30% of the variance of brain amyloid burden. When a threshold was used to classify subjects as PiB positive, the regression model was found to predict actual PiB positive individuals with a sensitivity of 0.918 and a specificity of 0.545. The number of APOE [Symbol: see text] 4 alleles and plasma apolipoprotein E level were found to contribute most to this model, and the relationship between these variables and brain amyloid burden was explored.Alzheimer's Disease Neuroimaging Initiative (ADNI)Canadian Institutes of Health ResearchFoundation for the National Institutes of HealthNational Institutes of HealthInnoMed, European Union of the Sixth Framework programNational Institutes for Health Research Biomedical Research Centre for Mental Health at the South London and Maudsley National Health Service Foundation TrustInstitute of Psychiatry, King's College Londo

A simple, high-throughput, colourimetric, field applicable loop-mediated isothermal amplification (HtLAMP) assay for malaria elimination.

BACKGROUND: To detect all malaria infections in elimination settings sensitive, high throughput and field deployable diagnostic tools are required. Loop-mediated isothermal amplification (LAMP) represents a possible field-applicable molecular diagnostic tool. However, current LAMP platforms are limited by their capacity for high throughput. METHODS: A high-throughput LAMP (HtLAMP) platform amplifying mitochondrial targets using a 96-well microtitre plate platform, processing 85 samples and 11 controls, using hydroxynaphtholblue as a colourimetric indicator was optimized for the detection of malaria parasites. Objective confirmation of visually detectable colour change results was made using a spectrophotometer. A dilution series of laboratory-cultured 3D7 Plasmodium falciparum parasites was used to determine the limit of detection of the HtLAMP assay, using P. falciparum (HtLAMP-Pf) and Plasmodium genus (HtLAMP-Pg) primers, on whole blood and filter paper, and using different DNA extraction protocols. The diagnostic accuracy of HtLAMP was validated using clinical samples from Papua New Guinea, Malaysia, Ghana and The Gambia and its field applicability was evaluated in Kota Marudu district hospital, Sabah, Malaysia. RESULTS: The HtLAMP assay proved to be a simple method generating a visually-detectable blue and purple colour change that could be objectively confirmed in a spectrophotometer at a wavelength of 600 nm. When compared with PCR, overall HtLAMP-Pg had a sensitivity of 98 % (n = 260/266, 95 % CI 95-99) and specificity 83 % (n = 15/18, 95 % CI 59-96). HtLAMP-Pf had a sensitivity of 97 % (n = 124/128, 95 % CI 92-99) and specificity of 96 % (n = 151/157, 95 % CI 92-99). A validation study in a regional hospital laboratory demonstrated ease of performance and interpretation of the HtLAMP assay. HtLAMP-Pf performed in this field setting had a sensitivity of 100 % (n = 17/17, 95 % CI 80-100) and specificity of 95 % (n = 123/128, 95 % CI 90-98) compared with multiplex PCR. HtLAMP-Pf also performed well on filter paper samples from asymptomatic Ghanaian children with a sensitivity of 88 % (n = 23/25, 95 % CI 69-97). CONCLUSION: This colourimetric HtLAMP assay holds much promise as a field applicable molecular diagnostic tool for the purpose of malaria elimination