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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    Guide-free Cas9 from pathogenic Campylobacter jejuni bacteria causes severe damage to DNA
    Saha, Chinmoy ; Mohanraju, Prarthana ; Stubbs, Andrew ; Dugar, Gaurav ; Hoogstrate, Youri ; Kremers, Gert Jan ; Cappellen, Wiggert A. Van; Horst-Kreft, Deborah ; Laffeber, Charlie ; Lebbink, Joyce H.G. ; Bruens, Serena ; Gaskin, Duncan ; Beerens, Dior ; Klunder, Maarten ; Joosten, Rob ; Demmers, Jeroen A.A. ; Gent, Dik Van; Mouton, Johan W. ; Spek, Peter J. Van Der; Oost, John Van Der; Baarlen, Peter Van; Louwen, Rogier - \ 2020
    Science Advances 6 (2020)25. - ISSN 2375-2548

    CRISPR-Cas9 systems are enriched in human pathogenic bacteria and have been linked to cytotoxicity by an unknown mechanism. Here, we show that upon infection of human cells, Campylobacter jejuni secretes its Cas9 (CjeCas9) nuclease into their cytoplasm. Next, a native nuclear localization signal enables CjeCas9 nuclear entry, where it catalyzes metal-dependent nonspecific DNA cleavage leading to cell death. Compared to CjeCas9, native Cas9 of Streptococcus pyogenes (SpyCas9) is more suitable for guide-dependent editing. However, in human cells, native SpyCas9 may still cause some DNA damage, most likely because of its ssDNA cleavage activity. This side effect can be completely prevented by saturation of SpyCas9 with an appropriate guide RNA, which is only partially effective for CjeCas9. We conclude that CjeCas9 plays an active role in attacking human cells rather than in viral defense. Moreover, these unique catalytic features may therefore make CjeCas9 less suitable for genome editing applications.

    Genome editing by natural and engineered CRISPR-associated nucleases
    Wu, Wen Y. ; Lebbink, Joyce H.G. ; Kanaar, Roland ; Geijsen, Niels ; Oost, John van der - \ 2018
    Nature Chemical Biology 14 (2018)7. - ISSN 1552-4450 - p. 642 - 651.

    Over the last decade, research on distinct types of CRISPR systems has revealed many structural and functional variations. Recently, several novel types of single-polypeptide CRISPR-associated systems have been discovered including Cas12a/Cpf1 and Cas13a/C2c2. Despite distant similarities to Cas9, these additional systems have unique structural and functional features, providing new opportunities for genome editing applications. Here, relevant fundamental features of natural and engineered CRISPR-Cas variants are compared. Moreover, practical matters are discussed that are essential for dedicated genome editing applications, including nuclease regulation and delivery, target specificity, as well as host repair diversity.

    Semi-quantitative proteomics of mammalian cells upon short-term exposure to nonionizing electromagnetic fields
    Kuzniar, Arnold ; Laffeber, Charlie ; Eppink, Berina ; Bezstarosti, Karel ; Dekkers, Dick ; Woelders, Henri ; Zwamborn, A.P.M. ; Demmers, Jeroen ; Lebbink, Joyce H.G. ; Kanaar, Roland - \ 2017
    PLoS ONE 12 (2017)2. - ISSN 1932-6203

    The potential effects of non-ionizing electromagnetic fields (EMFs), such as those emitted by power-lines (in extremely low frequency range), mobile cellular systems and wireless networking devices (in radio frequency range) on human health have been intensively researched and debated. However, how exposure to these EMFs may lead to biological changes underlying possible health effects is still unclear. To reveal EMF-induced molecular changes, unbiased experiments (without a priori focusing on specific biological processes) with sensitive readouts are required. We present the first proteome-wide semi-quantitative mass spectrometry analysis of human fibroblasts, osteosarcomas and mouse embryonic stem cells exposed to three types of non-ionizing EMFs (ELF 50 Hz, UMTS 2.1 GHz and WiFi 5.8 GHz). We performed controlled in vitro EMF exposures of metabolically labeled mammalian cells followed by reliable statistical analyses of differential protein-and pathway-level regulations using an array of established bioinformatics methods. Our results indicate that less than 1% of the quantitated human or mouse proteome responds to the EMFs by small changes in protein abundance. Further network-based analysis of the differentially regulated proteins did not detect significantly perturbed cellular processes or pathways in human and mouse cells in response to ELF, UMTS or WiFi exposure. In conclusion, our extensive bioinformatics analyses of semi-quantitative mass spectrometry data do not support the notion that the short-time exposures to non-ionizing EMFs have a consistent biologically significant bearing on mammalian cells in culture.

    Pathogenicity of Bovine Neonatal Pancytopenia-associated vaccine-induced alloantibodies correlates with Major Histocompatibility Complex class 1 expression
    Benedictus, L. ; Luteijn, Rutger D. ; Otten, H. ; Lebbink, Robert Jan ; Kooten, P.J.S. van; Wiertz, E.J.H.J. ; Rutten, Victor P.M.G. ; Koets, A.P. - \ 2015
    Scientific Reports 5 (2015). - ISSN 2045-2322
    Bovine Neonatal Pancytopenia (BNP), a fatal bleeding syndrome of neonatal calves, is caused by maternal alloantibodies absorbed from colostrum and is characterized by lymphocytopenia, thrombocytopenia and bone marrow hypoplasia. An inactivated viral vaccine is the likely source of alloantigens inducing BNP-associated alloantibodies in the dam. In this study the specificity of BNP alloantibodies was assessed and was linked to the pathology of BNP. We demonstrated that Major Histocompatibility Complex class I (MHC I) and Very Late Antigen-3, an integrin α3/β1 heterodimer, were the major targets of BNP alloantibodies. However, alloantibody binding to various bovine cell types correlated with MHC I expression, rather than integrin β1 or α3 expression. Likewise, alloantibody-dependent complement-mediated cell lysis correlated strongly with MHC I expression. Examination of several tissues of third trimester bovine foetuses revealed that cells, shown to be affected in calves with BNP, were characterized by high MHC class I expression and high levels of alloantibody binding. We conclude that in spite of the heterogeneous specificity of BNP associated maternal alloantibodies, MHC I-specific antibodies mediate the pathogenicity of BNP in the calf and that cells with high MHC I expression were preferentially affected in BNP.
    Kansen voor vrouwelijk talent : over carrières en barrières van vrouwen bij Wageningen UR
    Lebbink, S. ; Steuten, C. ; Neefjes, M. - \ 2013
    Wageningen : Wageningen UR, Wetenschapswinkel (Rapport / Wageningen UR, Wetenschapswinkel 292) - 42 p.
    vrouwen - universiteiten - positie van de vrouw - gelijke behandeling van de vrouw - gelderland - carrièreontwikkeling - carrièreplanning - women - universities - woman - female equality - gelderland - career development - career planning
    Dit rapport inventariseert de situatie van de doorstroming van vrouwelijke wetenschappers aan Wageningen UR. Zo blijkt er binnen Wageningen University een dik glazen plafond te bestaan, te meten aan de doorstroom van de ene functieschaal naar de volgende. In dit rapport beschrijven we een aantal oorzaken van dit probleem. Tenslotte geven we adviezen voor wat bestuurders en leidinggevenden kunnen doen.
    Verslag Veldwerkplaats Ontwikkeling en behoud van gradiënten in het stuifzandlandschap, (dz11-b)
    Klein Lebbink, E. ; Riksen, M.J.P.M. ; Sparrius, L. ; Nijssen, M. - \ 2011
    Bosschap - 4 p.
    Effecten van brand in een voedselarm dennenbos
    Kemmers, R.H. ; Dirkse, G.M. ; Mekkink, P. - \ 2005
    Vakblad Natuur Bos Landschap 2 (2005)3. - ISSN 1572-7610 - p. 6 - 10.
    naaldbossen - zandgronden - bosbranden - vegetatie - bodemchemie - nitraten - coniferous forests - sandy soils - forest fires - vegetation - soil chemistry - nitrates
    Door atmosferische stikstofdepositie zijn veel dennenbossen van de arme zandgronden vermest en in hun successie verstoord. Bij Kootwijk is in een voedselarm dennenbos onderzocht of de overmaat aan stikstof die in de strooisellaag lag opgeslagen door brand kan worden teruggedrongen en de bosontwikkeling zo kan worden teruggezet naar een voedselarme pionierfase. Op grond van de resultaten lijkt het twijfelachting dat brand effectief is als maatregel tegen vermesting. Hoofdlijnen uit Alterra rapport 1028; gevolgd door de visie van de beheerder (Staatsbosbeheer) in de persoon van Klein Lebbink
    Persistence of tertiary structure in 7.9M guanidinium chloride : the case of endo B-1,3-glucanase from Pyrococcus furiosus
    Chiaraluce, R. ; Oost, J. van der; Lebbink, J.H.G. ; Kaper, T. ; Consalvi, V. - \ 2002
    Biochemistry 41 (2002). - ISSN 0006-2960 - p. 14624 - 14632.
    The Pyrococcus furiosus endo--1,3-glucanase belongs to the subfamily of laminarinase, which can be classified as "all proteins" as confirmed by deconvolution of far-UV CD and FTIR spectra. The persistence of a significant amount of tertiary structure in 7.9 M GdmCl, as indicated by near-UV CD spectroscopy, accompanied by a red-shift of the maximum fluorescence emission wavelength is a peculiar property of this hyperthermophilic endoglucanase. The possibility to observe tertiary structure elements under extremely denaturing conditions is notable and is limited to only a few examples. The unusual resistance toward guanidinium chloride denaturation is paralleled by a notable stability at extremely low pH and at high temperature. The analysis of the protein spectral properties indicates that the secondary structure elements are preserved down to pH 1.0 and up to 90 C at pH 7.4 and pH 3.0. The study of the conditions that determine the persistence of residual structure at high denaturant concentration and the examination of these structures are particularly interesting because these state(s) may be preliminary or coincident with the coalescence of protein aggregates or to the formation of amyloid-like fibrils, and they may serve as seeds of protein folding.
    The Sulfolobus solfataricus Lrp-like protein LysM regulates lysine biosynthesis in response to lysine availability
    Brinkman, A.B. ; Bell, S.D. ; Lebbink, R.J. ; Vos, W.M. de; Oost, J. van der - \ 2002
    Journal of Biological Chemistry 277 (2002). - ISSN 0021-9258 - p. 29537 - 29549.
    Although the archaeal transcription apparatus resembles the eukaryal RNA polymerase II system, many bacterial-like regulators can be found in archaea. Particularly, all archaeal genomes sequenced to date contain genes encoding homologues of Lrp (leucine-responsive regulatory protein). Whereas Lrp-like proteins in bacteria are involved in regulation of amino acid metabolism, their physiological role in archaea is unknown. Although several archaeal Lrp-like proteins have been characterized recently, no target genes apart from their own coding genes have been discovered yet, and no ligands for these regulators have been identified so far. In this study, we show that the Lrp-like protein LysM from Sulfolobus solfataricus is involved in the regulation of lysine and possibly also arginine biosynthesis, encoded by the lys gene cluster. Exogenous lysine is the regulatory signal for lys gene expression and specifically serves as a ligand for LysM by altering its DNA binding affinity. LysM binds directly upstream of the TFB-responsive element of the intrinsically weak lysW promoter, and DNA binding is favored in the absence of lysine, when lysWXJK transcription is maximal. The combined in vivo and in vitro data are most compatible with a model in which the bacterial-like LysM activates the eukarya-like transcriptional machinery. As with transcriptional activation by Escherichia coli Lrp, activation by LysM is apparently dependent on a co-activator, which remains to be identified.
    Characterization of a unique nucleopolyhedrovirus gene, orf 17/18 of Spodoptera exigua MNPV, with a homolog in a distantly related granulovirus
    IJkel, W.F.J. ; Lebbink, R.J. ; Goldbach, R.W. ; Vlak, J.M. ; Zuidema, D. - \ 2002
    In: Proceedings of the 21st Annual meeting of the American Society for Virology, Lexington, 2002 Lexington : - p. 134 - 134.
    The ß-glucosidase CelB from Pyrococcus furiosus: Production by Escherichia coli, purification, and in vitro evolution
    Lebbink, J.H.G. ; Kaper, T. ; Kengen, S.W.M. ; Oost, J. van der; Vos, W.M. de - \ 2001
    Methods in Enzymology 330 (2001). - ISSN 0076-6879 - p. 364 - 379.
    Characterization of ß-glycosyl hydrolases from Pyrococcus furiosus
    Kaper, T. ; Verhees, C.H. ; Lebbink, J.H.G. ; Lieshout, J.F.T. van; Kluskens, L.D. ; Ward, D.E. - \ 2001
    Methods in Enzymology 330 (2001). - ISSN 0076-6879 - p. 329 - 346.
    Spodoptera exigua multicapsid nucleopolyhedrovirus encodes a 23 KDA ODV-specific protein linked to nucleocapsid
    IJkel, W.F.J. ; Lebbink, R.J. ; Brouw, M.L. op den; Goldbach, R.W. ; Vlak, J.M. ; Zuidema, D. - \ 2001
    In: 20th Annual Meeting of the American Society for Virology : ASV 2001, Wisconsin - 2001 Wisconsin : University of Wisconsin-Madison - p. 83 - 83.
    Identification of a novel occlusion derived virus-specific protein in Spodoptera exigua multicapsid nucleopolyhedrovirus
    IJkel, W.F.J. ; Lebbink, R.J. ; Brouw, M.L. op den; Goldbach, R.W. ; Vlak, J.M. ; Zuidema, D. - \ 2001
    Virology 284 (2001). - ISSN 0042-6822 - p. 170 - 181.
    Understanding the molecular basis of the distinct biological properties of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV), such as its narrow host range and high virulence, requires detailed information on the temporal expression and subcellular localization of SeMNPV gene products. The expression of two unique SeMNPV ORFs, 116 (Se116) and 117 (Se117), which show 45 mino acid similarity, was analyzed. Se116 and Se117 were expressed both in cultured cells and in larvae of S. exigua as polyadenylated transcripts of 0.80 and 0.75 kb, respectively. These transcripts initiated from ATCA(G/T)T promoter motifs, commonly found for baculovirus early genes. Se116 transcripts were detected with increasing abundance from 8 to 48 h p.i., whereas Se117 transcripts were present from 4 h p.i. and most abundantly at 24 h p.i. Western blot analysis of infected Se301 cells revealed 27- and 23-kDa proteins for Se116 and Se117, respectively. C-terminal GFP–fusion proteins of Se116 and Se117 were primarily localized in the nucleus of Se301 cells. When Se301 cells were infected with SeMNPV, both GFP–fusion proteins were localized in the virogenic stroma of the nucleus. While the function of the Se116 protein is still enigmatic, the Se117 protein appeared to be a structural protein associated with nucleocapsids of occlusion-derived SeMNPV virions but not of budded virus.
    Activity and stability of hyperthermophilic enzymes : a comparative study on two archaeal b-glycosidases
    Pouwels, J. ; Moracci, M. ; Cobucci-Ponzano, B. ; Perugino, G. ; Oost, J. van der; Kaper, T. ; Lebbink, J.H.G. ; Vos, W.M. de; Ciaramella, M. ; Rossi, M. - \ 2000
    Extremophiles 4 (2000). - ISSN 1431-0651 - p. 157 - 164.
    Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus b-glucosidase CelB by directed evolution
    Lebbink, J.H.G. ; Kaper, T. ; Bron, P. ; Oost, J. van der; Vos, W.M. de - \ 2000
    Biochemistry 39 (2000). - ISSN 0006-2960 - p. 3656 - 3665.
    The -glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus (CelB) is the most thermostable and thermoactive family 1 glycosylhydrolase described to date. To obtain more insight in the molecular determinants of adaptations to high temperatures and study the possibility of optimizing low-temperature activity of a hyperthermostable enzyme, we generated a library of random CelB mutants in Escherichia coli. This library was screened for increased activity on p-nitrophenyl--D-glucopyranoside at room temperature. Multiple CelB variants were identified with up to 3-fold increased rates of hydrolysis of this aryl glucoside, and 10 of them were characterized in detail. Amino acid substitutions were identified in the active-site region, at subunit interfaces, at the enzyme surface, and buried in the interior of the monomers. Characterization of the mutants revealed that the increase in low-temperature activity was achieved in different ways, including altered substrate specificity and increased flexibility by an apparent overall destabilization of the enzyme. Kinetic characterization of the active-site mutants showed that in all cases the catalytic efficiency at 20 C on p-nitrophenyl--D-glucose, as well as on the disaccharide cellobiose, was increased up to 2-fold. In most cases, this was achieved at the expense of -galactosidase activity at 20 C and total catalytic efficiency at 90 C. Substrate specificity was found to be affected by many of the observed amino acid substitutions, of which only some are located in the vicinity of the active site. The largest effect on substrate specificity was observed with the CelB variant N415S that showed a 7.5-fold increase in the ratio of p-nitrophenyl--D-glucopyranoside/p-nitrophenyl--D-galactopyranoside hydrolysis. This asparagine at position 415 is predicted to interact with active-site residues that stabilize the hydroxyl group at the C4 position of the substrate, the conformation of which is equatorial in glucose-containing substrates and axial in galactose-containing substrates.
    Comparative structural analysis and substrate specificity engineering of the hyperthermostable b-glucosidase CelB from Pyrococcus furiosus
    Kaper, T. ; Lebbink, J.H.G. ; Pouwels, J. ; Kopp, J. ; Schulz, G.E. ; Oost, J. van der; Vos, W.M. de - \ 2000
    Biochemistry 39 (2000). - ISSN 0006-2960 - p. 4963 - 4970.
    The substrate specificity of the -glucosidase (CelB) from the hyperthermophilic archaeon Pyrococcus furiosus, a family 1 glycosyl hydrolase, has been studied at a molecular level. Following crystallization and X-ray diffraction of this enzyme, a 3.3 Å resolution structural model has been obtained by molecular replacement. CelB shows a homo-tetramer configuration, with subunits having a typical ()8-barrel fold. Its active site has been compared to the one of the previously determined 6-phospho--glycosidase (LacG) from the mesophilic bacterium Lactococcus lactis. The overall design of the substrate binding pocket is very well conserved, with the exception of three residues that have been identified as a phosphate binding site in LacG. To verify the structural model and alter its substrate specificity, these three residues have been introduced at the corresponding positions in CelB (E417S, M424K, F426Y) in different combinations: single, double, and triple mutants. Characterization of the purified mutant CelB enzyme revealed that F426Y resulted in an increased affinity for galactosides, whereas M424K gave rise to a shifted pH optimum (from 5.0 to 6.0). Analysis of E417S revealed a 5-fold and a 3-fold increase of the efficiency of hydrolyzing o-nitrophenol--D-galactopyranoside-6-phosphate, in the single and triple mutants, respectively. In contrast, their activity on nonphosphorylated sugars was largely reduced (30-300-fold). The residue at position E417 in CelB seems to be the determining factor for the difference in substrate specificity between the two types of family 1 glycosidases.
    The transcriptional regulator LrpA from the hyperthermophilic archaeon Pyrococcus furiosus is negatively autoregulated
    Brinkman, A.B. ; Dahlke, I. ; Tuininga, J.E. ; Lammers, T. ; Dumay, V. ; Heus, E. de; Lebbink, J.H.G. ; Thomm, M. ; Vos, W.M. de; Oost, J. van der - \ 2000
    Journal of Biological Chemistry 275 (2000). - ISSN 0021-9258 - p. 38160 - 38169.
    The archaeal transcriptional initiation machinery closely resembles core elements of the eukaryal polymerase II system. However, apart from the established basal archaeal transcription system, little is known about the modulation of gene expression in archaea. At present, no obvious eukaryal-like transcriptional regulators have been identified in archaea. Instead, we have previously isolated an archaeal gene, the Pyrococcus furiosus lrpA, that potentially encodes a bacterial-like transcriptional regulator. In the present study, we have for the first time addressed the actual involvement of an archaeal Lrp homologue in transcription modulation. For that purpose, we have produced LrpA in Escherichia coli. In a cell-free P. furiosus transcription system we used wild-type and mutated lrpA promoter fragments to demonstrate that the purified LrpA negatively regulates its own transcription. In addition, gel retardation analyses revealed a single protein-DNA complex, in which LrpA appeared to be present in (at least) a tetrameric conformation. The location of the LrpA binding site was further identified by DNaseI and hydroxyl radical footprinting, indicating that LrpA binds to a 46-base pair sequence that overlaps the transcriptional start site of its own promoter. The molecular basis of the transcription inhibition by LrpA is discussed.
    Glutamate dehydrogenase from hyperthermophilic bacteria and archaea: determinants of thermostability and catalysis at extremely high temperatures
    Lebbink, J.H.G. ; Kengen, S.W.M. ; Oost, J. van der; Vos, W.M. de - \ 1999
    Journal of Molecular Catalysis. B, Enzymatic 7 (1999). - ISSN 1381-1177 - p. 133 - 145.
    Engineering activity and stability of Thermotoga maritima glutamate dehydrogenase. II: Construction of a 16-residue ion-pair network at the subunit interface
    Lebbink, J.H.G. ; Knapp, S. ; Oost, J. van der; Rice, D. ; Ladenstein, R. ; Vos, W.M. de - \ 1999
    Journal of Molecular Biology 289 (1999). - ISSN 0022-2836 - p. 357 - 369.
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