Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Editorial: Actinobacteria, a source of biocatalytic tools
Tischler, Dirk ; Berkel, Willem J.H. Van; Fraaije, Marco W. - \ 2019
Frontiers in Microbiology 10 (2019). - ISSN 1664-302X
Actinomycetes - Biocatalysis - Biotechnology - Extremophile actinobacteria - Germination - High GC genetics - Novel biocatalysts - Secondary metabolites
Two tyrosine residues, Tyr-108 and Tyr-503, are responsible for the deprotonation of phenolic substrates in vanillyl-alcohol oxidase
Ewing, Tom A. ; Nguyen, Quoc Thai ; Allan, Robert C. ; Gygli, Gudrun ; Romero, Elvira ; Binda, Claudia ; Fraaije, Marco W. ; Mattevi, Andrea ; Berkel, Willem J.H. Van - \ 2017
Journal of Biological Chemistry 292 (2017)35. - ISSN 0021-9258 - p. 14668 - 14679.
A number of oxidoreductases from the VAO/para-cresol methylhydroxylase flavoprotein family catalyze the oxidation of para-substituted phenols. One of the best-studied is vanillyl-alcohol oxidase (VAO) from the fungus Penicillium simplicissimum. For oxidation of phenols by VAO to occur, they must first be bound in the active site of the enzyme in their phenolate anion form. The crystal structure of VAO reveals that two tyrosine residues, Tyr-108 and Tyr-503, are positioned to facilitate this deprotonation. To investigate their role in catalysis, we created three VAO variants, Y108F, Y503F, and Y108F/Y503F, and studied their biochemical properties. Steady-state kinetics indicated that the presence of at least one of the tyrosine residues is essential for efficient catalysis by VAO. Stopped-flow kinetics revealed that the reduction of VAO by chavicol or vanillyl alcohol occurs at two different rates: kobs1, which corresponds to its reaction with the deprotonated form of the substrate, and kobs2, which corresponds to its reaction with the protonated form of the substrate. In Y108F, Y503F, and Y108F/Y503F, the relative contribution of kobs2 to the reduction is larger than in wild-type VAO, suggesting deprotonation is impaired in these variants. Binding studies disclosed that the competitive inhibitor isoeugenol is predominantly in its deprotonated form when bound to wild-type VAO, but predominantly in its protonated form when bound to the variants. These results indicate that Tyr-108 and Tyr-503 are responsible for the activation of substrates in VAO, providing new insights into the catalytic mechanism of VAO and related enzymes that oxidize para-substituted phenols.
The VAO/PCMH flavoprotein family
Ewing, Tom A. ; Fraaije, Marco W. ; Mattevi, Andrea ; Berkel, Willem J.H. van - \ 2017
Archives of Biochemistry and Biophysics 632 (2017). - ISSN 0003-9861 - p. 104 - 117.
Enzyme mechanism - Flavoprotein - Oxidoreductase - Phylogeny - Protein family
The VAO/PCMH flavoprotein family consists of structurally homologous flavin-dependent enzymes that catalyze a wide range of chemical reactions. Family members share an architecture consisting of a conserved FAD-binding domain and a more variable substrate-binding domain, which enables varying interactions with a range of substrates while maintaining the same cofactor-binding fold. Here, we provide an overview of the current state of our knowledge on the members of the VAO/PCMH family. Based on a phylogenetic analysis, we divide the family into 11 subgroups. We discuss the properties of these subgroups, focusing on recent developments in terms of the discovery of new family members and mechanistic advances. We also highlight open questions that will provide challenges for future research.
Directed dispersal by an abiotic vector : Wetland plants disperse their seeds selectively to suitable sites along the hydrological gradient via water
Soons, Merel B. ; Groot, Arjen de; Cuesta Ramirez, M.T. ; Fraaije, Rob G.A. ; Verhoeven, Jos T.A. ; Jager, Monique de - \ 2017
Functional Ecology 31 (2017)2. - ISSN 0269-8463 - p. 499 - 508.
Anemochory - Biodiversity - Evolutionary adaptations - Hydrochory - Hydrological niche - Plant functional traits - Riparian zones - Seed dispersal - Water dispersal - Wind dispersal
Plant species around the world invest in seed dispersal by producing large numbers of seeds, with a wide range of morphological adaptations that facilitate dispersal. Not all dispersed seeds reach suitable sites, however, and plants can significantly improve their fitness by increasing the proportion of seeds arriving at suitable sites for germination and establishment. Disproportionate dispersal to suitable sites is known as 'directed dispersal'. Yet, mechanisms of directed dispersal are only known for a limited number of animal-dispersed plant species. We tested the hypothesis that directed dispersal can also be driven by abiotic vectors, such as water or wind. We used a tiered approach, combining analyses of experimental, field and literature data on wetland plant species and evaluating the potential for evolution of directed dispersal with a spatially explicit individual-based model. The data collected demonstrate that wetland plants produce seeds with adaptations to promote transportation and deposition by water towards microsites along the hydrological gradient where they germinate and establish best. Aquatic species produce seeds that sink and are transported by water as bed load towards inundated sites. In contrast, shoreline species produce seeds that float for very long periods of time so that they are eventually entrapped by shoreline vegetation or deposited at the waterline. Our model simulations confirm that the patterns we observed in nature can evolve under natural selection through adaptations in seed buoyancy. For wind dispersal, the situation is more complex. Wind does not provide directed dispersal in the strictest sense but, rather, simply appears to be the best available dispersal vector for more terrestrial wetland plant species to reach drier areas in a wet environment. Synthesis. We show that directed dispersal towards specific, suitable microsites is not exclusive to animal-dispersed plant species, but may be far more common in plants - also in species dispersed by abiotic vectors, in particular water. As water and wind are very common dispersal vectors throughout the plant kingdom, directed dispersal (and not just dispersal distance) seems to be of general importance for the ecology of plants.
Special issue OxiZymes 2016
Berkel, Willem van; Fraaije, Marco ; Hollmann, Frank - \ 2016
Journal of Molecular Catalysis. B, Enzymatic 134 (2016). - ISSN 1381-1177 - p. 273 - 273.
Going against the flow: a case for upstream dispersal and detection of uncommon dispersal events
Wubs, E.R.J. ; Fraaije, Rob G.A. ; Groot, G.A. de; Erkens, R.H.J. ; Garsen, Annemarie G. ; Kleyheeg, Erik ; Raven, Bart M. ; Soons, Merel B. - \ 2016
Freshwater Biology 61 (2016)5. - ISSN 0046-5070 - p. 580 - 595.
1.Dispersal and colonisation are key processes determining species survival, and their importance is increasing as a consequence of ongoing habitat fragmentation, land-use change and climate change. Identification of long-distance dispersal events, including upstream dispersal, and of the dispersal mechanisms and resulting spatial dispersal patterns involved provides much-needed information for conservation in an era of rapid environmental change.
2.However, quantifying contemporary dispersal among populations is far from straightforward. We used the relatively well-defined, typically linear, spatial structure of streams, rivers and their associated riparian and aquatic plant populations to illustrate this. We performed a literature review on studies where dispersal and its directionality (upstream versus downstream) were explicitly quantified.
3.Upstream dispersal was detected in the majority (75%) of examined stream and riparian plant species and mediated mainly by waterfowl, but also by other animals and wind. However, upstream movements are generally less frequent than downstream. Upstream dispersal can occur in excess of tens and sometimes even hundreds of kilometres.
4.Most of the reviewed studies suffer from important methodological limitations that generate difficulties in detecting uncommon dispersal events. Major limitations include use of molecular ecological analyses based on unrealistic assumptions, and the inability to separate seed from pollen flow. On the basis of these findings, we outline a flexible research design using DNA-based assignment methods that allows quantification of contemporary dispersal in future studies. We suggest four key improvements: (i) assignment of propagules and/or seedlings; (ii) use of spatial models to inform sampling design; (iii) reducing the influence of unsampled populations and (iv) combined use of nuclear and uniparentally inherited DNA markers to separate gene flow (including pollen and sperm) in general from propagule-mediated dispersal. In combination with direct measurements of seed dispersal these facilitate empirical quantification of dispersal and the detection of uncommon dispersal events, allowing more realistic assessment of spatial population dynamics, relevant for sedentary and relatively immobile organisms.
Dispersal versus environmental filtering in a dynamic system: drivers of vegetation patterns and diversity along stream riparian gradients
Fraaije, R.G.A. ; Braak, C.J.F. ter; Verduyn, B. ; Verhoeven, Jos T.A. ; Soons, M.B. - \ 2015
community assembly - determinants of plant commiunity diversity and structure - directed dispersal - hydrological gradients - lowland streams - neutral versus niche - plant diversity - riparian vegetation - riparian zone - wetland restoration
1. Both environmental filtering and dispersal filtering are known to influence plant species distribution patterns and biodiversity. Particularly in dynamic habitats, however, it remains unclear whether environmental filtering (stimulated by stressful conditions) or dispersal filtering (during re-colonization events) dominates in community assembly, or how they interact. Such a fundamental understanding of community assembly is critical to the design of biodiversity conservation and restoration strategies. 2. Stream riparian zones are species-rich dynamic habitats. They are characterized by steep hydrological gradients likely to promote environmental filtering, and by spatiotemporal variation in the arrival of propagules likely to promote dispersal filtering. We quantified the contributions of both filters by monitoring natural seed arrival (dispersal filter) and experimentally assessing germination, seedling survival and growth of 17 riparian plant species (environmental filter) along riparian gradients of three lowland streams that were excavated to bare substrate for restoration. Subsequently, we related spatial patterns in each process to species distribution and diversity patterns after 1 and 2 years of succession. 3. Patterns in initial seed arrival were very clearly reflected in species distribution patterns in the developing vegetation and were more significant than environmental filtering. However, environmental filtering intensified towards the wet end of the riparian gradient, particularly through effects of flooding on survival and growth, which strongly affected community diversity and generated a gradient in the vegetation. Strikingly, patterns in seed arrival foreshadowed the gradient that developed in the vegetation; seeds of species with adult optima at wetter conditions dominated seed arrival at low elevations along the riparian gradient while seeds of species with drier optima arrived higher up. Despite previous assertions suggesting a dominance of environmental filtering, our results demonstrate that nonrandom dispersal may be an important driver of early successional riparian vegetation zonation and biodiversity patterns as well. 4. Synthesis: Our results demonstrate (and quantify) the strong roles of both environmental and dispersal filtering in determining plant community assemblies in early successional dynamic habitats. Furthermore, we demonstrate that dispersal filtering can already initiate vegetation gradients, a mechanism that may have been overlooked along many environmental gradients where interspecific interactions are (temporarily) reduced.
Early plant recruitment stages set the template for the development of vegetation patterns along a hydrological gradient
Fraaije, R.G.A. ; Braak, C.J.F. ter; Verduyn, B. ; Breeman, L.B.S. ; Verhoeven, J.T.A. ; Soons, M.B. - \ 2015
biodiversity - colonization - environmental filtering - lowland streams - niche segregation - plant community assembly - riparian zones - wetland restoration
1. Recruitment processes are critical components of a plant's life cycle. However, in comparison with later stages in the plant life cycle (e.g. competition among adults), relatively little is known about their contribution to the regulation of plant species distribution. Particularly little is known about the individual contributions of the three main recruitment processes—germination, seedling survival, and seedling growth—to community assembly, while quantitative information on these contributions is essential for a more mechanistic understanding of the regulation of plant species distribution and biodiversity. 2. Riparian zones along streams provide a globally-relevant case study for evaluating the importance of the different stages of plant recruitment. The natural hydrological gradients of stream riparian zones are currently being restored after a period of worldwide habitat degradation. To identify how recruitment contributes to vegetation patterns and biodiversity in riparian zones, we carried out field experiments at restored lowland streams. We quantified the germination of introduced seeds, and survival and growth of introduced seedlings of 17 riparian plant species across a gradient from the stream channel to upland. 3. The hydrological gradient of riparian zones acted as a strong environmental filter on all three recruitment processes, through imposing an abiotic limitation (excess water) at low elevations and a resource limitation (water shortage) at higher elevations. Other variables, such as soil organic matter content and nutrient availability, only affected recruitment marginally. 4. Species-specific patterns of environmental filtering initiated niche segregation along the riparian gradient during all three recruitment processes, but particularly during germination and seedling growth. These recruitment niches appeared strongly related to indicator values for adult distribution optima, suggesting that at least some riparian plant species may have evolutionary adaptations that promote recruitment under favourable hydrological conditions for adult growth and reproduction. 5. Our results suggest that strong environmental filtering during germination and seedling growth plays an important role in determining later adult distributions, by forming the spatial template on which all subsequent processes operate. In addition to well-known mechanisms, such as competitive exclusion at the adult stage, environmental filtering during early recruitment stages already strongly affect plant distribution and diversity.
Early plant recruitment stages set the template for the development of vegetation patterns along a hydrological gradient
Fraaije, Rob G.A. ; Braak, C.J.F. ter; Verduyn, Betty ; Breeman, Leonieke B.S. ; Verhoeven, Jos T.A. ; Soons, Merel B. - \ 2015
Functional Ecology 29 (2015)7. - ISSN 0269-8463 - p. 971 - 980.
Biodiversity - Colonization - Environmental filtering - Lowland streams - Niche segregation - Plant community assembly - Riparian zones - Wetland restoration

Recruitment processes are critical components of a plant's life cycle. However, in comparison with later stages in the plant life cycle (e.g. competition among adults), relatively little is known about their contribution to the regulation of plant species distribution. Particularly, little is known about the individual contributions of the three main recruitment processes - germination, seedling survival and seedling growth - to community assembly, while quantitative information on these contributions is essential for a more mechanistic understanding of the regulation of plant species distribution and biodiversity. Riparian zones along streams provide a globally-relevant case study for evaluating the importance of the different stages of plant recruitment. The natural hydrological gradients of stream riparian zones are currently being restored after a period of world-wide habitat degradation. To identify how recruitment contributes to vegetation patterns and biodiversity in riparian zones, we carried out field experiments at restored lowland streams. We quantified the germination of introduced seeds, and survival and growth of introduced seedlings of 17 riparian plant species across a gradient from the stream channel to upland. The hydrological gradient of riparian zones acted as a strong environmental filter on all three recruitment processes, through imposing an abiotic limitation (excess water) at low elevations and a resource limitation (water shortage) at higher elevations. Other variables, such as soil organic matter content and nutrient availability, only affected recruitment marginally. Species-specific patterns of environmental filtering initiated niche segregation along the riparian gradient during all three recruitment processes, but particularly during germination and seedling growth. These recruitment niches appeared strongly related to indicator values for adult distribution optima, suggesting that at least some riparian plant species may have evolutionary adaptations that promote recruitment under favourable hydrological conditions for adult growth and reproduction. Our results suggest that strong environmental filtering during germination and seedling growth plays an important role in determining later adult distributions, by forming the spatial template on which all subsequent processes operate. In addition to well-known mechanisms, such as competitive exclusion at the adult stage, environmental filtering during early recruitment stages already strongly affect plant distribution and diversity.

Dispersal versus environmental filtering in a dynamic system : Drivers of vegetation patterns and diversity along stream riparian gradients
Fraaije, R.G.A. ; Braak, C.J.F. ter; Verduyn, Betty ; Verhoeven, J.T.A. ; Soons, M.B. - \ 2015
Journal of Ecology 103 (2015)6. - ISSN 0022-0477 - p. 1634 - 1646.
Community assembly - Determinants of plant community diversity and structure - Directed dispersal - Hydrological gradients - Lowland streams - Neutral versus niche - Plant diversity - Riparian vegetation - Riparian zone - Wetland restoration

Both environmental filtering and dispersal filtering are known to influence plant species distribution patterns and biodiversity. Particularly in dynamic habitats, however, it remains unclear whether environmental filtering (stimulated by stressful conditions) or dispersal filtering (during recolonization events) dominates in community assembly, or how they interact. Such a fundamental understanding of community assembly is critical to the design of biodiversity conservation and restoration strategies. Stream riparian zones are species-rich dynamic habitats. They are characterized by steep hydrological gradients likely to promote environmental filtering, and by spatiotemporal variation in the arrival of propagules likely to promote dispersal filtering. We quantified the contributions of both filters by monitoring natural seed arrival (dispersal filter) and experimentally assessing germination, seedling survival and growth of 17 riparian plant species (environmental filter) along riparian gradients of three lowland streams that were excavated to bare substrate for restoration. Subsequently, we related spatial patterns in each process to species distribution and diversity patterns after 1 and 2 years of succession. Patterns in initial seed arrival were very clearly reflected in species distribution patterns in the developing vegetation and were more significant than environmental filtering. However, environmental filtering intensified towards the wet end of the riparian gradient, particularly through effects of flooding on survival and growth, which strongly affected community diversity and generated a gradient in the vegetation. Strikingly, patterns in seed arrival foreshadowed the gradient that developed in the vegetation; seeds of species with adult optima at wetter conditions dominated seed arrival at low elevations along the riparian gradient, while seeds of species with drier optima arrived higher up. Despite previous assertions suggesting a dominance of environmental filtering, our results demonstrate that non-random dispersal may be an important driver of early successional riparian vegetation zonation and biodiversity patterns as well. Synthesis. Our results demonstrate (and quantify) the strong roles of both environmental and dispersal filtering in determining plant community assemblies in early successional dynamic habitats. Furthermore, we demonstrate that dispersal filtering can already initiate vegetation gradients, a mechanism that may have been overlooked along many environmental gradients where interspecific interactions are (temporarily) reduced. Our results demonstrate and quantify the strong roles of both environmental and dispersal filtering in determining plant community assemblies in early successional dynamic habitats. Furthermore, we demonstrate that dispersal filtering can already initiate vegetation gradients, a mechanism that may have been overlooked along many environmental gradients where interspecific interactions are (temporarily) reduced.

Morphodynamic regime change in a reconstructed lowland stream
Eekhout, J.P.C. ; Fraaije, R. ; Hoitink, A.J.F. - \ 2014
Earth Surface Dynamics 2 (2014). - ISSN 2196-6311 - p. 279 - 293.
With the aim to establish and understand morphological changes in response to channel reconstruction, a detailed monitoring plan was implemented in a lowland stream called Lunterse Beek, located in the Netherlands. Over a period of almost 2 years, the monitoring programme included serial morphological surveys, continuous discharge and water level measurements, and riparian vegetation mapping, from photographs and field surveys. Morphological processes occurred mainly in the initial period, before riparian vegetation developed. The initial period was largely dominated by upstream sediment supply, which was associated with channel incision upstream from the study area. Herbaceous vegetation started to develop approximately 7 months after channel reconstruction. The monitoring period included two growing seasons. A clear increase of riparian vegetation cover from first to the second year was observed. Detailed morphological and hydrological data show a marked difference in morphological behaviour between the pre-vegetation and post-vegetation stage. A linear regression procedure was applied to relate morphological activity to time-averaged Shields stress. In the initial stage after channel reconstruction, with negligible riparian vegetation, channel morphology adjusted, showing only a weak response to the discharge hydrograph. In the subsequent period, morphological activity in the channel showed a clear relation with discharge variation. The two stages of morphological response to the restoration measures may be largely associated with the upstream sediment supply in the initial period. Riparian vegetation may have played a substantial role in stabilizing the channel banks and floodplain area, gradually restricting the morphological adjustments to the channel bed.
Bayer-Villiger Oxidation
Gonzalo, G. de; Berkel, W.J.H. van; Fraaije, M.W. - \ 2014
In: Science of Synthesis. Biocatalysis in Organic Synthesis 3 / Faber, K., Fessner, W.D., Turner, N.J., Thieme - ISBN 9783131746610 - p. 187 - 233.
Oxidations Using Alcohol Oxidases
Ewing, T.A. ; Fraaije, M.W. ; Berkel, W.J.H. van - \ 2014
In: Science of Synthesis. Biocatalysis in Organic Synthesis 3 / Faber, K., Fessner, W., Turner, NJ, Thieme - ISBN 9783131746610 - p. 157 - 185.
The Oxidation of Thiols by Flavoprotein Oxidases: a Biocatalytic Route to Reactive Thiocarbonyls.
Ewing, T.A. ; Dijkman, W.P. ; Vervoort, J.J.M. ; Fraaije, M.W. ; Berkel, W.J.H. van - \ 2014
Angewandte Chemie-International Edition 53 (2014)48. - ISSN 1433-7851 - p. 13206 - 13209.
vanillyl-alcohol oxidase - streptomyces-coelicolor - catalytic mechanism - alditol oxidase - sulfhydryl oxidase - discovery - quinones - flavins - enzyme
Flavoprotein oxidases are a diverse class of biocatalysts, most of which catalyze the oxidation of C[BOND]O, C[BOND]N, or C[BOND]C bonds. Flavoprotein oxidases that are known to catalyze the oxidation of C[BOND]S bonds are rare, being limited to enzymes that catalyze the oxidative cleavage of thioethers. Herein, we report that various flavoprotein oxidases, previously thought to solely act on alcohols, also catalyze the oxidation of thiols to thiocarbonyls. These results highlight the versatility of enzymatic catalysis and provide a potential biocatalytic route to reactive thiocarbonyl compounds, which have a variety of applications in synthetic organic chemistry.
Risk assessment on the impact of environmental usage of triazoles on the development and spread of resistance to medical triazoles in Aspergillus species
Kleinkauf, N. ; Verweij, P.E. ; Arendrup, M.C. ; Donnelly, P.J. ; Cuenca-Estrella, M. ; Fraaije, B. ; Melchers, W.J.G. ; Adriaenssens, N. ; Kema, G.H.J. ; Ullmann, A. ; Bowyer, P. ; Denning, D.W. - \ 2013
Stockholm, Sweden : European Centre for Disease Prevention and Control (ECDC) (ECDC Technical Report ) - ISBN 9789291934447 - 17 p.
In recent years, triazole resistance in human Aspergillus diseases appears to have been increasing in several European countries. However, current data on the prevalence of resistance are based on a small number of studies which are only available from a few European countries. If present, triazole resistance can severely limit treatment options since alternatives, which are only available in intravenous form, have been shown to be associated with more side effects and poorer outcomes. Triazole resistance in Aspergillus spp. can evolve during therapy. Several point mutations, particularly in the cyp51A gene, have been associated with the development of resistance. Increasingly however, resistant isolates are also being detected in azole-naive patients. These isolates tend to have a particular genetic alteration consisting of a 34-base pair tandem repeat in the promoter coupled with a point mutation in the cyp51A target gene. This leads to an amino-acid substitution at codon 98 (TR34/L98H) causing multi-azole resistance. In patients whose Aspergillus isolates have developed resistance during azole therapy wildtype isolates, closely related genetically to the resistant isolates, have regularly been recovered from samples taken before the start of therapy or during an earlier phase. To date however, no isogenic isolate with a wild-type phenotype has been recovered from patients infected with an Aspergillus strain carrying the TR34/L98H genetic alteration. This suggests a possible environmental origin of the resistant fungus. This particular resistance mechanism has been observed most frequently in clinical isolates in the Netherlands where it has also been found in the environment. Moreover, the resistance mechanism has been demonstrated in clinical isolates in eight other European countries. Azole fungicides are widely used for crop protection and material preservation in Europe. They protect crops from disease, ensure yields and prevent fungal contamination of produce. It has been proposed that triazole resistance has evolved in the environment and could be driven by the selective pressure of azole fungicides. Although evidence supporting this hypothesis is growing, the link between the environmental use of azole fungicides and the development of triazole resistance in Aspergillus spp. is not yet proven. Triazole therapy has become the established treatment for invasive aspergillosis and is widely used in the treatment of allergic aspergillosis and chronic pulmonary aspergillosis. Antifungal therapy for invasive pulmonary aspergillosis is usually prescribed for a minimum of 6–12 weeks, but often may need to be continued for months depending on the period of immunosuppression. Treatment of allergic aspergillosis and chronic pulmonary aspergillosis may need to continue for years or even throughout a patient’s lifetime. We estimated the burden of allergic, chronic and invasive aspergillosis using population statistics and published literature. Of the 733 million inhabitants in the European region1 [1], at any one time 2 100 000 patients may be suffering from allergic aspergillosis and 240 000 from chronic aspergillosis, that would be an indication for antifungal therapy. For invasive aspergillosis, we have estimated an annual incidence of 63 250 cases, complicating multiple underlying conditions including leukaemia, transplantation, chronic obstructive pulmonary disease (COPD) and medical intensive care. The inability to treat these patients with triazoles due to multi-azole resistance would have significant impact on patient management and associated health costs. Early and thorough investigation of this emerging public health problem is warranted in order to avoid the development and spread of resistance. This report examines current evidence for the environmental origin of resistance in Aspergillus spp. and makes recommendations for further steps to assess the risks and consequences of the environmental usage of azole derivatives. Improved surveillance of clinical isolates, including antifungal susceptibility testing, is the key to a better understanding of the magnitude of this emerging problem. Furthermore, the diagnosis of Aspergillus diseases needs to be improved and molecular methods allowing detection of resistance in culture-negative specimens must be further developed and implemented in laboratory practice. Finally, further environmental and laboratory studies are needed to confirm the environmental hypothesis
Characterization of molecular mechanisms underlying the multi-drug-resistant phenotypes of Mycosphaerella graminicola field isolates
Fillinger, S. ; Omrane, S. ; Sghyer, H. ; Lanen, C. ; Aoini, L. ; Kema, G.H.J. - \ 2013
In: Modern fungicides and antifungal compounds VII. Proceedings of the 17th International Reinhardsbrunn Symposium. - Braunschweig : Deutsche Phytomedizinische Gesellschaft - ISBN 9783941261136 - p. 111 - 116.
Multidrug resistance (MDR) is a common trait developed by many organisms to counteract chemicals and/or drugs used against them. The basic MDR mechanism is relying on an overexpressed efflux transport system that actively expulses the toxic agent outside the cell. In fungi, MDR has been extensively studied in Saccharomyces cerevisiae and Candida albicans, but also plant pathogenic fungi are concerned by this phenomenon. In this study, we confirmed a drug efflux based resistance mechanism in two isolates of the septoria leaf blotch agent Mycosphaerella graminicola. Using next generation sequencing approaches, we could demonstrate that the MgMFS1 gene was strongly overexpressed in both isolates. The mutation(s) responsible for the MDR phenotype was mapped to a 38.5 kb genomic fragment on chromosome VII, covering also the MgMFS1 gene. Precise genetic mapping will then identify the mdr mutation(s) leading to MfMFS1 overexpressio Characterization of molecular mechanisms underlying the multi-drug-resistant phenotypes of Mycosphaerella graminicola field isolates. (PDF Download Available). Available from: http://www.researchgate.net/publication/271765795_Characterization_of_molecular_mechanisms_underlying_the_multi-drug-resistant_phenotypes_of_Mycosphaerella_graminicola_field_isolates [accessed Jun 15, 2015]
Simultaneous real-time PCR quantification of Fusarium asiaticum, F ussurianum and F vorosii, representing the Asian clade of the F graminearum species complex
Fernandez-Ortuno, D. ; Waalwijk, C. ; Lee, T.A.J. van der; Fan, J. ; West, J.S. ; Fraaije, B.A. - \ 2013
International Journal of Food Microbiology 166 (2013)1. - ISSN 0168-1605 - p. 148 - 154.
head blight pathogen - gibberella-zeae - genealogical concordance - southern brazil - wheat - diversity - barley - quantification - populations - mycotoxins
Due to the repeated discovery of new members of the Fusarium graminearum species complex (FGSC), some of the F. graminearum sensu stricto (s.s.)-specific qPCR assays developed to date have since been shown to be non-specific. In this study, a probe-based qPCR method was developed, targeting a sterol 14-alpha demethylase (CYP51) paralogue, CYP51C unique to the genus Fusarium, for the simultaneous detection of F. asiaticum, F. ussurianum and F. vorosii. Specificity of the assay was demonstrated for a wide range of Fusarium species, including all tested FGSC members (n=6), originating from different hosts and geographic regions. Alongside a previously published assay for detection of F. graminearum, we were able to show that members of the Asian clade of the FGSC (i.e. F. asiaticum, F. ussurianum and F. vorosii) were the primary etiological agent in wheat seeds samples originating from Central-East China. The grain samples from the UK tested negative for the presence of the FGSC's Asian clade and positive for presence of F. graminearum. It is likely that only F. graminearum s.s. is present in the UK, but the presence of other FGSC members cannot be ruled out and need further investigation
Beekdalbreed Hermeanderen : bouwstenen voor de ‘leidraad voor innovatief beek- en beekdalherstel’
Verdonschot, P.F.M. ; Besse, A.A. ; Brouwer, J.H.F. de; Eekhout, J.P.C. ; Fraaije, R. - \ 2012
Amersfoort : Stowa (Rapport / STOWA 2012-36) - 56
beekdalen - ecosystemen - waterlopen - herstelbeheer - ecologisch herstel - natuurtechniek - aquatische ecologie - morfologie - brook valleys - ecosystems - streams - restoration management - ecological restoration - ecological engineering - aquatic ecology - morphology
Het doel van het project Beekdalbreed Hermeanderen was om in de praktijk in beekherstelprojecten een gedempt afvoer- en een stabiel en gevarieerd habitatpatroon te realiseren door morfologische en hydrologische maatregelen in samenhang uit te voeren. In 6 beekdalbreed uitgevoerde hermeanderingsprojecten en in 7 projecten, waarbij dood hout is ingebracht, is onderzocht welke hydrologische, morfologische, terrestrisch- en aquatisch-ecologische effecten optraden.
Multiple pathways guide oxygen diffusion into flavoenzyme active sites
Baron, R. ; Riley, C. ; Chenprakhon, P. ; Thotsaporn, K. ; Winter, R.T. ; Alfieri, A. ; Forneris, F. ; Berkel, W.J.H. van; Chaiyen, P. ; Fraaije, M.W. ; Mattevi, A. ; McCammon, J.A. - \ 2009
Proceedings of the National Academy of Sciences of the United States of America 106 (2009)26. - ISSN 0027-8424 - p. 10603 - 10608.
x-ray crystallography - molecular-dynamics - streptomyces-coelicolor - cholesterol oxidase - alditol oxidase - protein - o-2 - enzyme - fluorescence - simulations
Dioxygen (O2) and other gas molecules have a fundamental role in a variety of enzymatic reactions. However, it is only poorly understood which O2 uptake mechanism enzymes employ to promote efficient catalysis and how general this is. We investigated O2 diffusion pathways into monooxygenase and oxidase flavoenzymes, using an integrated computational and experimental approach. Enhanced-statistics molecular dynamics simulations reveal spontaneous protein-guided O2 diffusion from the bulk solvent to preorganized protein cavities. The predicted protein-guided diffusion paths and the importance of key cavity residues for oxygen diffusion were verified by combining site-directed mutagenesis, rapid kinetics experiments, and high-resolution X-ray structures. This study indicates that monooxygenase and oxidase flavoenzymes employ multiple funnel-shaped diffusion pathways to absorb O2 from the solvent and direct it to the reacting C4a atom of the flavin cofactor. The difference in O2 reactivity among dehydrogenases, monooxygenases, and oxidases ultimately resides in the fine modulation of the local environment embedding the reactive locus of the flavin
Identification of a gatekeeper residue that prevents dehydrogenases from acting as oxidases
Leferink, N.G.H. ; Fraaije, M.W. ; Joosten, H.J. ; Schaap, P.J. ; Mattevi, A. ; Berkel, W.J.H. van - \ 2009
Journal of Biological Chemistry 284 (2009)7. - ISSN 0021-9258 - p. 4392 - 4397.
gamma-lactone dehydrogenase - monomeric sarcosine oxidase - vanillyl-alcohol oxidase - cholesterol oxidase - l-galactono-1,4-lactone dehydrogenase - streptomyces-coelicolor - substrate-specificity - oxygen reactivity - alditol oxidase - enzyme
The oxygen reactivity of flavoproteins is poorly understood. Here we show that a single Ala to Gly substitution in L-galactono-1,4-lactone dehydrogenase (GALDH) turns the enzyme into a catalytically competent oxidase. GALDH is an aldonolactone oxidoreductase with a vanillyl-alcohol oxidase (VAO) fold. We found that nearly all oxidases in the VAO family contain either a Gly or Pro at a structurally conserved position near the C4a locus of the isoalloxazine moiety of the flavin, whereas dehydrogenases prefer another residue at this position. Mutation of the corresponding residue in GALDH (Ala113Gly) resulted in a striking 400 fold increase in oxygen reactivity, while the cytochrome c reductase activity is retained. The activity of the A113G variant shows a linear dependence on oxygen concentration (kox = 3.5 x 10e5 M-1 s-1), similar to most other flavoprotein oxidases. The Ala113Gly replacement does not change the reduction potential of the flavin, but creates space for molecular oxygen to react with the reduced flavin. In the wild-type enzyme Ala113 acts as a gatekeeper, preventing oxygen to access the isoalloxazine nucleus. The presence of such an oxygen access gate seems to be a key factor for the prevention of oxidase activity within the VAO family, and is absent in members that act as oxidases.
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