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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.

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    On the role of vaccine dose and antigenic distance in the transmission dynamics of Highly Pathogenic Avian Influenza (HPAI) H5N1 virus and its selected mutants in vaccinated animals
    Sitaras, Ioannis - \ 2017
    Wageningen University. Promotor(en): M.C.M. Jong, co-promotor(en): B. Peeters. - Wageningen : Wageningen University - ISBN 9789463438063 - 209
    avian influenza viruses - avian influenza - disease transmission - vaccines - vaccination - dosage - antigenic variation - mutants - mutations - immunity - vaccine development - virology - epidemiology - aviaire influenzavirussen - aviaire influenza - ziekteoverdracht - vaccins - vaccinatie - dosering - antigene variatie - mutanten - mutaties - immuniteit - vaccinontwikkeling - virologie - epidemiologie

    Influenza virus infections can cause high morbidity and mortality rates among animals and humans, and result in staggering direct and indirect financial losses amounting to billions of US dollars. Ever since it emerged in 1996 in Guangdong province, People’s Republic of China, one particular highly pathogenic avian influenza (HPAI) H5N1 virus has spread globally, and is responsible for massive losses of poultry, as well as human infections. For these reasons, HPAI H5N1 is considered as one of the viruses possible to cause a future influenza pandemic.

    One of the main reasons why influenza is a recurring problem is its ability to constantly evolve through the selection of mutants that are able to avoid immunity (be it natural or acquired). Due to the accumulation of mutations during genome replication, diverse/variant influenza genome sequences co-exist in a virus pool (quasispecies). These sequences can contain mutations that are able to confer selective advantages to the influenza virus given the opportunity. As a consequence, whenever a situation arises that places the virus under any type of pressure that the dominant virus sequence cannot cope with (i.e. immune pressure, selective receptor binding, etc.), the virus with the genome sequence that allows it to better adapt to that particular pressure becomes selected and takes over.

    Because of the influenza virus’s high rate of mutations, a global surveillance network is in place to monitor changes in circulating strains among humans that would warrant an update of the vaccines used. For human influenza strains, vaccines are updated frequently (every one or two years) and a similar situation holds true for racehorse vaccination. For avian influenza vaccination, however, the situation is different. In most countries, vaccination against avian influenza is not used, and in the countries where vaccines are used (either as routine or emergency measures), they are not updated as frequently as human vaccines are. In addition, in many instances vaccination against avian influenza viruses has met with some spectacular failures, since it failed to produce a level of immunity that would protect against circulating field strains. These vaccination failures have often been attributed to the fact that without constant vaccine updating (as is done for human influenza), the vaccines used are not able to keep up with continuously evolving antigenic variants selected in the field, and thus to protect poultry against them. In addition, since it is known that immune pressure resulting from vaccination can be a driving force in the evolution of influenza viruses and the selection of immune-escape mutants, there is a school of thought that posits that vaccination against avian influenza is not only a very expensive affair (especially if vaccines need to be frequently updated), but can also lead to selection of mutants that are able to avoid vaccination-induced immunity.

    The research reported in this thesis started with addressing the gaps in the knowledge regarding the role of vaccination-induced immunity in the selection of immune-escape mutants of HPAI H5N1, and if there is a way for vaccines to still be able to protect against antigenically-distant variants of the vaccine seed strain, without the need for frequent vaccine updates.

    Our first step in studying influenza virus evolution and selection of immune-escape mutants was to investigate how antigenic pressure may drive the selection of such mutants, and what the effect of the selected mutations on the pathogenicity and transmissibility of the mutants may be. Although there exist a variety of methods to select for influenza virus mutations (i.e. monoclonal antibodies, site-directed mutagenesis, reverse genetics, etc.), none of them is representative of selection as it happens in a vaccinated animal. In Chapter 2, we discuss in detail a laboratory-based system we have developed, in which immune-escape mutants are selected using homologous polyclonal chicken sera, similar to how they are selected in the field due to vaccination- induced immune pressure. We find that selection takes place early on, and additional mutations are selected when immune pressure is increased. Antigenic distances between the selected mutants and their parent strains are also increased throughout the selection process, but not in a linear fashion. Our selection system proved to be robust and replicable, and to be representative of selection in the field, since the mutations we selected for are also found in naturally-selected field isolates, and the antigenic distances between our selected mutants and their parent strains are similar to antigenic distances between vaccine strains and field isolates.

    We continued our research by addressing the roles played by vaccine dose (and resulting immunity) and antigenic distance between vaccine and challenge strains, in the transmission of HPAI H5N1 viruses, by employing transmission experiments using vaccinated chickens (Chapter 3). To our surprise, we found that the effect of antigenic distances between vaccine and challenge strains on transmission is very small compared to the effect of vaccine dose. We then quantified, for the first time, the minimum level of immunity and minimum percentage of the vaccinated population exhibiting said immunity, in order for vaccines to be able to protect against transmission even of strains that are antigenically distant to the vaccine seed strain. Transmission of such strains in well-vaccinated populations would allow for a scenario where vaccination- induced immunity may drive the selection of immune-escape mutants. Our results show that in order for vaccines to prevent transmission of antigenically distant strains (such as the ones resulting from selection due to immune pressure), the threshold level of immunity against these strains should be ≥23 haemagglutination inhibition units (HIU), in at least 86.5% of the vaccinated population. This level of immunity can be estimated by knowing the antigenic distance between the vaccine and challenge (field) strain, and the HI titre against the vaccine strain, which would then allow the approximate level of immunity against the field strain to be deduced. For example, assuming the HI titre against a vaccine strain is 210 HIU, and the distance with the challenge (field) strain is 24 HIU, according to our results the vaccine should be able to protect against the challenge strain, because the difference in HI titres should be around 26 HIU (i.e. above 23 HIU). These results, taken together with our previous work on selection of mutants, where we showed that the antigenic distances between our mutants and their parent strains are representative of distances found in the field, point to the fact that it is unlikely that vaccination-induced immunity can lead to selection of mutants able to escape it, given that a threshold level of immunity in a minimum percentage of the vaccinated population is achieved. As a consequence, we believe that constant vaccine updating may not be necessary for avian influenza viruses, as long as a threshold level of immunity is maintained. This makes vaccination a more attractive control measure, both from a health perspective and a financial one, than just applying biosecurity measures.

    To examine the effect the mutations in the haemagglutinin protein of our selected mutants may have in their transmission among chickens vaccinated with the parent strain, we used reverse genetics techniques to insert the HA gene of our most antigenically distant mutant into the parent strain backbone (Chapter 4). We vaccinated animals with a sub-optimal dose of vaccine, and we concluded that the mutations we selected for did not allow the mutant to avoid even low levels of immunity, such as the ones resulting from a sub-optimal vaccine dose (which resembles a poor field vaccination scenario). At the same time, the HA mutations we selected for did not appear to have a negative effect either on the pathogenicity of the mutant, or its ability to transmit to unvaccinated animals, since both parameters were comparable to the parent strain.

    Finally, we studied the role inter-animal variation in immunity – as measured by HI titres – has in the accuracy of antigenic cartography calculations (Chapter 5). We found that using sera from more than one animal significantly increased the accuracy of antigenic distance calculations, since it takes into account individual differences in immune responses to vaccination, an inevitable phenomenon documented in both humans and animals. In addition, we increased the accuracy of antigenic maps by avoiding the use of dimension-reducing algorithms as is currently done. By not reducing the dimensionality of virus positioning in space, our maps retain the original geometry between strains or sera, leading to more accurate positioning (Chapters 2 and 5). We hope that improving the accuracy of antigenic cartography can lead to a more precise surveillance of influenza evolution and better informed decisions regarding the need to update vaccines.

    Taken collectively, our results can improve field vaccination outcomes, since they provide guidelines on how to increase vaccination efficiency in stopping transmission of even antigenically-distant strains. In addition, our method for selecting for immune- escape mutants can be a valuable addition to research on influenza virus evolution. Moreover, policy making decisions regarding vaccination against any type of influenza can also benefit from our improvement on antigenic cartography accuracy, saving unnecessary costs in vaccine updating, and reducing morbidity and mortality of both animals and humans.

    Isolation, characterization and engineering of Bacillus smithii : a novel thermophilic platform organism for green chemical production
    Bosma, E.F. - \ 2015
    Wageningen University. Promotor(en): Willem de Vos; John van der Oost, co-promotor(en): Richard van Kranenburg. - Wageningen : Wageningen University - ISBN 9789462575073 - 220
    bacillus smithii - bacillus (bacteria) - biobrandstoffen - chemicaliën uit biologische grondstoffen - thermofielen - metabolische profilering - genoomanalyse - mutaties - isolatie - bioengineering - karakterisering - bacillus smithii - bacillus (bacteria) - biofuels - biobased chemicals - thermophiles - metabolic profiling - genome analysis - mutations - isolation - bioengineering - characterization

    Due to the globally increasing demand for chemicals and fuels and the high environmental impact and limited amount of fossil resources, there is a growing interest in green chemicals and fuels derived from renewable resources. As described in Chapter 1, one of the most feasible alternatives on the short term is microbial conversion of the sugars in biomass to fuels and chemicals in a biorefinery. To be economically and ethically feasible, non-food biomass should be used as a resource, which is often difficult with currently used production organisms. Also, to be economically feasible, the costs of green chemicals and fuels need to be further reduced to be below the costs of products based on fossil resources. To do so, other organisms than the currently most-used platform organisms such as Escherichia coli and Saccharomyces cerevisiae should be used. Ideally, this alternative organism is genetically accessible, has high productivity, titre and yield, is flexible in carbon source, robust, moderately thermophilic, acidophilic, facultatively anaerobic and has little nutritional requirements. The organisms that come closest to these criteria are thermophilic bacilli, which form a diverse class of organisms in the family of Bacillaceae. This thesis describes the isolation, characterization and metabolic engineering of Bacillus smithii, a novel potential thermophilic platform organism.

    Chapter 2 provides more detail on the use of thermophilic microorganisms as platform organisms for green chemical production in a biorefinery concept. As commercially available enzyme mixtures used in the simultaneous saccharification and fermentation (SSF) of biomass have their optimum temperature around 50-60°C, using a moderately thermophilic organism would reduce the costs of the SSF process compared to when using mesophiles by reducing the amount of required enzyme. Also, thermophilic processes are less prone to contaminations, and substrate and product solubility are increased. Several successful examples of the application of facultatively anaerobic thermophiles for green chemical production from lignocellulose in an SSF setting are for example Bacillus coagulans for lactic acid production and Bacillus licheniformis for 2,3-butanediol production. However, whereas strongly developed genetic toolboxes are available for current mesophilic production organisms, these tools are still in their infancy for thermophilic organisms. Such tools are required to optimize production and to study metabolism. Thermophilic organisms show a wide variety in metabolism and in many cases the metabolism of these organisms is still poorly understood, hampering full optimization. Chapter 2 furthermore provides an overview of transformation, integration and counter-selection methods currently used for thermophiles. Although several deletion mutants have been constructed using these methods, not all of them are entirely markerless and most are not suited as high-throughput engineering tools, stressing the need for further research in this area.

    Despite several facultatively anaerobic thermophiles being described as genetically accessible, this feature is still one the major bottlenecks in developing these organisms into platform organisms. Therefore, in Chapter 3, we set out to isolate a facultatively anaerobic, moderately thermophilic bacterium that was genetically accessible and produced high titers of organic acids. A total of 267 strains of different thermophilic bacilli species were isolated from compost and screened for C5 and C6 sugar utilization and acid production. The 44 best strains were screened for genetic accessibility via electroporation. Only 3 strains tested positive for this, namely Geobacillus thermodenitrificans strains ET 144-2 and ET 251 and B. smithii strain ET 138. In subsequent evaluations in lab-scale bioreactors at 55°C and pH 6.5 on glucose, the two G. thermodenitrificans strains performed poorly whereas B. smithii performed well with high titers, yields and productivity of mainly lactate. In similar lab-scale reactors, this strain also performed well on xylose and at pH 5.5 and was still able to perform for 48 at pH 4.5. The electroporation protocol for this strain was optimized, resulting in a maximum efficiency of 5x103 colonies per µg plasmid pNW33n. Two other B. smithii strains, among which the type strain DSM 4216T, were also shown to be transformable with pNW33n. This is the first time that genetic accessibility is described for B. smithii and it is the first step towards developing it into a platform organism, for which it appears to be suitable based on its efficient C5 and C6 sugar utilization and acid production profile.

    In order to become a platform organism and to study its atypical metabolism, a genetic toolbox needs to be established for B. smithii. Chapter 5 describes the development of a markerless gene deletion method for B. smithii. For strains ET 138 and DSM 4216T, the ldhL gene was markerlessly removed via double homologous recombination using plasmid pNW33n. Despite the replicative nature of this plasmid at 55°C, mixtures of single and double crossovers were readily obtained. A pure double crossover deletion mutant was obtained after several transfers on a more defined medium containing acetate or lactate and PCR-based screenings. To eliminate the possibility of mixed genotypes, we subsequently developed a lacZ-counter-selection system, which is based on the toxicity of high X-gal concentrations in the presence of the plasmid-encoded lacZ gene. Using this method, the sporulation-specific sigma factor sigF and pyruvate dehydrogenase complex E1-α pdhA were consecutively removed from the B. smithii ET 138 genome in a markerless way. An initial evaluation of the growth and production profiles of the mutant strains in tubes showed that removal of the ldhL gene eliminates l-lactate production and causes a severe decrease in anaerobic growth and production capacities. B. smithii mutants lacking the sigF gene were unable to sporulate and removal of the pdhA gene eliminated acetate production and rendered the strains auxotrophic for acetate.

    DNA : the recipe book for all the processes in the plant : all cells have the same generic information
    Heuvelink, E. ; Kierkels, T. - \ 2015
    In Greenhouses : the international magazine for greenhouse growers 4 (2015)4. - ISSN 2215-0633 - p. 12 - 13.
    dna - plantenveredeling - genetische modificatie - transfer rna - messenger rna - ribosomen - eiwitten - aminozuren - enzymen - mutaties - dna - plant breeding - genetic engineering - transfer rna - messenger rna - ribosomes - proteins - amino acids - enzymes - mutations
    It’s sometimes called a blueprint: DNA, the carrier of genetic information. But the term recipe book covers it better. It explains how the plant can respond to changing conditions. Plant breeders take advantage of natural variations in DNA. Genetic modification can make their job easier.
    Wat is erfelijkheid?
    Maurice - Van Eijndhoven, M.H.T. ; Oldenbroek, Kor - \ 2015
    Zeldzaam huisdier 40 (2015)3. - ISSN 0929-905X - p. 10 - 12.
    heritability - rassen (dieren) - dierveredeling - dna - eigenschappen - spermatozoön - eicellen - bevruchting - genen - allelen - homozygoten - heterozygoten - mutaties - genetische merkers - heritability - breeds - animal breeding - dna - properties - spermatozoa - ova - fertilization - genes - alleles - homozygotes - heterozygotes - mutations - genetic markers
    Eigenschappen van dieren zijn in meer of mindere mate erfelijk. Ze gaan over van ouders op nakomelingen. Maar ervaren fokkers weten dat in de fokkerij 1+1 geen 2 is. Welke wetmatigheden en welke toevalligheden spelen een rol in de erfelijkheid? Wat heeft het DNA-onderzoek ons daar recentelijk over geleerd en wat kunnen we daarmee?
    A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells
    Angelo, L. D'; Piazzi, G. ; Pacilli, A. ; Prossomariti, A. ; Fazio, C. ; Montanaro, L. ; Graziani, G. ; Fogliano, V. ; Munarini, A. ; Bianchi, F. ; Belluzzi, A. ; Bazzoli, F. ; Ricciardiello, L. - \ 2014
    Carcinogenesis 35 (2014)10. - ISSN 0143-3334 - p. 2314 - 2320.
    activated protein-kinase - colon-cancer - liver metastasis - drug-resistance - carcinoma cells - in-vitro - growth - therapy - inhibition - mutations
    Colorectal cancer (CRC) is one of the major causes of cancer death worldwide. The development of novel anti-CRC agents able to overcome drug resistance and/or off-target toxicity is of pivotal importance. The mammalian target of rapamycin (mTOR) plays a critical role in CRC, regulating protein translation and controlling cell growth, proliferation, metabolism and survival. The aim of this study was to explore the effect of a combination of three natural compounds, eicosapentaenoic acid-free fatty acid (EPA-FFA), epigallocatechin-3-gallate (EGCG) and proanthocyanidins (grape seed [GS] extract) at low cytotoxic concentrations on CRC cells and test their activity on mTOR and translational regulation. The CRC cell lines HCT116 and SW480 were treated for 24 h with combinations of EPA-FFA (0-150 mu M), EGCG (0-175 mu M) and GS extract (0-15 mu M) to evaluate the effect on cell viability. The low cytotoxic combination of EPA-FFA 150 mu M, EGCG 175 mu M and GS extract 15 mu M completely inhibited the mTOR signaling in HCT116 and SW480 cells, reaching an effect stronger than or comparable to that of the mTOR inhibitor Rapamycin in HCT116 or SW480 cells, respectively. Moreover, the treatment led to changes of protein translation of ribosomal proteins, c-Myc and cyclin D1. In addition, we found a reduction of clonal capability in both cell lines, with block of cell cycle in G(0)G(1) and induction of apoptosis. Our data suggest that the low cytotoxic combination of EPA-FFA, EGCG and GS extract, tested for the first time here, inhibits mTOR signaling and thus could be considered for CRC treatment.
    Colorectal cancer risk variants on 11q23 and 15q13 are associated with unexplained adenomatous polyposis
    Hes, F.J. ; Ruano, D. ; Nieuwenhuis, M. ; Tops, C.M.J. ; Schrumpf, M. ; Nielsen, M. ; Huijts, P.E. ; Wijnen, J. ; Wagner, A. ; Gomet Garcia, E.B. ; Sijmons, R.H. ; Menko, F.H. ; Letteboer, T.G. ; Hoogerbrugge, N. ; Harryvan, J.L. ; Kampman, E. ; Morreau, H. ; Vasen, H.F. ; Wezel, T.G. van - \ 2014
    Journal of Medical Genetics 51 (2014)1. - ISSN 0022-2593 - p. 55 - 60.
    genome-wide association - susceptibility loci - genetic-variants - apc - mutations - hereditary - families - metaanalysis - mechanisms - phenotype
    Background Colorectal adenomatous polyposis is associated with a high risk of colorectal cancer (CRC) and is frequently caused by germline mutations in APC or MUTYH. However, in about 20–30% of patients no underlying gene defect can be identified. In this study, we tested if recently identified CRC risk variants play a role in patients with >10 adenomas. Methods We analysed a total of 16 SNPs with a reported association with CRC in a cohort of 252 genetically unexplained index patients with >10 colorectal adenomas and 745 controls. In addition, we collected detailed clinical information from index patients and their first-degree relatives (FDRs). Results We found a statistically significant association with two of the variants tested: rs3802842 (at chromosome 11q23, OR=1.60, 95% CI 1.3 to 2.0) and rs4779584 (at chromosome 15q13, OR=1.50, 95% CI 1.2 to 1.9). The majority of index patients (84%) had between 10 and 100 adenomas and 15% had >100 adenomas. Only two index patients (1%), both with >100 adenomas, had FDRs with polyposis. Forty-one per cent of the index patients had one or more FDRs with CRC. Conclusions These SNPs are the first common, low-penetrant variants reported to be associated with adenomatous polyposis not caused by a defect in the APC, MUTYH, POLD1 and POLE genes. Even though familial occurrence of polyposis was very rare, CRC was over-represented in FDRs of polyposis patients and, if confirmed, these relatives will therefore benefit from surveillance.
    Sensitivities of baseline isolates and boscalid-resistant mutants of Alternaria alternata from pistachio to fluopyram, penthiopyrad, and fluxapyroxad
    Avenot, H.F. ; Biggelaar, H. van den; Morgan, D.P. ; Moral, J. ; Joosten, M.H.A.J. ; Michailides, T.J. - \ 2014
    Plant Disease 98 (2014)2. - ISSN 0191-2917 - p. 197 - 205.
    molecular characterization - botrytis-cinerea - inhibiting fungicides - california pistachio - didymella-bryoniae - sdhi fungicides - late blight - mutations - azoxystrobin - populations
    Resistance of Alternaria alternata to boscalid, the first succinate dehydrogenase inhibitor (SDHI) fungicide labeled on pistachio, has become a common occurrence in California pistachio orchards and affects the performance of this fungicide. In this study, we established the baseline sensitivities of A. alternata to the new SDHIs fluopyram, fluxapyroxad, and penthiopyrad and assessed their cross resistance patterns with boscalid. Examination of the effective fungicide concentration that inhibits mycelial growth to 50% relative to the control (EC50) for 50 baseline isolates revealed that the majority were sensitive to boscalid, penthiopyrad, fluopyram, and fluxapyroxad. Analysis of EC50 values for boscalid for 117 A. alternata isolates originating from boscalid-exposed orchards showed that 44, 3, 1, and 69 isolates had sensitive, reduced sensitivity, moderately resistant, and highly resistant boscalid phenotypes, respectively. Molecular investigation of the occurrence of known SDH mutations showed that, among the 69 isolates highly resistant to boscalid, 44, 2, 14, and 1 isolates possessed the mutations leading to the H277Y, H277R, H134R, and H133R amino acid substitutions in AaSDHB, AaSDHB, AaSDHC, and AaSDHD subunits, respectively. Some SDHB or SDHC mutants displayed highly sensitive, sensitive, or reduced sensitivity phenotypes toward penthiopyrad or fluxapyroxad, whereas other had low, moderate, or high levels of resistance to these fungicides. In contrast, all the SDHB mutants were sensitive to fluopyram, while 10, 5, and 1 SDHC mutants had sensitive, reduced sensitivity, and moderately resistant fluopyram phenotypes, respectively. The SDHD mutant had reduced sensitivity to fluopyram and penthiopyrad but was highly resistant to fluxapyroxad. The discrepancies of cross-resistance patterns between SDHIs suggest that their binding sites in complex II may differ slightly and that additional mechanisms of resistance to these compounds are likely involved. Ultimately, the findings of this study should lead to the rational and sustained deployment of new SDHIs in Alternaria late blight spray programs.
    Defining the role of common variation in the genomic and biological architecture of adult human height
    Wood, A.R. ; Esko, T. ; Yang, J. ; Dhonukshe-Rutten, R.A.M. ; Groot, C.P.G.M. de - \ 2014
    Nature Genetics 46 (2014). - ISSN 1061-4036 - p. 1173 - 1186.
    genetic-variation - complex traits - heritability - mutations - snps
    Using genome-wide data from 253,288 individuals, we identified 697 variants at genome-wide significance that together explained one-fifth of the heritability for adult height. By testing different numbers of variants in independent studies, we show that the most strongly associated ~2,000, ~3,700 and ~9,500 SNPs explained ~21%, ~24% and ~29% of phenotypic variance. Furthermore, all common variants together captured 60% of heritability. The 697 variants clustered in 423 loci were enriched for genes, pathways and tissue types known to be involved in growth and together implicated genes and pathways not highlighted in earlier efforts, such as signaling by fibroblast growth factors, WNT/ß-catenin and chondroitin sulfate–related genes. We identified several genes and pathways not previously connected with human skeletal growth, including mTOR, osteoglycin and binding of hyaluronic acid. Our results indicate a genetic architecture for human height that is characterized by a very large but finite number (thousands) of causal variants.
    What cost mitochondria? The maintenance of functional mitochondrial DNA within and across generations
    Aanen, D.K. ; Spelbrink, J.N. ; Beekman, M. - \ 2014
    Philosophical Transactions of the Royal Society B. Biological sciences 369 (2014)1646. - ISSN 0962-8436
    hereditary optic neuropathy - cytoplasmic inheritance - deletion mutants - evolution - mtdna - mutations - selection - disease - genomes - accumulation
    The peculiar biology of mitochondrial DNA (mtDNA) potentially has detrimental consequences for organismal health and lifespan. Typically, eukaryotic cells contain multiple mitochondria, each with multiple mtDNA genomes. The high copy number of mtDNA implies that selection on mtDNA functionality is relaxed. Furthermore, because mtDNA replication is not strictly regulated, within-cell selection may favour mtDNA variants with a replication advantage, but a deleterious effect on cell fitness. The opportunities for selfish mtDNA mutations to spread are restricted by various organism-level adaptations, such as uniparental transmission, germline mtDNA bottlenecks, germline selection and, during somatic growth, regular alternation between fusion and fission of mitochondria. These mechanisms are all hypothesized to maintain functional mtDNA. However, the strength of selection for maintenance of functional mtDNA progressively declines with age, resulting in age-related diseases. Furthermore, organismal adaptations that most probably evolved to restrict the opportunities for selfish mtDNA create secondary problems. Owing to predominantly maternal mtDNA transmission, recombination among mtDNA from different individuals is highly restricted or absent, reducing the scope for repair. Moreover, maternal inheritance precludes selection against mtDNA variants with male-specific effects. We finish by discussing the consequences of life-history differences among taxa with respect to mtDNA evolution and make a case for the use of microorganisms to experimentally manipulate levels of selection
    Whole-genome sequencing of 234 bulls facilitates mapping of monogenic and complex traits in cattle
    Daetwyler, H.D. ; Capitan, A. ; Pausch, H. ; Stothard, P. ; Binsbergen, R. van; Brondum, R.F. ; Liao, X. ; Djari, A. ; Rodriguez, S.C. ; Grohs, C. ; Esquerré, D. ; Bouchez, O. ; Rossignol, M.N. ; Klopp, C. ; Rocha, D. ; Fritz, S. ; Eggen, A. ; Bowman, P.J. ; Coote, D. ; Chamberlain, A.J. ; Anderson, C.L. ; Tassel, C.P. ; Hulsegge, B. ; Goddard, M.E. ; Guldbrandsten, B. ; Lund, M.S. ; Veerkamp, R.F. ; Boichard, D.A. ; Fries, R. ; Hayes, B.J. - \ 2014
    Nature Genetics 46 (2014). - ISSN 1061-4036 - p. 858 - 865.
    boophilus-microplus resistance - mitotic chromosomes - genotype imputation - holstein calves - dairy-cattle - milk-yield - bos-taurus - condensin - mutations - gene
    The 1000 bull genomes project supports the goal of accelerating the rates of genetic gain in domestic cattle while at the same time considering animal health and welfare by providing the annotated sequence variants and genotypes of key ancestor bulls. In the first phase of the 1000 bull genomes project, we sequenced the whole genomes of 234 cattle to an average of 8.3-fold coverage. This sequencing includes data for 129 individuals from the global Holstein-Friesian population, 43 individuals from the Fleckvieh breed and 15 individuals from the Jersey breed. We identified a total of 28.3 million variants, with an average of 1.44 heterozygous sites per kilobase for each individual. We demonstrate the use of this database in identifying a recessive mutation underlying embryonic death and a dominant mutation underlying lethal chrondrodysplasia. We also performed genome-wide association studies for milk production and curly coat, using imputed sequence variants, and identified variants associated with these traits in cattle.
    A mutation breeding program to improve the quality of the oil crop Crambe abyssinica
    Cheng, J. - \ 2014
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Elma Salentijn; Robert van Loo. - Wageningen : Wageningen University - ISBN 9789461739629 - 180
    crambe abyssinica - plantenveredeling - moleculaire veredeling - veredelingsprogramma's - mutaties - olieleverende planten - brandstofgewassen - biobased economy - crambe abyssinica - plant breeding - molecular breeding - breeding programmes - mutations - oil plants - fuel crops - biobased economy
    Since C. abyssinica is considered to be an ideal crop for industrial oil, further improving the value of crambe oil is of importance. The value of crambe oil can be improved by increasing the C22:1 content or reducing polyunsaturated fatty acids (PUFA). To improve crambe oil by a non-GM approach, a mutant population (12,480 lines) was established from the EMS-treated seeds of C. abyssinica cv. ‘Galactica’. Furthermore, the mutants in CaFAD2 were detected by 454-amplicon sequencing and Illumina sequencing.
    Common and rare single nucleotide polymorphisms in the LDLR gene are present in a black South African population and associate with low-density lipoprotein cholesterol levels
    Zyl, T. ; Jerling, J.C. ; Conradie, K.R. ; Feskens, E.J.M. - \ 2014
    Journal of Human Genetics 59 (2014). - ISSN 1434-5161 - p. 88 - 94.
    coronary-heart-disease - plasma-lipid levels - receptor gene - familial hypercholesterolemia - messenger-rna - human genome - mutations - risk - stabilization - expression
    The LDL receptor has an essential role in regulating plasma LDL-C levels. Genetic variation in the LDLR gene can be associated with either lower or moderately raised plasma levels of LDL-C, or may cause familial hypercholesterolemia. The prevalence of single-nucleotide polymorphisms (SNPs) in the LDLR in the black South African population is not known and therefore, we aimed to determine the genotypic variation of the LDLR in the study population as well as to define the association of the different genotypes with plasma LDL-C levels. A random selection of 1860 apparently healthy black South African volunteers aged 35–60 years was made in a cross-sectional study. Novel SNPs were identified in a subset of 30 individuals by means of automated sequencing before screening the entire cohort by means of the Illumina VeraCode GoldenGate Genotyping Assay on a BeadXpress Reader system. Twenty-five SNPs were genotyped, two of which were novel. A very rare SNP, rs17249141, in the promoter region was significantly associated with lower levels of LDL-C. Four other SNPs (rs2738447, rs14158, rs2738465 and rs3180023) were significantly associated with increased levels of LDL-C. We can conclude that some of the various SNPs identified do indeed associate with LDL-C levels
    Antiphase light and temperature cycles affect PHYTOCHROME B-controlled ethylene sensitivity and biosynthesis, limiting leaf movement and growth of Arabidopsis.
    Bours, R.M.E.H. ; Zanten, M. van; Pierik, R. ; Bouwmeester, H.J. ; Krol, A.R. van der - \ 2013
    Plant Physiology 163 (2013)2. - ISSN 0032-0889 - p. 882 - 895.
    gene family - thermoperiodic responses - night temperature - circadian-rhythms - identify ethylene - stem elongation - thaliana - gibberellins - morphology - mutations
    In the natural environment, days are generally warmer than the night, resulting in a positive day/night temperature difference (+DIF). Plants have adapted to these conditions, and when exposed to antiphase light and temperature cycles (cold photoperiod/warm night [-DIF]), most species exhibit reduced elongation growth. To study the physiological mechanism of how light and temperature cycles affect plant growth, we used infrared imaging to dissect growth dynamics under +DIF and -DIF in the model plant Arabidopsis (Arabidopsis thaliana). We found that -DIF altered leaf growth patterns, decreasing the amplitude and delaying the phase of leaf movement. Ethylene application restored leaf growth in -DIF conditions, and constitutive ethylene signaling mutants maintain robust leaf movement amplitudes under -DIF, indicating that ethylene signaling becomes limiting under these conditions. In response to -DIF, the phase of ethylene emission advanced 2 h, but total ethylene emission was not reduced. However, expression analysis on members of the 1-aminocyclopropane-1-carboxylic acid (ACC) synthase ethylene biosynthesis gene family showed that ACS2 activity is specifically suppressed in the petiole region under -DIF conditions. Indeed, petioles of plants under -DIF had reduced ACC content, and application of ACC to the petiole restored leaf growth patterns. Moreover, acs2 mutants displayed reduced leaf movement under +DIF, similar to wild-type plants under -DIF. In addition, we demonstrate that the photoreceptor PHYTOCHROME B restricts ethylene biosynthesis and constrains the -DIF-induced phase shift in rhythmic growth. Our findings provide a mechanistic insight into how fluctuating temperature cycles regulate plant growth.
    The C-terminal domain of chikungunya virus nsP2 independently governs viral RNA replication, cytopathicity, and inhibition of interferon signaling
    Fros, J.J. ; Maten, E. van der; Vlak, J.M. ; Pijlman, G.P. - \ 2013
    Journal of Virology 87 (2013)18. - ISSN 0022-538X - p. 10394 - 10400.
    semliki-forest-virus - nonstructural protein-2 - nuclear-localization - infection - transcription - alphaviruses - translation - expression - mutations - cells
    Alphavirus nonstructural protein 2 (nsP2) has pivotal roles in viral RNA replication, host cell shutoff, and inhibition of antiviral responses. Mutations that individually rendered other alphaviruses noncytopathic were introduced into chikungunya virus nsP2. Results show that (i) nsP2 mutation P718S only in combination with KR649AA or adaptive mutation D711G allowed noncytopathic replicon RNA replication, (ii) prohibiting nsP2 nuclear localization abrogates inhibition of antiviral interferon-induced JAK-STAT signaling, and (iii) nsP2 independently affects RNA replication, cytopathicity, and JAK-STAT signaling
    Functional analysis of the omega-6 fatty acid desaturase (CaFAD2) gene family of the oil seed crop Crambe abyssinica using RNAi-mediated gene silencing
    Cheng, J. ; Zhu, L. ; Salentijn, E.M.J. ; Huang, B. ; Gruber, J. ; Dechesne, A.C. ; Krens, F.A. ; Qi, W. ; Visser, R.G.F. ; Loo, E.N. van - \ 2013
    BMC Plant Biology 13 (2013). - ISSN 1471-2229
    high oleic-acid - erucic-acid - brassica-napus - fad2 gene - fae1 gene - carrier protein - expression - suppression - metabolism - mutations
    Background Crambe abyssinica produces high erucic acid (C22:1, 55-60 %) in the seed oil, which can be further increased by reduction of polyunsaturated fatty acid (PUFA) levels. The omega-6 fatty acid desaturase enzyme (FAD2) is known to be involved in PUFA biosynthesis. In crambe, three CaFAD2 genes, CaFAD2-C1, CaFAD2-C2 and CaFAD2-C3 are expressed. Results The individual effect of each CaFAD2 gene on oil composition was investigated through studying transgenic lines (CaFAD2-RNAi) for differential expression levels in relation to the composition of seed-oil. Six first generation transgenic plants (T1) showed C18:1 increase (by 6% to 10.5 %) and PUFA reduction (by 8.6% to 10.2 %). The silencing effect in these T1-plants ranged from the moderate silencing (40% to 50% reduction) of all three CaFAD2 genes to strong silencing (95% reduction) of CaFAD2-C3 alone. The progeny of two T1-plants (WG4-4 and WG19-6) was further analysed. Four or five transgene insertions are characterized in the progeny (T2) of WG19-6 in contrast to a single insertion in the T2 progeny of WG4-4. For the individual T2-plants of both families (WG19-6 and WG4-4), seed-specific silencing of CaFAD2-C1 and CaFAD2-C2 was observed in several individual T2-plants but, on average in both families, the level of silencing of these genes was not significant. A significant reduction in expression level (P <0.01) in both families was only observed for CaFAD2-C3 together with significantly different C18:1 and PUFA levels in oil. Conclusions CaFAD2-C3 expression is highly correlated to levels of C18:1 (r = -0.78) and PUFA (r = 0.75), which suggests that CaFAD2-C3 is the most important one for changing the oil composition of crambe.
    Onderzoek naar de resistentie van de bruine rat in Nederland - 2012
    Lee, T.A.J. van der; Gent-Pelzer, M.P.E. van; Schilder, H. ; Huis in 'T Veld, J.W.H. ; Meerburg, B.G. - \ 2013
    Lelystad : Wageningen UR Livestock Research (Rapport / Wageningen UR Livestock Research 690) - 21
    rattus norvegicus - resistentie tegen rodenticiden - feces - genetische verandering - mutaties - rattus norvegicus - rodenticide resistance - faeces - genetic change - mutations
    Development and application of a quick test for resistance of brown rats against rodenticides.
    Mechanism-Based Covalent Neuraminidase Inhibitors with Broad Spectrum Influenza Antiviral Activity
    Kim, J.H. ; Resende, R. ; Wennekes, T. ; Chen, N. ; Bance, N. ; Buchini, S. ; Watts, A.G. ; Pilling, P. ; Streltsov, V.A. ; Petric, M. ; Liggins, R. ; Barrett, S. ; McKimm-Breschkin, J.L. ; Niikura, M. ; Withers, S.G. - \ 2013
    Science 340 (2013)6128. - ISSN 0036-8075 - p. 71 - 75.
    virus neuraminidase - drug design - h1n1 virus - resistant - sialidase - 4-guanidino-neu5ac2en - sensitivity - oseltamivir - mutations - variant
    Influenza antiviral agents play important roles in modulating disease severity and in controlling pandemics while vaccines are prepared, but the development of resistance to agents like the commonly used neuraminidase inhibitor oseltamivir may limit their future utility. We report here a new class of specific, mechanism-based anti-influenza drugs that function via the formation of a stabilized covalent intermediate in the influenza neuraminidase enzyme, and confirm this mode of action via structural and mechanistic studies. These compounds function in cell-based assays and in animal models, with efficacies comparable to that of the neuraminidase inhibitor zanamivir and with broad spectrum activity against drug-resistant strains in vitro. The similarity of their structure to that of the natural substrate and their mechanism-based design make these attractive antiviral candidates
    Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data
    Bryant, J.M. ; Schürch, A.C. ; Deutekom, H. van; Harris, S.R. ; Beer, J.L. de; Jager, V.C.L. de; Kremer, K. ; Hijum, S.A.F.T. van; Siezen, R.J. ; Borgdorff, M. ; Bentley, S.D. ; Parkhill, J. ; Soolingen, D. van - \ 2013
    Bmc Infectious Diseases 13 (2013)1. - ISSN 1471-2334
    resistance - strains - mutations - evolution - epidemiology - complex - gene
    BACKGROUND: Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of the rate of change in the genome over relevant timescales is required. METHODS: We attempted to estimate a molecular clock by sequencing 199 isolates from epidemiologically linked tuberculosis cases, collected in the Netherlands spanning almost 16 years. RESULTS: Multiple analyses support an average mutation rate of ~0.3 SNPs per genome per year. However, all analyses revealed a very high degree of variation around this mean, making the confirmation of links proposed by epidemiology, and inference of novel links, difficult. Despite this, in some cases, the phylogenetic context of other strains provided evidence supporting the confident exclusion of previously inferred epidemiological links. CONCLUSIONS: This in-depth analysis of the molecular clock revealed that it is slow and variable over short time scales, which limits its usefulness in transmission studies. However, the superior resolution of whole genome sequencing can provide the phylogenetic context to allow the confident exclusion of possible transmission events previously inferred via traditional DNA fingerprinting techniques and epidemiological cluster investigation. Despite the slow generation of variation even at the whole genome level we conclude that the investigation of tuberculosis transmission will benefit greatly from routine whole genome sequencing
    Identification of a complete set of functional markers for the selection of er1 powdery mildew resistance in Pisum sativum L.
    Pavan, S.N.C. ; Schiavulli, A. ; Appiano, M. ; Miacola, C. ; Visser, R.G.F. ; Bai, Y. ; Lotti, C. ; Ricciardi, L. - \ 2013
    Molecular Breeding 31 (2013)1. - ISSN 1380-3743 - p. 247 - 253.
    resolution melting analysis - scar markers - pea - gene - specificity - sensitivity - mutations - barley - plants - snps
    Powdery mildew is the most widespread disease of pea (Pisum sativum L.) and causes severe economic losses worldwide. Recessively inherited er1 powdery mildew resistance, successfully used for decades in pea breeding programs, has recently been shown to originate from the loss of function of the PsMLO1 gene. Five er1 alleles, each corresponding to a different PsMLO1 null mutation, have been characterized to date in pea germplasm. In order to aid er1 selection, we aimed to identify functional markers which target PsMLO1 polymorphisms directly responsible for the resistant phenotype. Highly informative cleaved amplified polymorphic sequence (CAPS), derived cleaved amplified polymorphic sequence (dCAPS), sequence tagged site (STS) and highresolution melting (HRM) markers were developed which enable the selection of each of the five er1 alleles. Taken together, the results described here provide a powerful tool for breeders, overcoming limitations of previously reported er1-linked markers due to the occurrence of recombination with the resistance locus and/or the lack of polymorphism between parental genotypes. The HRM marker er1-5/HRM54 reported here, targeting a mutagenesisinduced er1 allele recently described by us, does not require manual processing after PCR amplification, and is therefore suitable for large-scale breeding programs based on high-throughput automated screening.
    Promoter propagation in prokaryotes
    Matus-Garcia, M. ; Nijveen, H. ; Passel, M.W.J. van - \ 2012
    Nucleic acids research 40 (2012)20. - ISSN 0305-1048 - p. 10032 - 10040.
    horizontal gene-transfer - escherichia-coli - adaptive evolution - lactococcus-lactis - sequences - genomes - networks - identification - mutations - families
    Transcriptional activation or 'rewiring' of silent genes is an important, yet poorly understood, phenomenon in prokaryotic genomes. Anecdotal evidence coming from experimental evolution studies in bacterial systems has shown the promptness of adaptation upon appropriate selective pressure. In many cases, a partial or complete promoter is mobilized to silent genes from elsewhere in the genome. We term hereafter such recruited regulatory sequences as Putative Mobile Promoters (PMPs) and we hypothesize they have a large impact on rapid adaptation of novel or cryptic functions. Querying all publicly available prokaryotic genomes (1362) uncovered >4000 families of highly conserved PMPs (50 to 100 long with =80% nt identity) in 1043 genomes from 424 different genera. The genomes with the largest number of PMP families are Anabaena variabilis (28 families), Geobacter uraniireducens (27 families) and Cyanothece PCC7424 (25 families). Family size varied from 2 to 93 homologous promoters (in Desulfurivibrio alkaliphilus). Some PMPs are present in particular species, but some are conserved across distant genera. The identified PMPs represent a conservative dataset of very recent or conserved events of mobilization of non-coding DNA and thus they constitute evidence of an extensive reservoir of recyclable regulatory sequences for rapid transcriptional rewiring
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