Current factor VIII (FVIII) products display a half-life (t1/2) of ∼ 8-12 hours, requiring frequent intravenous injections for prophylaxis and treatment of patients with hemophilia A. rFVIIIFc is a ...recombinant fusion protein composed of a single molecule of FVIII covalently linked to the Fc domain of human IgG1 to extend circulating rFVIII t1/2. This first-in-human study in previously treated subjects with severe hemophilia A investigated safety and pharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose of rFVIII at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Most adverse events were unrelated to study drug. None of the study subjects developed anti-rFVIIIFc antibodies or inhibitors. Across dose levels, compared with rFVIII, rFVIIIFc showed 1.54- to 1.70-fold longer elimination t1/2, 1.49- to 1.56-fold lower clearance, and 1.48- to 1.56-fold higher total systemic exposure. rFVIII and rFVIIIFc had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was ∼ 1.53- to 1.68-fold longer than rFVIII across dose levels. Each subject showed prolonged exposure to rFVIIIFc relative to rFVIII. Thus, rFVIIIFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia A. This trial was registered at www.clinicaltrials.gov as NCT01027377.
Oxalic acid is an important virulence factor for disease caused by the fungal necrotrophic pathogen Sclerotinia sclerotiorum, yet calcium oxalate (CaOx) crystals have not been widely reported. B. ...carinata stems were infected with S. sclerotiorum and observed using light microscopy. Six hours post inoculation (hpi), CaOx crystals were evident on 46% of stem sections and by 72 hpi on 100%, demonstrating that the secretion of oxalic acid by S. sclerotiorum commences before hyphal penetration. This is the first time CaOx crystals have been reported on B. carinata infected with S. sclerotiorum. The shape of crystals varied as infection progressed. Long tetragonal rods were dominant 12 hpi (68% of crystal-containing samples), but by 72 hpi, 50% of stems displayed bipyramidal crystals, and only 23% had long rods. Scanning electron microscopy from 24 hpi revealed CaOx crystals in all samples, ranging from tiny irregular crystals (< 0.5 μm) to large (up to 40 μm) highly organized arrangements. Crystal morphology encompassed various forms, including tetragonal prisms, oval plates, crystal sand, and druses. Large conglomerates of CaOx crystals were observed in the hyphal mass 72 hpi and these are proposed as a strategy of the fungus to hold and detoxify Ca2+ions. The range of crystal morphologies suggests that S. sclerotiorum growth and infection controls the form taken by CaOx crystals.
•High-level resistance to Sclerotina identified across diverse cruciferous spp.•Stem and leaf resistance are under separate genetic control.•Species value as a resistance source depends upon number ...of genotypes tested.•Resistances identified allow new disease-resistant commercial cruciferous crops.
Current management of Sclerotinia rot (Sclerotinia sclerotiorum) relies heavily upon cultural and chemical control options that provide, at best, only partial, sometimes sporadic control and can be cost prohibitive. Effective host resistance to S. sclerotiorum is urgently needed if Sclerotinia rot is to be successfully managed across diverse oilseed, forage and vegetable crucifer crops worldwide. Differences in resistance across 127 diverse cruciferous genotypes to S. sclerotiorum in one experiment, and a set of 55 Brassica napus lines carrying one or more B. carinata B genome introgressions in a second experiment, were assessed by field-inoculation of stems and natural ascospore infection. Reactions on B. fruticulosa, B. oxyrrhina, B. parachinensis, B. tournefortii, Camelina sativa, Carrichtera annua, Diplotaxis tenuifolia, Eruca vesicaria, Hirschfeldia incana, Raphanus raphanistrum, Raphanus sativus, Rapistrum rugosum, Sinapis arvensis and Sisymbrium irio to this pathogen are reported for the first time. Among these, and across other species previously explored, including B. carinata, B. napus, B. juncea, B. rapa, B. cretica, B. incana, B. insularis, B. nigra, and B. montana, responses ranged from highly resistant (stem lesion length 2mm) to highly susceptible (155mm). Against a highly virulent and prevailing pathogen pathotype, R. raphanistrum RRA 41, B. napus Mystic, B. carinata BRA 926/78, B. carinata 054113, R. sativus Krasnodar. Market B, B. carinata PI 193459 and R. raphanistrum WARR 20 all showed very high-level resistance, with stem lesion length <5mm; with a further 29 genotypes across these species and/or B. incana, B. rapa var. rosularis, B. oleracea vars acephala and italica, and S. arvensis, that showed slightly less but still high resistance (lesions 5–10mm). Natural air-borne ascospore leaf infection occurred in 89% of plots, yet some forage and/or vegetable crucifers, such as B. rapa var. chinensis Ivory and R. sativus Oriental radish, showed high-level resistance to such infections. B. napus with introgression(s) from B. carinata, ranged from highly resistant (lesions <2mm) to very susceptible (>67mm). There appeared to be separate genetic control for stem vs. leaf resistance, crucial to the understanding needed in developing resistant cultivars across different cruciferous crop types. While this study highlighted individual genotypes that offer great potential for improving resistance to Sclerotinia rot in commercial cruciferous crops, it also demonstrated that assessment of the overall value of a species is only possible when a significant number of genotypes within the species are tested. Overall, while genotypes with high-level stem or leaf resistance identified are of particularly significant value for developing new disease-resistant cultivars of oilseed, forage and vegetable crucifers, even genotypes with moderate levels of resistance, if deployed commercially, offer prospects of improving current integrated disease management strategies compared with current reliance upon cultural and/or chemical controls utilized in conjunction with cultivars lacking any effective resistance. As resistance was identified for the first time across many of these diverse cruciferous species, it is likely that some resistances identified constitute new sources and/or types of host resistance not previously identified.
Sclerotinia stem rot (SSR, Sclerotinia sclerotiorum) is a damaging disease of oilseed brassicas world-wide. Host resistance is urgently needed to achieve control, yet the factors that contribute to ...stem resistance are not well understood. This study investigated the mechanisms of resistance to SSR.
Stems of 5-week-old Brassica carinata, B. juncea and B. napus of known resistance were infected via filter paper discs impregnated with S. sclerotiorum mycelium under controlled conditions. Transverse sections of the stem and portions of the stem surface were examined using optical and scanning electron microscopy. The association of anatomical features with the severity of disease (measured by mean lesion length) was determined.
Several distinct resistance mechanisms were recorded for the first time in these Brassica-pathogen interactions, including hypersensitive reactions and lignification within the stem cortex, endodermis and in tissues surrounding the lesions. Genotypes showing a strong lignification response 72 h post-infection (hpi) tended to have smaller lesions. Extensive vascular invasion by S. sclerotiorum was observed only in susceptible genotypes, especially in the vascular fibres and xylem. Mean lesion length was negatively correlated with the number of cell layers in the cortex, suggesting progress of S. sclerotiorum is impeded by more cell layers. Hyphae in the centre of lesions became highly vacuolate 72 hpi, reflecting an ageing process in S. sclerotiorum hyphal networks that was independent of host resistance. The infection process of S. sclerotiorum was analogous in B. carinata and B. napus. Infection cushions of the highly virulent isolate of S. sclerotiorum MBRS-1 were grouped together in dense parallel bundles, while hyphae in the infection cushions of a less aggressive isolate WW-3 were more diffuse, and this was unaffected by host genotype.
A variety of mechanisms contribute to host resistance against S. sclerotiorum across the three Brassica species. These complex interactions between pathogen and host help to explain variable expressions of resistance often observed in the field.
Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is an important disease of oilseed brassicas, yet the susceptibility of Australian varieties is unknown. Fifty-five historic, current ...and potential new Australian canola and mustard varieties were field-screened to determine their relative levels of resistance to SSR. Mean lesion length following stem inoculation with a highly virulent isolate (MBRS1) of the prevailing S. sclerotiorum pathotype (76) ranged from 3.0mm in the B. napus cultivar Mystic to 202.6mm (P<0.001). Three recently developed B. juncea varieties or breeding lines, Sahara, JB0T-908982 and Xceed X121 CL, were extremely susceptible to S. sclerotiorum (mean lesion lengths 90.6, 132.3 and 202.6mm, respectively). Histological study showed that the high level of resistance in Mystic was associated with strong deposition of lignin in stem cortical cell walls to form a barrier between the invading pathogen and the vascular tissues. Lack of association between mean lesion length and the year of varietal release (R2=0.005) shows that there has been no improvement in level of resistance to SSR in Australian canola and mustard varieties over the last two decades. Although the very high susceptibility of a few B. juncea varieties demonstrated the value of SSR resistance present in B. napus varieties, this level of resistance is inadequate to prevent ongoing, severe yield losses from SSR under conditions conducive for disease development. Breeding programs can immediately utilise the SSR resistance in Mystic, and other recently identified resistances. This will enable a shift from the current dependence on fungicidal control to reliance on cost-effective, sustainable host resistance as the basis for better management of SSR.
Field resistances against Sclerotinia rot (SR) (Sclerotinia sclerotiorum) were determined in 52 Chinese genotypes of Brassica oleracea var. capitata, 14 Indian Brassica juncea genotypes carrying wild ...weedy Brassicaceae introgression(s) and four carrying B‐genome introgression, 22 Australian commercial Brassica napus varieties, and 12 B. napus and B. juncea genotypes of known resistance. All plants were individually inoculated by securing an agar disc from a culture of S. sclerotiorum growing on a glucose‐rich medium to the stem above the second internode with Parafilm tape. Mean stem lesion length across tested genotypes ranged from <1 to >68 mm. While there was considerable diversity within the germplasm sets from each country, overall, 65% of the B. oleracea var. capitata genotypes from China showed the highest levels of stem resistance, a level comparable with the highest resistance ever recorded for oilseed B. napus or B. juncea from China or Australia. One Indian B. juncea line carrying weedy introgression displayed a significant level of both stem and leaf resistance. However, the vast majority of commercial Australian oilseed B. napus varieties fell within the most susceptible 40% of genotypes tested for stem disease. There was no correlation between expressions of stem versus leaf resistance, suggesting their independent inheritance. A few Chinese B. oleracea var. capitata genotypes that expressed combined extremely high‐level stem (≤1 mm length) and leaf (≤0.5 mean number of infections/plant) resistance will be particularly significant for developing new SR‐resistant horticultural and oilseed Brassica varieties.
Oxalic acid is an important virulence factor for disease caused by the fungal necrotrophic pathogen Sclerotinia sclerotiorum, yet calcium oxalate (CaOx) crystals have not been widely reported. B. ...carinata stems were infected with S. sclerotiorum and observed using light microscopy. Six hours post inoculation (hpi), CaOx crystals were evident on 46% of stem sections and by 72 hpi on 100%, demonstrating that the secretion of oxalic acid by S. sclerotiorum commences before hyphal penetration. This is the first time CaOx crystals have been reported on B. carinata infected with S. sclerotiorum. The shape of crystals varied as infection progressed. Long tetragonal rods were dominant 12 hpi (68% of crystal-containing samples), but by 72 hpi, 50% of stems displayed bipyramidal crystals, and only 23% had long rods. Scanning electron microscopy from 24 hpi revealed CaOx crystals in all samples, ranging from tiny irregular crystals (< 0.5 mu m) to large (up to 40 mu m) highly organized arrangements. Crystal morphology encompassed various forms, including tetragonal prisms, oval plates, crystal sand, and druses. Large conglomerates of CaOx crystals were observed in the hyphal mass 72 hpi and these are proposed as a strategy of the fungus to hold and detoxify Ca2+ions. The range of crystal morphologies suggests that S. sclerotiorum growth and infection controls the form taken by CaOx crystals.
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