Genetic and molecular basis of botrydial biosynthesis: connecting cytochrome P450-encoding genes to biosynthetic intermediates

Over two hundred species of plants can be infected by the phytopathogenic fungus Botrytis cinerea under a range of different environmental conditions. In response to these, the fungus produces unique terpenoid and polyketide metabolites. Parts of the plants may be killed by the phytotoxin botrydial, enabling the fungus to feed on the dead cells. In this paper, we describe the genetic and molecular basis of botrydial biosynthesis and the function of the five genes of the genome of B. cinerea that together constitute the botrydial biosynthetic gene cluster. Genes BcBOT3 and BcBOT4, encoding two cytochrome P450 monooxygenases, were inactivated by homologous recombination and were shown to catalyze regio- and stereospecific hydroxylations at the carbons C-10 and C-4, respectively, of the presilphiperfolan-8β-ol skeleton. The null mutants, bcbot3Δ and bcbot4Δ, accumulated key intermediates in the botrydial biosynthesis enabling the complete genetic and molecular basis of the botrydial biosynthetic pathway to be established. Furthermore, the bcbot4Δ mutant overproduced a significant number of polyketides, which included, in addition to known botcinins, botrylactones and cinbotolide A, two new botrylactones and two new cinbotolides, cinbotolides B and C

Characterization of a Netrin-4 Like Protein in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The netrin family of proteins has homeostatic roles in vertebrate development and angiogenesis, and pathophysiological roles in the progression and metastasis of cancer. We have previously characterized a netrin-1-like protein in Tetrahymena thermophila, and have shown that vertebrate netrin-1, netrin-3, and netrin-4 all serve as chemorepellents in this organism. We are currently using Western blotting and immunofluorescence to further characterize the netrin-like proteins in Tetrahymena. Western blotting with our anti-netrin-4 antibody shows a band that is clearly visible in whole cell extract, but shows little reactivity with secreted protein, indicating that most of our netrin-4-like protein remains within the cell. Western blotting of whole cell extract with anti-netrin-1, netrin-3, and netrin-4 antibodies shows a clear band measuring 50 kD that stains with all three antibodies. Some lower molecular weight bands are also evident in all three blots, possibly due to proteolytic activity. Immunolocalization with an anti-netrin-4 antibody shows some colocalization with netrin-1, netrin-3, and ER Tracker™. Our anti-netrin-4 antibody localizes to the oral groove, basal bodies, and nuclei of cells, indicating a possible structural role for the netrin-4-like protein in this organism. Further research will involve determining the primary amino acid sequence of the 50 kD protein and comparing it with the Tetrahymena thermophila proteome database to help ascertain the physiological role of this protein

Crab Waist Scheme Luminosity and Background Diagnostic at DAFNE

TUPTPF029International audienceTest of the crab waist scheme, undergoing at the Frascati DAFNE accelerator complex, needs a fast and accurate measurement of the luminosity, as well as a full characterization of the background conditions. Three different monitors, a Bhabha calorimeter, a Bhabha GEM tracker and a gamma bremsstrahlung proportional counter have been designed, tested and installed on the accelerator at the end of January 2008. Results from beam-test measurements, comparison with the Monte Carlo simulation and preliminary data collected during the SIDDHARTA run are presented

Vestibular Perception following Acute Unilateral Vestibular Lesions.

Little is known about the vestibulo-perceptual (VP) system, particularly after a unilateral vestibular lesion. We investigated vestibulo-ocular (VO) and VP function in 25 patients with vestibular neuritis (VN) acutely (2 days after onset) and after compensation (recovery phase, 10 weeks). Since the effect of VN on reflex and perceptual function may differ at threshold and supra-threshold acceleration levels, we used two stimulus intensities, acceleration steps of 0.5°/s(2) and velocity steps of 90°/s (acceleration 180°/s(2)). We hypothesised that the vestibular lesion or the compensatory processes could dissociate VO and VP function, particularly if the acute vertiginous sensation interferes with the perceptual tasks. Both in acute and recovery phases, VO and VP thresholds increased, particularly during ipsilesional rotations. In signal detection theory this indicates that signals from the healthy and affected side are still fused, but result in asymmetric thresholds due to a lesion-induced bias. The normal pattern whereby VP thresholds are higher than VO thresholds was preserved, indicating that any 'perceptual noise' added by the vertigo does not disrupt the cognitive decision-making processes inherent to the perceptual task. Overall, the parallel findings in VO and VP thresholds imply little or no additional cortical processing and suggest that vestibular thresholds essentially reflect the sensitivity of the fused peripheral receptors. In contrast, a significant VO-VP dissociation for supra-threshold stimuli was found. Acutely, time constants and duration of the VO and VP responses were reduced - asymmetrically for VO, as expected, but surprisingly symmetrical for perception. At recovery, VP responses normalised but VO responses remained shortened and asymmetric. Thus, unlike threshold data, supra-threshold responses show considerable VO-VP dissociation indicative of additional, higher-order processing of vestibular signals. We provide evidence of perceptual processes (ultimately cortical) participating in vestibular compensation, suppressing asymmetry acutely in unilateral vestibular lesions

Agroforestry systems of high nature and cultural value in Europe: provision of commercial goods and other ecosystem services

Land use systems that integrate woody vegetation with livestock and/or crops and are recognised for their biodiversity and cultural importance can be termed high nature and cultural value (HNCV) agroforestry. In this review, based on the literature and stakeholder knowledge, we describe the structure, components and management practices of ten contrasting HNCV agroforestry systems distributed across five European bioclimatic regions. We also compile and categorize the ecosystem services provided by these agroforestry systems, following the Common International Classification of Ecosystem Services. HNCV agroforestry in Europe generally enhances biodiversity and regulating ecosystem services relative to conventional agriculture and forestry. These systems can reduce fire risk, compared to conventional forestry, and can increase carbon sequestration, moderate the microclimate, and reduce soil erosion and nutrient leaching compared to conventional agriculture. However, some of the evidence is location specific and a better geographical coverage is needed to generalize patterns at broader scales. Although some traditional practices and products have been abandoned, many of the studied systems continue to provide multiple woody and non-woody plant products and high-quality food from livestock and game. Some of the cultural value of these systems can also be captured through tourism and local events. However there remains a continual challenge for farmers, landowners and society to fully translate the positive social and environmental impacts of HNCV agroforestry into market prices for the products and services

Genome analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared t

Aganirsen Antisense Oligonucleotide Eye Drops Inhibit Keratitis-Induced Corneal Neovascularization and Reduce Need for Transplantation: The I-CAN Study.

OBJECTIVE: Eye drops of aganirsen, an antisense oligonucleotide preventing insulin receptor substrate-1 expression, inhibited corneal neovascularization in a previous dose-finding phase II study. We aimed to confirm these results in a phase III study and investigated a potential clinical benefit on visual acuity (VA), quality of life (QoL), and need for transplantation. DESIGN: Multicenter, double-masked, randomized, placebo-controlled phase III study. PARTICIPANTS: Analysis of 69 patients with keratitis-related progressive corneal neovascularization randomized to aganirsen (34 patients) or placebo (35 patients). Patients applied aganirsen eye drops (86 μg/day/eye) or placebo twice daily for 90 days and were followed up to day 180. MAIN OUTCOME MEASURES: The primary end point was VA. Secondary end points included area of pathologic corneal neovascularization, need for transplantation, risk of graft rejection, and QoL. RESULTS: Although no significant differences in VA scores between groups were observed, aganirsen significantly reduced the relative corneal neovascularization area after 90 days by 26.20% (P = 0.014). This improvement persisted after 180 days (26.67%, P = 0.012). Aganirsen tended to lower the transplantation need in the intent-to-treat (ITT) population at day 180 (P = 0.087). In patients with viral keratitis and central neovascularization, a significant reduction in transplantation need was achieved (P = 0.048). No significant differences between groups were observed in the risk of graft rejection. However, aganirsen tended to decrease this risk in patients with traumatic/viral keratitis (P = 0.162) at day 90. The QoL analyses revealed a significant improvement with aganirsen in composite and near activity subscores (P = 0.039 and 0.026, respectively) at day 90 in the per protocol population. Ocular and treatment-related treatment-emergent adverse events (TEAEs) were reported in a lower percentage with aganirsen compared with placebo. Only 3 serious TEAEs (2 with aganirsen and 1 with placebo) were considered treatment-related. CONCLUSIONS: This first phase III study on a topical inhibitor of corneal angiogenesis showed that aganirsen eye drops significantly inhibited corneal neovascularization in patients with keratitis. The need for transplantation was significantly reduced in patients with viral keratitis and central neovascularization. Topical application of aganirsen was safe and well tolerated