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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    The Arabidopsis lectin receptor kinase LecRK-I.9 enhances resistance to Phytophthora infestans in Solanaceous plants
    Bouwmeester, K. ; Han, M. ; Blanco-Portales, R. ; Song, W. ; Weide, R. ; Guo, L.Y. ; Vossen, E.A.G. van der; Govers, F. - \ 2014
    Plant Biotechnology Journal 12 (2014)1. - ISSN 1467-7644 - p. 10 - 16.
    interfamily transfer - potato cultivars - plasma-membrane - tomato ve1 - activation - expression - infection - proteins - lacking - gene
    Phytophthora species are notorious plant pathogens which cause a variety of devastating crop diseases. Phytophthora pathogens secrete a plethora of effector proteins, several of which are known to interact with receptors in the host cell thereby either activating or suppressing defense responses. Unlike animals, plants lack an adaptive immune system; however, they are not defenseless and have acquired other mechanisms to withstand pathogens. Receptor proteins play important roles in sensing alterations at the plant cell wall and in mediating responses upon pathogen attack. This paper focuses on the Arabidopsis lectin receptor kinase LecRK-I.9, a mediator of cell wall – plasma membrane (CW-PM) adhesions that is known to bind in vitro to the Phytophthora infestans effector IPI-O via the cell attachment motif RGD. T-DNA mutants deficient in LecRK-I.9 and transgenic Arabidopsis lines expressing ipiO1 were found to behave as phenocopies. Both show a ‘gain-of-susceptibility’ phenotype towards the Arabidopsis pathogen Phytophthora brassicae and are disturbed in callose deposition. Overall, the results suggest that destabilizing the CW-PM continuum is a strategy for Phytophthora to promote infection. As countermeasure, the host may want to strengthen CW-PM adhesions, and the novel resistance component LecRK-I.9 apparently functions in this process
    A SCARECROW-RETINOBLASTOMA Protein Network Controls Protective Quiescence in the Arabidopsis Root Stem Cell Organizer
    Cruz-Ramirez, A. ; Diaz Trivino, S. ; Wachsman, G. ; Du, Y. ; Arteága-Vázquez, M. ; Zhang Hongtao, ; Benjamins, R. ; Blilou, I. ; Neef, A.B. ; Chandler, V. ; Scheres, B. - \ 2013
    PloS Biology 11 (2013)11. - ISSN 1545-7885 - 12 p.
    thaliana root - replication stress - cycle progression - clonal analysis - self-renewal - dna-damage - in-vivo - division - meristem - gene
    Quiescent long-term somatic stem cells reside in plant and animal stem cell niches. Within the Arabidopsis root stem cell population, the Quiescent Centre (QC), which contains slowly dividing cells, maintains surrounding short-term stem cells and may act as a long-term reservoir for stem cells. The RETINOBLASTOMA-RELATED (RBR) protein cell-autonomously reinforces mitotic quiescence in the QC. RBR interacts with the stem cell transcription factor SCARECROW (SCR) through an LxCxE motif. Disruption of this interaction by point mutation in SCR or RBR promotes asymmetric divisions in the QC that renew short-term stem cells. Analysis of the in vivo role of quiescence in the root stem cell niche reveals that slow cycling within the QC is not needed for structural integrity of the niche but allows the growing root to cope with DNA damage
    Single Mutation in Shine-Dalgarno-Like Sequence Present in the Amino Terminal of Lactate Dehydrogenase of Plasmodium Effects the Production of an Eukaryotic Protein Expressed in a Prokaryotic System
    Cicek, M. ; Mutlu, O. ; Erdemir, A. ; Ozkan, E. ; Saricay, Y. ; Turgut-Balik, D. - \ 2013
    Molecular Biotechnology 54 (2013)2. - ISSN 1073-6085 - p. 602 - 608.
    human malaria parasite - escherichia-coli - messenger-rna - recombinant proteins - bacillus-subtilis - ribosomal-rna - q-beta - falciparum - genome - gene
    One of the most important step in structure-based drug design studies is obtaining the protein in active form after cloning the target gene. In one of our previous study, it was determined that an internal Shine-Dalgarno-like sequence present just before the third methionine at N-terminus of wild type lactate dehydrogenase enzyme of Plasmodium falciparum prevent the translation of full length protein. Inspection of the same region in P. vivax LDH, which was overproduced as an active enzyme, indicated that the codon preference in the same region was slightly different than the codon preference of wild type PfLDH. In this study, 5'-GGAGGC-3' sequence of P. vivax that codes for two glycine residues just before the third methionine was exchanged to 5'-GGAGGA-3', by mimicking P. falciparum LDH, to prove the possible effects of having an internal SD-like sequence when expressing an eukaryotic protein in a prokaryotic system. Exchange was made by site-directed mutagenesis. Results indicated that having two glycine residues with an internal SD-like sequence (GGAGGA) just before the third methionine abolishes the enzyme activity due to the preference of the prokaryotic system used for the expression. This study emphasizes the awareness of use of a prokaryotic system to overproduce an eukaryotic protein.
    Phyllotaxis and Rhizotaxis in Arabidopsis Are Modified by Three PLETHORA Transcription Factors
    Hofhuis, H. ; Laskowski, M. ; Du, Y.J. ; Prasad, K. ; Grigg, S. ; Pinon, V. ; Scheres, B. - \ 2013
    Current Biology 23 (2013)11. - ISSN 0960-9822 - p. 956 - 962.
    lateral root initiation - auxin transport - thaliana - growth - arf19 - gene - expression - nph4/arf7 - proteins - system
    Background: The juxtaposition of newly formed primordia in the root and shoot differs greatly, but their formation in both contexts depends on local accumulation of the signaling molecule auxin. Whether the spacing of lateral roots along the main root and the arrangement of leaf primordia at the plant apex are controlled by related underlying mechanisms has remained unclear. Results: Here, we show that, in Arabidopsis thaliana, three transcriptional regulators implicated in phyllotaxis, PLETHORA3 (PLT3), PLT5, and PLT7, are expressed in incipient lateral root primordia where they are required for primordium development and lateral root emergence. Furthermore, all three PLT proteins prevent the formation of primordia close to one another, because, in their absence, successive lateral root primordia are frequently grouped in close longitudinal or radial clusters. The triple plt mutant phenotype is rescued by PLT-vYFP fusion proteins, which are expressed in the shoot meristem as well as the root, but not by expression of PLT7 in the shoot alone. Expression of all three PLT genes requires auxin response factors ARF7 and ARF19, and the reintroduction of PLT activity suffices to rescue lateral root formation in arf7,arf19. Conclusions: Intriguingly PLT 3, PLT5, and PLT7 not only control the positioning of organs at the shoot meristem but also in the root; a striking observation that raises many evolutionary questions.
    Evolution of uni- and bifactorial sexual compatibility systems in fungi
    Nieuwenhuis, B.P.S. ; Billiard, S. ; Vuilleumier, S. ; Petit, E. ; Hood, M.E. ; Giraud, T. - \ 2013
    Heredity 111 (2013)6. - ISSN 0018-067X - p. 445 - 455.
    mating-type locus - schizophyllum-commune - microbotryum-violaceum - wood-decay - cryptococcus-neoformans - transposable elements - self-incompatibility - tilletia-indica - ustilago-maydis - gene
    Mating systems, that is, whether organisms give rise to progeny by selfing, inbreeding or outcrossing, strongly affect important ecological and evolutionary processes. Large variations in mating systems exist in fungi, allowing the study of their origin and consequences. In fungi, sexual incompatibility is determined by molecular recognition mechanisms, controlled by a single mating-type locus in most unifactorial fungi. In Basidiomycete fungi, however, which include rusts, smuts and mushrooms, a system has evolved in which incompatibility is controlled by two unlinked loci. This bifactorial system probably evolved from a unifactorial system. Multiple independent transitions back to a unifactorial system occurred. It is still unclear what force drove evolution and maintenance of these contrasting inheritance patterns that determine mating compatibility. Here, we give an overview of the evolutionary factors that might have driven the evolution of bifactoriality from a unifactorial system and the transitions back to unifactoriality. Bifactoriality most likely evolved for selfing avoidance. Subsequently, multiallelism at mating-type loci evolved through negative frequency-dependent selection by increasing the chance to find a compatible mate. Unifactoriality then evolved back in some species, possibly because either selfing was favoured or for increasing the chance to find a compatible mate in species with few alleles. Owing to the existence of closely related unifactorial and bifactorial species and the increasing knowledge of the genetic systems of the different mechanisms, Basidiomycetes provide an excellent model for studying the different forces that shape breeding systems.
    Towards a new classification system for legumes: Progress report from the 6th International Legume Conference
    Pontes Coelho Borges, L.M. ; Bruneau, A. ; Cardoso, D. ; Crisp, M. ; Delgado-Salinas, A. ; Doyle, J.J. ; Egan, A. ; Herendeen, P.S. ; Hughes, C. ; Kenicer, G. ; Klitgaard, B. ; Koenen, E. ; Lavin, M. ; Lewis, G. ; Luckow, M. ; Mackinder, B. ; Malecot, V. ; Miller, J.T. ; Pennington, R.T. ; Queiroz, L.P. de; Schrire, B. ; Simon, M.F. ; Steele, K. ; Torke, B. ; Wieringa, J.J. ; Wojciechowski, M.F. ; Boatwright, S. ; Estrella, M. de la; Mansano, V.D. ; Prado, D.E. ; Stirton, C. ; Wink, M. - \ 2013
    South African Journal of Botany 89 (2013). - ISSN 0254-6299 - p. 3 - 9.
    caesalpinioid legumes - phylogeny - leguminosae - evolution - diversification - dipsacales - sequences - lineages - gene - rbcl
    Legume systematists have been making great progress in understanding evolutionary relationships within the Leguminosae (Fabaceae), the third largest family of flowering plants. As the phylogenetic picture has become clearer, so too has the need for a revised classification of the family. The organization of the family into three subfamilies and 42 tribes is outdated and evolutionarily misleading. The three traditionally recognized subfamilies, Caesalpinioideae, Mimosoideae, and Papilionoideae, do not adequately represent relationships within the family. The occasion of the Sixth International Legume Conference in Johannesburg, South Africa in January 2013, with its theme "Towards a new classification system for legumes," provided the impetus to move forward with developing a new classification. A draft classification, based on current phylogenetic results and a set of principles and guidelines, was prepared in advance of the conference as the basis for discussion. The principles, guidelines, and draft classification were presented and debated at the conference. The objectives of the discussion were to develop consensus on the principles that should guide the development of the classification, to discuss the draft classification's strengths and weaknesses and make proposals for its revision, and identify and prioritize phylogenetic deficiencies that must be resolved before the classification could be published. This paper describes the collaborative process by a large group of legume systematists, publishing under the name Legume Phylogeny Working Group, to develop a new phylogenetic classification system for the Leguminosae. The goals of this paper are to inform the broader legume community, and others, of the need for a revised classification, and spell out clearly what the alternatives and challenges are for a new classification system for the family. (C) 2013 SAAB. Published by Elsevier B.V. All rights reserved.
    Powdery Mildew Resistance in Tomato by Impairment of SIPMR4 and SIDMR1
    Huibers, R.P. ; Loonen, A.E.H.M. ; Dongli Gao, Dongli ; Ackerveken, G. van den; Visser, R.G.F. ; Bai, Y. - \ 2013
    PLoS ONE 8 (2013)6. - ISSN 1932-6203
    zinc-finger nucleases - disease resistance - arabidopsis mutants - targeted mutagenesis - gene - plants - pathogenesis - mutation
    Genetic dissection of disease susceptibility in Arabidopsis to powdery and downy mildew has identified multiple susceptibility (S) genes whose impairment results in disease resistance. Although several of these S-genes have been cloned and characterized in more detail it is unknown to which degree their function in disease susceptibility is conserved among different plant species. Moreover, it is unclear whether impairment of such genes has potential in disease resistance breeding due to possible fitness costs associated with impaired alleles. Here we show that the Arabidopsis PMR4 and DMR1, genes encoding a callose synthase and homoserine kinase respectively, have functional orthologs in tomato with respect to their S-gene function. Silencing of both genes using RNAi resulted in resistance to the tomato powdery mildew fungus Oidium neolycopersici. Resistance to O. neolycopersici by SlDMR1 silencing was associated with severely reduced plant growth whereas SlPMR4 silencing was not. SlPMR4 is therefore a suitable candidate gene as target for mutagenesis to obtain alleles that can be deployed in disease resistance breeding of tomato.
    Dissecting structural and nucleotide genome-wide variation in inbred Iberian pigs
    Esteve-Codina, A. ; Paudel, Y. ; Ferretti, L. ; Megens, H.J.W.C. ; Groenen, M. - \ 2013
    BMC Genomics 14 (2013). - ISSN 1471-2164
    copy number variation - meat quality traits - positive selection - next-generation - snp discovery - evolution - gene - samples - region - interleukin-2
    Background In contrast to international pig breeds, the Iberian breed has not been admixed with Asian germplasm. This makes it an important model to study both domestication and relevance of Asian genes in the pig. Besides, Iberian pigs exhibit high meat quality as well as appetite and propensity to obesity. Here we provide a genome wide analysis of nucleotide and structural diversity in a reduced representation library from a pool (n=9 sows) and shotgun genomic sequence from a single sow of the highly inbred Guadyerbas strain. In the pool, we applied newly developed tools to account for the peculiarities of these data. Results A total of 254,106 SNPs in the pool (79.6 Mb covered) and 643,783 in the Guadyerbas sow (1.47 Gb covered) were called. The nucleotide diversity (1.31x10-3 per bp in autosomes) is very similar to that reported in wild boar. A much lower than expected diversity in the X chromosome was confirmed (1.79x10-4 per bp in the individual and 5.83x10-4 per bp in the pool). A strong (0.70) correlation between recombination and variability was observed, but not with gene density or GC content. Multicopy regions affected about 4% of annotated pig genes in their entirety, and 2% of the genes partially. Genes within the lowest variability windows comprised interferon genes and, in chromosome X, genes involved in behavior like HTR2C or MCEP2. A modified Hudson-Kreitman-Aguadé test for pools also indicated an accelerated evolution in genes involved in behavior, as well as in spermatogenesis and in lipid metabolism. Conclusions This work illustrates the strength of current sequencing technologies to picture a comprehensive landscape of variability in livestock species, and to pinpoint regions containing genes potentially under selection. Among those genes, we report genes involved in behavior, including feeding behavior, and lipid metabolism. The pig X chromosome is an outlier in terms of nucleotide diversity, which suggests selective constraints. Our data further confirm the importance of structural variation in the species, including Iberian pigs, and allowed us to identify new paralogs for known gene families.
    Arabidopsis semidwarfs evolved from independent mutations in GA20ox1, ortholog to green revolution dwarf alleles in rice and barley
    Barboza, L. ; Effgen, S. ; Alonso-Blanco, C. ; Kooke, R. ; Keurentjes, J.J.B. ; Koornneef, M. - \ 2013
    Proceedings of the National Academy of Sciences of the United States of America 110 (2013)39. - ISSN 0027-8424 - p. 15818 - 15823.
    quantitative trait loci - gibberellin biosynthesis - natural variation - thaliana - gene - populations - model - polymorphism - association - adaptation
    Understanding the genetic bases of natural variation for developmental and stress-related traits is a major goal of current plant biology. Variation in plant hormone levels and signaling might underlie such phenotypic variation occurring even within the same species. Here we report the genetic and molecular basis of semidwarf individuals found in natural Arabidopsis thaliana populations. Allelism tests demonstrate that independent loss-offunction mutations at GA locus 5 (GA5), which encodes gibberellin 20-oxidase 1 (GA20ox1) involved in the last steps of gibberellin biosynthesis, are found in different populations from southern, western, and northern Europe; central Asia; and Japan. Sequencing of GA5 identified 21 different loss-of-function alleles causing semidwarfness without any obvious general tradeoff affecting plant performance traits. GA5 shows signatures of purifying selection, whereas GA5 loss-of-function alleles can also exhibit patterns of positive selection in specific populations as shown by Fay and Wu’s H statistics. These results suggest that antagonistic pleiotropy might underlie the occurrence of GA5 loss-of-function mutations in nature. Furthermore, because GA5 is the ortholog of rice SD1 and barley Sdw1/Denso green revolution genes, this study illustrates the occurrence of conserved adaptive evolution between wild A.thaliana and domesticated plants
    Identification of Cisplatin-Regulated Metabolic Pathways in Pluripotent Stem Cells
    Stechow, L. von; Ruiz-Aracama, A. ; Water, B. ; Peijnenburg, A. ; Danen, E. ; Lommen, A. - \ 2013
    PLoS ONE 8 (2013)10. - ISSN 1932-6203 - 13 p.
    dna-damage response - platinum anticancer drugs - mass-spectrometry - modified nucleosides - arginine metabolism - profiling reveals - cancer - resistance - gene - proline
    The chemotherapeutic compound, cisplatin causes various kinds of DNA lesions but also triggers other pertubations, such as ER and oxidative stress. We and others have shown that treatment of pluripotent stem cells with cisplatin causes a plethora of transcriptional and post-translational alterations that, to a major extent, point to DNA damage response (DDR) signaling. The orchestrated DDR signaling network is important to arrest the cell cycle and repair the lesions or, in case of damage beyond repair, eliminate affected cells. Failure to properly balance the various aspects of the DDR in stem cells contributes to ageing and cancer. Here, we performed metabolic profiling by mass spectrometry of embryonic stem (ES) cells treated for different time periods with cisplatin. We then integrated metabolomics with transcriptomics analyses and connected cisplatin-regulated metabolites with regulated metabolic enzymes to identify enriched metabolic pathways. These included nucleotide metabolism, urea cycle and arginine and proline metabolism. Silencing of identified proline metabolic and catabolic enzymes indicated that altered proline metabolism serves as an adaptive, rather than a toxic response. A group of enriched metabolic pathways clustered around the metabolite S-adenosylmethionine, which is a hub for methylation and transsulfuration reactions and polyamine metabolism. Enzymes and metabolites with pro- or anti-oxidant functions were also enriched but enhanced levels of reactive oxygen species were not measured in cisplatin-treated ES cells. Lastly, a number of the differentially regulated metabolic enzymes were identified as target genes of the transcription factor p53, pointing to p53-mediated alterations in metabolism in response to genotoxic stress. Altogether, our findings reveal interconnecting metabolic pathways that are responsive to cisplatin and may serve as signaling modules in the DDR in pluripotent stem cells.
    Local Auxin Sources Orient the Apical-Basal Axis in Arabidopsis Embryos
    Robert, H.S. ; Grones, P. ; Stepanova, A.N. ; Robles, L.M. ; Lokerse, A.S. ; Alonso, J.M. ; Weijers, D. ; Friml, J. - \ 2013
    Current Biology 23 (2013)24. - ISSN 0960-9822 - p. 2506 - 2512.
    plant development - cotyledon development - seed development - pin proteins - biosynthesis - gene - polarity - embryogenesis - expression - efflux
    Establishment of the embryonic axis foreshadows the main body axis of adults both in plants and in animals, but underlying mechanisms are considered distinct. Plants utilize directional, cell-to-cell transport of the growth hormone auxin [1 and 2] to generate an asymmetric auxin response that specifies the embryonic apical-basal axis [3, 4, 5 and 6]. The auxin flow directionality depends on the polarized subcellular localization of PIN-FORMED (PIN) auxin transporters [7 and 8]. It remains unknown which mechanisms and spatial cues guide cell polarization and axis orientation in early embryos. Herein, we provide conceptually novel insights into the formation of embryonic axis in Arabidopsis by identifying a crucial role of localized tryptophan-dependent auxin biosynthesis [ 9, 10, 11 and 12]. Local auxin production at the base of young embryos and the accompanying PIN7-mediated auxin flow toward the proembryo are required for the apical auxin response maximum and the specification of apical embryonic structures. Later in embryogenesis, the precisely timed onset of localized apical auxin biosynthesis mediates PIN1 polarization, basal auxin response maximum, and specification of the root pole. Thus, the tight spatiotemporal control of distinct local auxin sources provides a necessary, non-cell-autonomous trigger for the coordinated cell polarization and subsequent apical-basal axis orientation during embryogenesis and, presumably, also for other polarization events during postembryonic plant life [ 13 and 14].
    Potential of proton-pumping rhodopsins: engineering photosystems into microorganisms
    Claassens, N.J.H.P. ; Volpers, M. ; Martins Dos Santos, V.A.P. ; Oost, J. van der; Vos, W.M. de - \ 2013
    Trends in Biotechnology 31 (2013)11. - ISSN 0167-7799 - p. 633 - 642.
    escherichia-coli - functional expression - microbial rhodopsins - carbon-dioxide - schizosaccharomyces-pombe - gloeobacter rhodopsin - purple membrane - proteorhodopsin - bacteriorhodopsin - gene
    A wide range of proton-pumping rhodopsins (PPRs) have been discovered in recent years. Using a synthetic biology approach, PPR photosystems with different features can be easily introduced in nonphotosynthetic microbial hosts. PPRs can provide hosts with the ability to harvest light and drive the sustainable production of biochemicals or biofuels. PPRs use light energy to generate an outward proton flux, and the resulting proton motive force can subsequently power cellular processes. Recently, the introduction of PPRs in microbial production hosts has successfully led to light-driven biotechnological conversions. In this review, we discuss relevant features of natural PPRs, evaluate reported biotechnological applications of microbial production hosts equipped with PPRs, and provide an outlook on future developments
    Targeted Resequencing of 9p in Acute Lymphoblastic Leukemia Yields Concordant Results with Array CGH and Reveals Novel Genomic Alterations
    Sarhadi, V.K. ; Lahti, L.M. ; Scheinin, I. ; Tyybäkinoja, A. ; Savola, S. ; Usvasalo, A. ; Räty, R. ; Elonen, E. ; Saarinen-Pihkala, U.M. ; Knuutila, S. - \ 2013
    Genomics 102 (2013)3. - ISSN 0888-7543 - p. 182 - 188.
    gene - cancer - heterozygosity - association - expression - deletions - growth
    Genetic alterations of the short arm of chromosome 9 are frequent in acute lymphoblastic leukemia. We performed targeted sequencing of 9p region in 35 adolescent and adult acute lymphoblastic leukemia patients and sought to investigate the sensitivity of detecting copy number alterations in comparison with array comparative genomic hybridization (aCGH), and besides, to detect novel genetic anomalies. We found a high concordance of copy number variations (CNVs) as detected by next generation sequencing (NGS) and aCGH. By both methodologies, the recurrent deletion at CDKN2A/B locus was identified, whereas NGS revealed additional, small regions of CNVs, seen more frequently in adult patients, while aCGH was better at detecting larger CNVs. Also, by NGS, we detected novel structural variations, novel SNVs and small insertion/deletion variants. Our results show that NGS, in addition to detecting mutations and other genetic aberrations, can be used to study CNVs
    Urinary excretion of copper, zinc and iron with and without D-penicillamine administration in relation to hepatic copper concentration in dogs
    Fieten, H. ; Hugen, S. ; Ingh, T.S.G.A.M. van den; Hendriks, W.H. ; Vernooij, J.C.M. ; Bode, P. ; Watson, A.L. ; Leegwater, P.A.J. ; Rothuizen, J. - \ 2013
    The Veterinary Journal 197 (2013)2. - ISSN 1090-0233 - p. 468 - 473.
    wilsons-disease - diagnosis - metabolism - challenge - toxicosis - gene
    Hereditary copper-associated hepatitis in dogs resembles Wilson’s disease, a copper storage disease in humans. Values for urinary copper excretion are well established in the diagnostic protocol of Wilson’s disease, whereas in dogs these have not been evaluated. The objectives of this study were to characterize both basal and D-penicillamine induced urinary copper, zinc and iron excretion in dogs in relation to hepatic copper concentration. Beagles, Beagle-Bedlington terrier cross-breeds homozygous for the COMMD1 gene mutation that causes copper toxicosis, and Labrador retrievers with normal or increased hepatic copper concentrations were investigated. The hepatic copper phenotype was determined by histological evaluation of liver biopsies and measurement of the hepatic copper concentration by instrumental neutron activation analysis. Urinary excretion of copper, iron and zinc was measured via inductively coupled plasma optical emission spectrometry under basal conditions and after oral administration of a single dose (20 mg/kg bodyweight) of the chelator D-penicillamine. There was a rapid increase in urinary excretion of copper and zinc, but not iron after D-penicillamine administration. This increase was not different between dogs with high or normal hepatic copper concentrations. D-penicillamine-induced urinary copper excretion and the copper/creatinine ratio did not correlate with hepatic copper concentrations in the dogs studied, although basal urinary copper/zinc ratios did correlate with hepatic copper concentrations in Labrador retrievers. The latter parameter may be useful in diagnostic and follow-up protocols for copper-associated hepatitis in Labrador retrievers.
    Evidence for regulation of columnar habit in apple encodes a putative 2og-fe(ii) oxygenase
    Wolters, P.J. ; Schouten, H.J. ; Riccardo, V. ; Si-Ammour, A. ; Baldi, P. - \ 2013
    New Phytologist 200 (2013)4. - ISSN 0028-646X - p. 993 - 999.
    malus-x-domestica - growth habit - arabidopsis - gene - transformation - activation - expression - abscission - genome - spur
    Understanding the genetic mechanisms controlling columnar-type growth in the apple mutant Wijcik will provide insights on how tree architecture and growth are regulated in fruit trees. In apple, columnar-type growth is controlled by a single major gene at the Columnar (Co) locus. By comparing the genomic sequence of the Co region of Wijcik with its wild-type McIntosh, a novel non-coding DNA element of 1956 bp specific to Pyreae was found to be inserted in an intergenic region of Wijcik. Expression analysis of selected genes located in the vicinity of the insertion revealed the upregulation of the MdCo31 gene encoding a putative 2OG-Fe(II) oxygenase in axillary buds of Wijcik. Constitutive expression of MdCo31 in Arabidopsis thaliana resulted in compact plants with shortened floral internodes, a phenotype reminiscent of the one observed in columnar apple trees. We conclude that MdCo31 is a strong candidate gene for the control of columnar growth in Wijcik.
    Overexpression of angiopoietin-like protein 4 protects against atherosclerosis development
    Georgiadi, A. ; Wang, Y. ; Stienstra, R. ; Tjeerdema, N. ; Janssen, A. ; Stalenhoef, A. ; Vliet, A. van der; Roos, J.A. de; Tamsma, J.T. ; Smit, J.W. ; Tan, N.S. ; Müller, M.R. ; Rensen, P.C. ; Kersten, A.H. - \ 2013
    Arteriosclerosis Thrombosis and Vascular Biology 33 (2013)7. - ISSN 1079-5642 - p. 1529 - 1537.
    low-density-lipoprotein - mouse peritoneal-macrophages - foam cell-formation - transgenic mice - lipase - angptl4 - expression - gene - hyperlipoproteinemia - hyperlipidemia
    Objective—Macrophage foam cells play a crucial role in several pathologies including multiple sclerosis, glomerulosclerosis, and atherosclerosis. Angiopoietin-like protein 4 (Angptl4) was previously shown to inhibit chyle-induced foam cell formation in mesenteric lymph nodes. Here we characterized the regulation of Angptl4 expression in macrophages and examined the impact of Angptl4 on atherosclerosis development. Approach and Results—Macrophage activation elicited by pathogen-recognition receptor agonists decreased Angptl4 expression, whereas lipid loading by intralipid and oxidized low-density lipoprotein increased Angptl4 expression. Consistent with an antilipotoxic role of Angptl4, recombinant Angptl4 significantly decreased uptake of oxidized low-density lipoprotein by macrophages, via lipolysis-dependent and -independent mechanisms. Angptl4 protein was detectable in human atherosclerotic lesions and localized to macrophages. Transgenic overexpression of Angptl4 in atherosclerosis-prone apolipoprotein E*3-Leiden mice did not significantly alter plasma cholesterol and triglyceride levels. Nevertheless, Angptl4 overexpression reduced lesion area by 34% (P
    Dietary Supplement Use and Colorectal Adenoma Risk in Individuals with Lynch Syndrome: The GEOLynch Cohort Study
    Heine-Bröring, R.C. ; Winkels, R.M. ; Botma, A. ; Duijnhoven, F.J.B. van; Jung, A.Y. ; Kampman, E. - \ 2013
    PLoS ONE 8 (2013)6. - ISSN 1932-6203
    cancer - prevention - validity - families - vitamin - gene - omega-3-fatty-acids - questionnaire - metaanalysis - population
    Background and Aims: Individuals with Lynch syndrome have a high lifetime risk of developing colorectal tumors. In this prospective cohort study of individuals with Lynch syndrome, we examined associations between use of dietary supplements and occurrence of colorectal adenomas. Materials and Methods: Using data of 470 individuals with Lynch syndrome in a prospective cohort study, associations between dietary supplement use and colorectal adenoma risk were evaluated by calculating hazard ratios (HR) and 95% confidence intervals (CI) using cox regression models adjusted for age, sex, and number of colonoscopies during person time. Robust sandwich covariance estimation was used to account for dependency within families. Results: Of the 470 mismatch repair gene mutation carriers, 122 (26.0%) developed a colorectal adenoma during an overall median person time of 39.1 months. 40% of the study population used a dietary supplement. Use of any dietary supplement was not statistically significantly associated with colorectal adenoma risk (HR = 1.18; 95% CI 0.80-1.73). Multivitamin supplement use (HR = 1.15; 95% CI 0.72-1.84), vitamin C supplement use (HR = 1.57; 95% CI 0.93-2.63), calcium supplement use (HR = 0.69; 95% CI 0.25-1.92), and supplements containing fish oil (HR = 1.60; 95% CI 0.79-3.23) were also not associated with occurrence of colorectal adenomas. Conclusion: This prospective cohort study does not show inverse associations between dietary supplement use and occurrence of colorectal adenomas among individuals with Lynch syndrome. Further research is warranted to determine whether or not dietary supplement use is associated to colorectal adenoma and colorectal cancer risk in MMR gene mutation carriers.
    Complete Sequence of pSAM7, an IncX4 Plasmid Carrying a Novel blaCTX-M-14b Transposition Unit Isolated from Escherichia coli and Enterobacter cloacae from Cattle
    Stokes, M.O. ; Abuoun, M. ; Umur, S. ; Wu, G. ; Partridge, S.R. ; Mevius, D.J. ; Coldham, N.G. ; Fielder, M.D. - \ 2013
    Antimicrobial Agents and Chemotherapy 57 (2013)9. - ISSN 0066-4804 - p. 4590 - 4594.
    spectrum-beta-lactamases - klebsiella-pneumoniae - identification - gene - bacteria - strains - ctx-m-9 - humans - family
    The same plasmid carrying blaCTX-M-14b was identified from an Escherichia coli isolate and an Enterobacter cloacae isolate collected from cattle in the United Kingdom by complete plasmid sequencing. This 35,341-bp plasmid, pSAM7, had an IncX4 backbone that is 99% identical to that of pJIE143 from a human isolate in Australia. PCR screening identified pSAM7-like plasmids in three other E. coli isolates of different multilocus sequence types isolated from cattle on different farms in the United Kingdom.
    Validation of a real-time PCR based method for detection of Clostridium botulinum types C, D and their mosaic variants C-D and D-C in a multicenter collaborative trial
    Woudstra, C. ; Skarin, H. ; Anniballi, F. ; Auricchio, B. ; Medici, D. De; Bano, L. ; Drigo, I. ; Hansen, T. ; Löfström, Ch. ; Hamidjaja, R. ; Rotterdam, B. van; Koene, M.G.J. - \ 2013
    Anaerobe 22 (2013). - ISSN 1075-9964 - p. 31 - 37.
    quantitative detection - clinical-samples - toxin - gene - neurotoxins - strains - food
    Two real-time PCR arrays based on the GeneDisc(®) cycler platform (Pall-GeneDisc Technologies) were evaluated in a multicenter collaborative trial for their capacity to specifically detect and discriminate Clostridium botulinum types C, D and their mosaic variants C-D and D-C that are associated with avian and mammalian botulism. The GeneDisc(®) arrays developed as part of the DG Home funded European project 'AnibioThreat' were highly sensitive and specific when tested on pure isolates and naturally contaminated samples (mostly clinical specimen from avian origin). Results of the multicenter collaborative trial involving eight laboratories in five European Countries (two laboratories in France, Italy and The Netherlands, one laboratory in Denmark and Sweden), using DNA extracts issued from 33 pure isolates and 48 naturally contaminated samples associated with animal botulism cases, demonstrated the robustness of these tests. Results showed a concordance among the eight laboratories of 99.4%-100% for both arrays. The reproducibility of the tests was high with a relative standard deviation ranging from 1.1% to 7.1%. Considering the high level of agreement achieved between the laboratories these PCR arrays constitute robust and suitable tools for rapid detection of C. botulinum types C, D and mosaic types C-D and D-C. These are the first tests for C. botulinum C and D that have been evaluated in a European multicenter collaborative trial.
    An integrative model of the control of ovule primordia formation
    Galbiati, F. ; Sihna Roy, D. ; Simonini, S. ; Cucinotta, M. ; Ceccato, L. ; Cuesta, C. ; Simaskova, M. ; Benkova, E. ; Kamiuchi, Y. ; Aida, M. ; Weijers, D. ; Simon, R. ; Masiero, S. ; Colombo, L. - \ 2013
    The Plant Journal 76 (2013)3. - ISSN 0960-7412 - p. 446 - 455.
    dependent auxin gradients - arabidopsis-thaliana - transcription factor - pattern-formation - early sporogenesis - direct target - gene - cytokinin - gynoecium - aintegumenta
    Upon hormonal signaling, ovules develop as lateral organs from the placenta. Ovule numbers ultimately determine the number of seeds that develop, and thereby contribute to the final seed yield in crop plants. We demonstrate here that CUP-SHAPED COTYLEDON 1 (CUC1), CUC2 and AINTEGUMENTA (ANT) have additive effects on ovule primordia formation. We show that expression of the CUC1 and CUC2 genes is required to redundantly regulate expression of PINFORMED1 (PIN1), which in turn is required for ovule primordia formation. Furthermore, our results suggest that the auxin response factor MONOPTEROS (MP/ARF5) may directly bind ANT, CUC1 and CUC2 and promote their transcription. Based on our findings, we propose an integrative model to describe the molecular mechanisms of the early stages of ovule development.
    Arabidopsis COBRA-LIKE 10, a GPI-anchored protein, mediates directional growth of pollen tubes
    Li, S. ; Ge, F.R. ; Xu, M. ; Zhao, X.Y. ; Huang, G.Q. ; Zhou, L.Z. ; Wang, J.G. ; Kombrink, A. ; McCormick, S. ; Zhang, X.S. ; Zhang, Y. - \ 2013
    The Plant Journal 74 (2013)3. - ISSN 0960-7412 - p. 486 - 497.
    male gametophyte development - oriented cell expansion - membrane-proteins - polarized growth - tip growth - in-vitro - thaliana - gene - guidance - encodes
    Successful reproduction of flowering plants requires constant communication between female tissues and growing pollen tubes. Female cells secrete molecules and peptides as nutrients or guidance cues for fast and directional tube growth, which is executed by dynamic changes of intracellular activities within pollen tubes. Compared with the extensive interest in female cues and intracellular activities of pollen tubes, how female cues are sensed and interpreted intracellularly in pollen is poorly understood. We show here that COBL10, a glycosylphosphatidylinositol (GPI)-anchored protein, is one component of this pollen tube internal machinery. Mutations in COBL10 caused gametophytic male sterility due to reduced pollen tube growth and compromised directional sensing in the female transmitting tract. Deposition of the apical pectin cap and cellulose microfibrils was disrupted in cobl10 pollen tubes. Pollen tube localization of COBL10 at the apical plasma membrane is critical for its function and relies on proper GPI processing and its C-terminal hydrophobic residues. GPI-anchored proteins are widespread cell sensors in mammals, especially during egg-sperm communication. Our results that COBL10 is critical for directional growth of pollen tubes suggest that they play critical roles in cell-cell communications in plants.
    Enhancing pterin and para-aminobenzoate content is not sufficient to successfully biofortify potato tubers and Arabidopsis thaliana plants with folate
    Blancquaert, D. ; Storozhenko, S. ; Daele, W. ; Stove, C. ; Visser, R.G.F. ; Lambert, W. ; Straeten, D. van der - \ 2013
    Journal of Experimental Botany 64 (2013)12. - ISSN 0022-0957 - p. 3899 - 3909.
    time quantitative pcr - folic-acid - tomato fruit - gene - expression - transformation - homocysteine - promoter - disease - fortification
    Folates are important cofactors in one-carbon metabolism in all living organisms. Since only plants and micro- organisms are capable of biosynthesizing folates, humans depend entirely on their diet as a folate source. Given the low folate content of several staple crop products, folate deficiency affects regions all over the world. Folate biofortification of staple crops through enhancement of pterin and para-aminobenzoate levels, precursors of the folate biosynthesis pathway, was reported to be successful in tomato and rice. This study shows that the same strategy is not sufficient to enhance folate content in potato tubers and Arabidopsis thaliana plants and concludes that other steps in folate biosynthesis and/or metabolism need to be engineered to result in substantial folate accumulation. The findings provide a plausible explanation why, more than half a decade after the proof of concept in rice and tomato, successful folate biofortification of other food crops through enhancement of para-aminobenzoate and pterin content has not been reported thus far. A better understanding of the folate pathway is required in order to determine an engineering strategy that can be generalized to most staple crops.
    A Trichome-Specific Linoleate Lipoxygenase Expressed During Pyrethrin Biosynthesis in Pyrethrum
    Ramirez, A.M. ; Yang, T. ; Bouwmeester, H.J. ; Jongsma, M.A. - \ 2013
    Lipids 48 (2013)10. - ISSN 0024-4201 - p. 1005 - 1015.
    chrysanthemum-cinerariaefolium - tanacetum-cinerariifolium - jasmonic acid - gene - arabidopsis - seedlings - defense - pathway - stress - growth
    The lipid precursor alcohols of pyrethrins—jasmolone, pyrethrolone and cinerolone—have been proposed as sharing parts of the oxylipin pathway with jasmonic acid. This implies that one of the first committed steps of pyrethrin biosynthesis is catalyzed by a lipoxygenase, catalyzing the hydroperoxidation of linolenic acid at position 13. Previously, we showed that the expression and activity of chrysanthemyl diphosphate synthase (TcCDS), the enzyme catalyzing the first committed step in the biosynthesis of the acid moiety of pyrethrins, is trichome-specific and developmentally regulated in flowers. In the present study we characterized the expression pattern of 25 lipoxygenase EST contigs, and subsequently carried out the molecular cloning of two pyrethrum lipoxygenases, TcLOX1 and TcLOX2, that have a similar pattern to TcCDS. Only recombinant TcLOX1 catalyzed the peroxidation of the linolenic acid substrate. Just as TcCDS, TcLOX1, are exclusively expressed in trichomes. Phylogenetic analysis showed that the enzyme shared the highest homology with chloroplast-localized 13-type-lipoxygenases that are involved in maintaining basal levels of jasmonate.
    NON-SMOKY GLYCOSYLTRANSFERASE1 Prevents the Release of Smoky Aroma from Tomato Fruit
    Tikunov, Y.M. ; Molthoff, J.W. ; Vos, R.C.H. de; Beekwilder, M.J. ; Houwelingen, A.M.M.L. van; Hooft, J.J.J. van der; Nijenhuis-de Vries, M.A. ; Labrie, C.W. ; Verkerke, W. ; Geest, H.C. van de; Víquez Zamora, A.M. ; Presa, S. ; Rambla Nebot, J.L. ; Granell, A. ; Hall, R.D. ; Bovy, A.G. - \ 2013
    The Plant Cell 25 (2013)8. - ISSN 1040-4651 - p. 3067 - 3078.
    mass spectrometry - small molecules - salicylic-acid - key enzyme - flavor - volatiles - biosynthesis - components - odor - gene
    Phenylpropanoid volatiles are responsible for the key tomato fruit (Solanum lycopersicum) aroma attribute termed “smoky.” Release of these volatiles from their glycosylated precursors, rather than their biosynthesis, is the major determinant of smoky aroma in cultivated tomato. Using a combinatorial omics approach, we identified the NON-SMOKY GLYCOSYLTRANSFERASE1 (NSGT1) gene. Expression of NSGT1 is induced during fruit ripening, and the encoded enzyme converts the cleavable diglycosides of the smoky-related phenylpropanoid volatiles into noncleavable triglycosides, thereby preventing their deglycosylation and release from tomato fruit upon tissue disruption. In an nsgt1/nsgt1 background, further glycosylation of phenylpropanoid volatile diglycosides does not occur, thereby enabling their cleavage and the release of corresponding volatiles. Using reverse genetics approaches, the NSGT1-mediated glycosylation was shown to be the molecular mechanism underlying the major quantitative trait locus for smoky aroma. Sensory trials with transgenic fruits, in which the inactive nsgt1 was complemented with the functional NSGT1, showed a significant and perceivable reduction in smoky aroma. NSGT1 may be used in a precision breeding strategy toward development of tomato fruits with distinct flavor phenotypes.
    Detoxification of a-tomatine by Cladosporium fulvum is required for full virulence on tomato
    Ökmen, B. ; Etalo, D.W. ; Joosten, M.H.A.J. ; Bouwmeester, H.J. ; Vos, R.C.H. de; Collemare, J.A.R. ; Wit, P.J.G.M. de - \ 2013
    New Phytologist 198 (2013)4. - ISSN 0028-646X - p. 1203 - 1214.
    f-sp lycopersici - saponin-detoxifying enzyme - fungal effector proteins - alpha-tomatine - septoria-lycopersici - mass-spectrometry - heterologous expression - liposome membranes - plant - gene
    -Tomatine is an antifungal glycoalkaloid that provides basal defense to tomato (Solanum lycopersicum). However, tomato pathogens overcome this basal defense barrier by the secretion of tomatinases that degrade -tomatine into the less fungitoxic compounds
    GK4, a G-protein-coupled receptor with a phosphatidylinositol phosphate kinase domain in Phytophthora infestans, is involved in sporangia development and virulence
    Hua, C. ; Meijer, H.J.G. ; Keijzer, J. de; Zhao, W. ; Wang, Y. ; Govers, F. - \ 2013
    Molecular Microbiology 88 (2013)2. - ISSN 0950-382X - p. 352 - 370.
    heterotrimeric g-protein - pollen-tube growth - escherichia-coli - nicotiana-tabacum - genome sequence - alpha-subunit - arabidopsis - gene - topology - fungi
    For dispersal and host infection plant pathogens largely depend on asexual spores. Pathogenesis and sporulation are complex processes that are governed by cellular signalling networks including G-protein and phospholipid signalling. Oomycetes possess a family of novel proteins called GPCR-PIPKs (GKs) that are composed of a seven-transmembrane spanning (7-TM) domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain. Based on this domain structure GKs are anticipated to link G-protein and phospholipid signal pathways; however, their functions are currently unknown. Expression analyses of the 12 GK genes in Phytophthora infestans and their orthologues in Phytophthora sojae, revealed differential expression during asexual development. PiGK1 and PiGK4 were fused to monomeric red fluorescent protein (mRFP) and ectopically expressed in P.¿infestans. In growing hyphae different subcellular distribution patterns were observed indicating that these two GKs act independently during development. We focused on the functional analyses of PiGK4. Its localization suggested involvement in cell differentiation and elongation and its 7-TM domain showed a canonical GPCR membrane topology. Silencing of GK4 and overexpression of full-length and truncated constructs in P.¿infestans revealed that PiGK4 is not only involved in spore germination and hyphal elongation but also in sporangia cleavage and infection.
    Discovery of T cell epitopes implementing HLA-peptidomics into a reverse immunology approach
    Hombrink, P. ; Hassan, C. ; Kester, M.G.D. ; Ru, A.H. ; Bergen, C.A.M. ; Nijveen, H. ; Drijfhout, J.W. ; Falkenburg, J.H.F. ; Heemskerk, M.H.M. ; Veelen, P.A. van - \ 2013
    The Journal of Immunology 190 (2013)8. - ISSN 0022-1767 - p. 3869 - 3877.
    minor histocompatibility antigens - versus-host-disease - restricted tissue distribution - identification - peptides - leukemia - gene - immunotherapy - lymphocytes - complexes
    T cell recognition of minor histocompatibility Ags (MiHA) plays an important role in the graft-versus-tumor effect of allogeneic stem cell transplantation. Selective infusion of T cells reactive for hematopoiesis-restricted MiHA presented in the context of HLA class I or II molecules may help to separate the graft-versus-tumor effects from graft-versus-host disease effects after allogeneic stem cell transplantation. Over the years, increasing numbers of MiHA have been identified by forward immunology approaches, and the relevance of these MiHA has been illustrated by correlation with clinical outcome. As the tissue distribution of MiHA affects the clinical outcome of T cell responses against these Ags, it would be beneficial to identify additional predefined MiHA that are exclusively expressed on hematopoietic cells. Therefore, several reverse immunology approaches have been explored for the prediction of MiHA. Thus far, these approaches frequently resulted in the identification of T cells directed against epitopes that are not naturally processed and presented. In this study we established a method for the identification of biologically relevant MiHA, implementing mass spectrometry–based HLA-peptidomics into a reverse immunology approach. For this purpose, HLA class I binding peptides were eluted from transformed B cells, analyzed by mass spectrometry, and matched with a database dedicated to identifying polymorphic peptides. This process resulted in a set of 40 MiHA candidates that were evaluated in multiple selection steps. The identification of LB-NISCH-1A demonstrated the technical feasibility of our approach. On the basis of these results, we present an approach that can be of value for the efficient identification of MiHA or other T cell epitopes.
    The Arabidopsis exocyst subunit SEC3A is essential for embryo development and accumulates in transient puncta at the plasma membrane
    Zhang, Y. ; Immink, G.H. ; Liu, C.M. ; Emons, A.M.C. ; Ketelaar, T. - \ 2013
    New Phytologist 199 (2013)1. - ISSN 0028-646X - p. 74 - 88.
    plant-cell growth - auxin transport - root hairs - complex - exocytosis - secretion - gene - expression - thaliana - yeast
    The exocyst is a protein complex that is essential for polarized secretion in mammals and fungi. Although the exocyst is essential for plant development, its precise function has not been elucidated. We studied the role of exocyst subunit SEC3A in plant development and its subcellular localization. T-DNA insertional mutants were identified and complemented with a SEC3A-green fluorescent protein (GFP) fusion construct. SEC3A-GFP localization was determined using confocal microscopy. sec3a mutants are defective in the globular to heart stage transition in embryogenesis. SEC3A-GFP has similar cell plate localization to the other plant exocyst subunits. In interphase cells, SEC3A-GFP localizes to the cytoplasm and to the plasma membrane, where it forms immobile, punctate structures with discrete lifetimes of 2-40 s. These puncta are equally distributed over the cell surface of root epidermal cells and tip growing root hairs. The density of puncta does not decrease after growth termination of these cells, but decreases strongly when exocytosis is inhibited by treatment with brefeldin A. SEC3A does not appear to be involved in polarized secretion for cell expansion in tip growing root hairs. The landmark function performed by SEC3 in mammals and yeast is likely to be conserved in plants
    Naturally occurring allele diversity allows potato cultivation in northern latitudes
    Kloosterman, B.A. ; Abelenda, J.A. ; Carretero Gomez, M. ; Oortwijn, M.E.P. ; Boer, J.M. de; Kowitwanich, K. ; Horvath, B.M. ; Eck, H.J. van; Smaczniak, C. ; Prat, S. ; Visser, R.G.F. ; Bachem, C.W.B. - \ 2013
    Nature 495 (2013)7440. - ISSN 0028-0836 - p. 246 - 250.
    flowering-time - arabidopsis - constans - tuberization - gene - plants - fkf1
    Potato (Solanum tuberosum L.) originates from the Andes and evolved short-day-dependent tuber formation as a vegetative propagation strategy. Here we describe the identification of a central regulator underlying a major-effect quantitative trait locus for plant maturity and initiation of tuber development. We show that this gene belongs to the family of DOF (DNA-binding with one finger) transcription factors1 and regulates tuberization and plant life cycle length, by acting as a mediator between the circadian clock and the StSP6A mobile tuberization signal2. We also show that natural allelic variants evade post-translational light regulation, allowing cultivation outside the geographical centre of origin of potato. Potato is a member of the Solanaceae family and isone of the world’s most important food crops. This annual plant originates from the Andean regions of South America3. Potato develops tubers from underground stems called stolons. Its equatorial origin makes potato essentially short-day dependent for tuberization and potato will not make tubers in the long-day conditions of spring and summer in the northern latitudes. When introduced in temperate zones, wild material will form tubers in the course of the autumnal shortening of day-length. Thus, one of the first selected traits in potato leading to a European potato type4 is likely to have been long-day acclimation for tuberization. Potato breeders can exploit the naturally occurring variation in tuberization onset and life cycle length, allowing varietal breeding for different latitudes, harvest times and markets.
    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
    Jasmonate and ethylene signaling mediate whitefly-induced interference with indirect plant defense in Arabidopsis thaliana
    Zhang, P.J. ; Broekgaarden, C. ; Zheng, S.J. ; Snoeren, T.A.L. ; Loon, J.J.A. van; Gols, R. ; Dicke, M. - \ 2013
    New Phytologist 197 (2013)4. - ISSN 0028-646X - p. 1291 - 1299.
    salicylic-acid - transcriptome changes - feeding guilds - tomato plants - herbivores - volatiles - insect - responses - gene - involvement
    Upon herbivore attack, plants activate an indirect defense, that is, the release of a complex mixture of volatiles that attract natural enemies of the herbivore. When plants are simultaneously exposed to two herbivore species belonging to different feeding guilds, one herbivore may interfere with the indirect plant defense induced by the other herbivore. However, little is understood about the mechanisms underlying such interference. Here, we address the effect of herbivory by the phloem-feeding whitefly Bemisia tabaci on the induced indirect defense of Arabidopsis thaliana plants to Plutella xylostella caterpillars, that is, the attraction of the parasitoid wasp Diadegma semiclausum. Assays with various Arabidopsis mutants reveal that B. tabaci infestation interferes with indirect plant defense induced by P. xylostella, and that intact jasmonic acid and ethylene signaling are required for such interference caused by B. tabaci. Chemical analysis of plant volatiles showed that the composition of the blend emitted in response to the caterpillars was significantly altered by co-infestation with whiteflies. Moreover, whitefly infestation also had a considerable effect on the transcriptomic response of the plant to the caterpillars. Understanding the mechanisms underlying a plant’s responses to multiple attackers will be important for the development of crop protection strategies in a multi-attacker context.
    Dietary Patterns and Colorectal Adenomas in Lynch Syndrome: The GEOLynch Cohort Study
    Botma, A. ; Vasen, H.F. ; Duijnhoven, F.J.B. van; Kleibeuker, J.H. ; Nagengast, F.M. ; Kampman, E. - \ 2013
    Cancer 119 (2013)3. - ISSN 0008-543X - p. 512 - 521.
    colon-cancer - endometrial cancer - risk - hnpcc - gene - families - questionnaire - consumption - population - recurrence
    BACKGROUND: Patients with Lynch syndrome (LS) have a high risk of developing colorectal cancer due to mutations in mismatch repair genes. Because dietary factors, alone and in combination, influence sporadic colorectal carcinogenesis, the association of dietary patterns with colorectal adenomas in LS patients was assessed. METHODS: In the GEOLynch cohort of 486 persons with LS, dietary information was collected, using a food frequency questionnaire. Dietary pattern scores were obtained by principal components analysis. Hazard ratios (HR) between dietary patterns and colorectal adenomas were calculated using Cox regression models. Robust sandwich variance estimates were used to control for dependency within families. Final models were adjusted for age, sex, smoking habits, colorectal adenoma history, and extent of colon resection. RESULTS: During a median follow-up of 20 months, colorectal adenomas were detected in 58 persons. Four dietary patterns were identified: a Prudent, Meat, Snack, and Cosmopolitan pattern. Individuals within the highest tertile of the Prudent pattern had a HR of 0.73 (95% confidence interval [CI], 0.32-1.66) for colorectal adenomas, compared with the lowest tertile. Those with high Meat pattern scores had a HR of 1.70 (95% CI, 0.83-3.52). A high Snack pattern was associated with an increased risk of colorectal adenomas (HR, 2.16; 95% CI, 1.03-4.49). A HR of 1.25 (95% CI, 0.61-2.55) was observed for persons in the highest tertile of the Cosmopolitan pattern. CONCLUSIONS: These findings suggest that dietary patterns may be associated with development of colorectal adenoma in patients with Lynch syndrome. The directions of these findings are corroborative with those observed in studies investigating sporadic colorectal cancer. Cancer 2013. (C) 2012 American Cancer Society.
    Visual Detection of Potato leafroll virus by One-step Reverse Transcription Loop-Mediated Isothermal Amplification of DNA with Hydroxynaphthol Blue Dye
    Ahmadi, S. ; Almasi, A.M. ; Fatehi, F. ; Struik, P.C. ; Moradi, A. - \ 2013
    Journal of Phytopathology 161 (2013). - ISSN 0931-1785 - p. 120 - 124.
    nucleic-acid amplification - rapid detection - rt-lamp - assay - tubers - spp. - gene
    Loop-mediated isothermal amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. We applied reverse transcription loop-mediated isothermal amplification (RT-LAMP) to visually detect Potato leafroll virus. One-step RT-LAMP was performed using RNA of PLRV-infected potato leaves and a set of primers (F3, B3, FIP, BIP, LF and LB) designed for RT-LAMP reaction of the coat protein (CP) gene of PLRV. Positive effects of RT-LAMP were detected by agarose gel electrophoresis and hydroxynaphthol blue (HNB) dye and were shown by a colour change from violet to sky blue. RT-LAMP with HNB dye proved to be a simple assay for the rapid detection of PLRV.
    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.
    Biochemical and functional characterization of recombinant fungal immunomodulatory proteins (rFIPs)
    Bastiaan-Net, S. ; Chanput, W. ; Hertz, A. ; Zwittink, R.D. ; Mes, J.J. ; Wichers, H.J. - \ 2013
    International Immunopharmacology 15 (2013)1. - ISSN 1567-5769 - p. 167 - 175.
    ganoderma-lucidium - murine macrophages - fip-fve - expression - lectins - cloning - gene - lz-8 - mushroom - sinense
    In this study two novel FIPs have been identified and characterized. The first is FIP-nha, identified in the ascomycete Nectria haematococca, and as such, FIP-nha would be the first FIP to be identified outside the order of Basidiomycota. The second is LZ-9, an LZ-8 like protein identified in Ganoderma lucidum. Recombinant FIP proteins were produced in Pichia pastoris and purified using His-affinity magnetic beads. The bioactive characteristics of FIP-nha and LZ-9 were compared to two other well-known FIP proteins, LZ-8 from G. lucidum and FIP-fve from Flammulina velutipes, which were produced and purified using the same method. The produced recombinant FIPs (rFIPs): rLZ-8, rLZ-9, rFIP-fve and rFIP-nha were investigated for their hemagglutinating activity which revealed that rLZ-8, rLZ-9 and rFIP-nha were able to agglutinate rabbit, mouse and sheep red blood cells while rFIP-fve only agglutinated rabbit red blood cells. None of the rFIPs were able to agglutinate human red blood cells unless the cells were trypsinized. In addition, all rFIPs were studied and compared to several lectins for their effect on Caco-2 intestinal cell layer integrity using transepithelial electrical resistance (TEER) measurement. rLZ-9 appeared to have the highest effect in lowering TEER, similar to one of the tested lectins. Testing of rFIPs for their activation of inflammation-related genes of THP-1 macrophages showed rFIP-fve to be the strongest inducer of pro-inflammatory cytokine transcription. These results indicate that each rFIP has a unique bioactive profile as well as each lectin, creating the basis for further studies to relate structure to biological activity.
    Identification of alg3 in the mushroom-forming fungus Schizophyllum commune and analysis of the ¿alg3 knockout mutant
    Berends, E. ; Lehle, L. ; Henquet, M. ; Hesselink, T. ; Wösten, H.A.B. ; Lugones, L.G. ; Bosch, H.J. - \ 2013
    Glycobiology 23 (2013)2. - ISSN 0959-6658 - p. 147 - 154.
    saccharomyces-cerevisiae - n-glycan - gene - glycoproteins - enzyme - mannosyltransferase - glucocerebrosidase - oligosaccharides - glycosylation - therapy
    Alg3 of Saccharomyces cerevisiae catalyzes the mannosyl transfer from Man-P-Dol to Man(5)GlcNAc(2)-PP-Dol resulting in the formation of Man(6)GlcNAc(2)-PP-Dol, which is then further processed to the final precursor oligosaccharide Glc(3)Man(9)GlcNAc(2) for N-glycosylation of proteins. Here, we identified the alg3 gene of the mushroom-forming fungus Schizophyllum commune by homology search. Its function was confirmed by the complementation of the Delta alg3 strain of S. cerevisiae. Inactivation of alg3 in S. commune resulted in the production of predominantly Man(3)GlcNAc(2) protein-linked N-glycans. No impact on growth nor a developmental phenotype due to the deletion was observed. This provides a first step toward engineering of a homogeneous, humanized N-glycosylation pattern for the production of therapeutic glycoproteins in mushrooms.
    Enhanced production of Aspergillus niger laccase-like multicopper oxidases through mRNA optimization of the glucoamylase expression system
    Tamayo Ramos, J.A. ; Barends, S. ; Lange, D. ; Jel, A. de; Verhaert, R.M. ; Graaff, L.H. de - \ 2013
    Biotechnology and Bioengineering 110 (2013)2. - ISSN 0006-3592 - p. 543 - 551.
    5'-untranslated region - protein expression - gene - translation - transformation - promoter - nidulans - cloning - oryzae
    In filamentous fungi, most of the strategies used for the improvement of protein yields have been based on an increase in the transcript levels of a target gene. Strategies focusing at the translational level have been also described, but are far less explored. Here the 5' untranslated sequence of the glaA mRNA, a widely used expression system for the expression of recombinant proteins, was modified by the introduction of different nucleotide elements that have positive role in the translation process. Five Aspergillus niger laccase-like multicopper oxidases (MCOs) coding genes were fused to the native glaA 5'UTR and the three synthetic versions (sUTR1, sUTR2, and sUTR3) as well, and placed under the control of the glucoamylase gene promoter. Afterwards, a total of 20 fungal transformations were done using A. niger N593 as a recipient strain and 50 transformants per transformation were isolated and analyzed. The result of the incorporation of the synthetic 5'UTRs on the overall productivity of the transformants was assessed, on one hand by monitoring the laccase activity of all the isolated transformants, and on the other hand by quantifying and comparing the activity of those secreting the highest level of each MCO. For this purpose, a high-throughput method for the screening and selection of the best producers was developed. Once the best transformants producing the highest yield of McoA, McoB, McoC, McoD, and McoJ laccases were selected, their production level was quantified in supernatants of liquid cultures. The results obtained in this work indicate that modifications in the native glaA 5'UTR can lead to improvements in protein yields. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc
    Functional analysis of inter-individual transcriptome differential expression in pig longissimus muscle
    Zhao, S. ; Hulsegge, B. ; Harders, F.L. ; Bossers, R. ; Keuning, E. ; Hoekman, A.J.W. ; Hoving-Bolink, A.H. ; Pas, M.F.W. te - \ 2013
    Journal of Animal Breeding and Genetics 130 (2013)1. - ISSN 0931-2668 - p. 72 - 78.
    meat quality - microarrays - discovery - traits - fibers - gene - pork
    Selection of pigs for increased meat production or improved meat quality changes muscle mass and muscle composition. This will be related to transcriptome expression profile changes in muscle tissue, generating inter-individual differences. This study investigated the differentially expressed genes in the transcriptome profiles of the longissimus muscle of 75 Large White–Duroc cross sows and castrates. The use of a common reference design enabled to investigate the inter-individual transcriptome expression profile differences between the animals as compared with the means of all animals. The aim of the study was to identify the biological processes related to these inter-individual differences. It was expected that these processes underlie the selection effects. In total, 908 transcripts were differentially expressed. Among them, 762 were mainly downregulated and 146 were mainly upregulated. Gene Ontology and Pathways analyses indicated that the differentially expressed genes belong to three groups of processes involved in protein synthesis and amino acid–protein metabolism, energy metabolism and muscle-specific structure and activity processes. Comparing the functional biological analysis results with previously reported data suggested that the protein synthesis, energy metabolism and muscle-specific structure would contribute to meat production and the meat quality
    The turbulent life of Sirevirus retrotransposons and the evolution of the maize genome: more than ten thousand elements tell the story
    Bousios, A. ; Kourmpetis, Y.I.A. ; Pavlidis, P. ; Minga, E. ; Tsaftaris, A. ; Darzentas, N. - \ 2012
    The Plant Journal 69 (2012)3. - ISSN 0960-7412 - p. 475 - 488.
    plant transposable elements - ty1/copia retrotransposons - ltr retrotransposons - rice genome - diversity - gene - arabidopsis - sequence - sorghum - rearrangement
    Sireviruses are one of the three genera of Copia long terminal repeat (LTR) retrotransposons, exclusive to and highly abundant in plants, and with a unique, among retrotransposons, genome structure. Yet, perhaps due to the few references to the Sirevirus origin of some families, compounded by the difficulty in correctly assigning retrotransposon families into genera, Sireviruses have hardly featured in recent research. As a result, analysis at this key level of classification and details of their colonization and impact on plant genomes are currently lacking. Recently, however, it became possible to accurately assign elements from diverse families to this genus in one step, based on highly conserved sequence motifs. Hence, Sirevirus dynamics in the relatively obese maize genome can now be comprehensively studied. Overall, we identified >10 600 intact and approximately 28 000 degenerate Sirevirus elements from a plethora of families, some brought into the genus for the first time. Sireviruses make up approximately 90% of the Copia population and it is the only genus that has successfully infiltrated the genome, possibly by experiencing intense amplification during the last 600 000 years, while being constantly recycled by host mechanisms. They accumulate in chromosome-distal gene-rich areas, where they insert in between gene islands, mainly in preferred zones within their own genomes. Sirevirus LTRs are heavily methylated, while there is evidence for a palindromic consensus target sequence. This work brings Sireviruses in the spotlight, elucidating their lifestyle and history, and suggesting their crucial role in the current genomic make-up of maize, and possibly other plant hosts
    Hedgehog signaling via a calcitonin receptor-like receptor can induce arterial differentiation independently of VEGF signaling in zebrafish
    Wilkinson, R.N. ; Koudijs, M.J. ; Patient, R.K. ; Ingham, P.W. ; Schulte-Merker, S. ; Eeden, F.J.M. van - \ 2012
    Blood : journal of the American Society of Hematology 120 (2012)2. - ISSN 0006-4971 - p. 477 - 488.
    endothelial-growth-factor - embryonic vascular development - hematopoietic stem-cells - aortic endothelium - sonic-hedgehog - pathway - notch - blood - gene - kinase
    Multiple signaling pathways control the specification of endothelial cells (ECs) to become arteries or veins during vertebrate embryogenesis. Current models propose that a cascade of Hedgehog (Hh), vascular endothelial growth factor (VEGF), and Notch signaling acts instructively on ECs to control the choice between arterial or venous fate. Differences in the pheno-types induced by Hh, VEGF, or Notch inhibition suggest that not all of the effects of Hh on arteriovenous specification are mediated by VEGF. We establish that full derepression of the Hh pathway in ptc1;ptc2 mutants converts the posterior cardinal vein into a second arterial vessel that manifests intact arterial gene expression, intersegmental vessel sprouting, and HSC gene expression. Importantly, although VEGF was thought to be absolutely essential for arterial fates, we find that normal and ectopic arterial differentiation can occur without VEGF signaling in ptc1; ptc2 mutants. Furthermore, Hh is able to bypass VEGF to induce arterial differentiation in ECs via the calcitonin receptor-like receptor, thus revealing a surprising complexity in the interplay between Hh and VEGF signaling during arteriovenous specification. Finally, our experiments establish a dual function of Hh during induction of runx1(+) HSCs. (Blood. 2012;120(2):477-488)
    ABA-deficiency results in reduced plant and fruit size in tomato
    Nitsch, L. ; Kohlen, W. ; Oplaat, C. ; Charnikhova, T. ; Cristescu, S. ; Michieli, P. ; Wolters-Arts, M. ; Bouwmeester, H.J. ; Mariani, C. ; Vriezen, W.H. ; Rieu, I. - \ 2012
    Journal of Plant Physiology 169 (2012)9. - ISSN 0176-1617 - p. 878 - 883.
    abscisic-acid biosynthesis - shoot growth - arabidopsis-thaliana - endogenous aba - ethylene - mutants - drought - stress - gene - expression
    Abscisic acid (ABA) deficient mutants, such as notabilis and flacca, have helped elucidating the role of ABA during plant development and stress responses in tomato (Solanum lycopersicum L.). However, these mutants have only moderately decreased ABA levels. Here we report on plant and fruit development in the more strongly ABA-deficient notabilis/flacca (not/flc) double mutant. We observed that plant growth, leaf-surface area, drought-induced wilting and ABA-related gene expression in the different genotypes were strongly correlated with the ABA levels and thus most strongly affected in the not/flc double mutants. These mutants also had reduced fruit size that was caused by an overall smaller cell size. Lower ABA levels in fruits did not correlate with changes in auxin levels, but were accompanied by higher ethylene evolution rates. This suggests that in a wild-type background ABA stimulates cell enlargement during tomato fruit growth via a negative effect on ethylene synthesis.
    A naturally occurring InDel variation in BraA.FLC.b(BrFLC2) associated with flowering time variation in Brassica rapa
    Wu, J. ; Wei, K.Y. ; Cheng, F. ; Li, S.K. ; Wang, Q. ; Jianjun Zhao, Jianjun ; Bonnema, A.B. ; Wang, X.W. - \ 2012
    BMC Plant Biology 12 (2012). - ISSN 1471-2229 - 9 p.
    locus-c flc - arabidopsis-thaliana - gene - vernalization - frigida - phenotype - mutants - protein
    Background: Flowering time is an important trait in Brassica rapa crops. FLOWERING LOCUS C (FLC) is a MADS-box transcription factor that acts as a potent repressor of flowering. Expression of FLC is silenced when plants are exposed to low temperature, which activates flowering. There are four copies of FLC in B. rapa. Analyses of different segregating populations have suggested that BraA.FLC.a (BrFLC1) and BraA.FLC.b (BrFLC2) play major roles in controlling flowering time in B. rapa. Results: We analyzed the BrFLC2 sequence in nine B. rapa accessions, and identified a 57-bp insertion/deletion (InDel) across exon 4 and intron 4 resulting in a non-functional allele. In total, three types of transcripts were identified for this mutated BrFLC2 allele. The InDel was used to develop a PCR-based marker, which was used to screen a collection of 159 B. rapa accessions. The deletion genotype was present only in oil-type B. rapa, including ssp. oleifera and ssp. tricolaris, and not in other subspecies. The deletion genotype was significantly correlated with variation in flowering time. In contrast, the reported splicing site variation in BrFLC1, which also leads to a non-functional locus, was detected but not correlated with variation in flowering time in oil-type B. rapa, although it was correlated with variation in flowering time in vegetable-type B. rapa. Conclusions: Our results suggest that the naturally occurring deletion mutation across exon 4 and intron 4 in BrFLC2 gene contributes greatly to variation in flowering time in oil-type B. rapa. The observed different relationship between BrFLC1 or BrFLC2 and flowering time variation indicates that the control of flowering time has evolved separately between oil-type and vegetable-type B. rapa groups.
    A bistable circuit involving SCARECROW-RETINOBLASTOMA integrates cues to inform asymmetic stem cell division
    Cruz-Ramirez, A. ; Diaz-Trivino, S. ; Blilou, I. ; Benjamins, R. ; Long, Y. ; Scheres, B.J.G. - \ 2012
    Cell 150 (2012)5. - ISSN 0092-8674 - p. 1002 - 1015.
    protein-protein interactions - arabidopsis root-meristem - tumor-suppressor - split luciferase - auxin transport - cycle - gene - differentiation - plants - growth
    In plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOMA-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a “flip flop” that constrains asymmetric cell division to the stem cell region.
    Cloning and characterization of a tuberous root-specific promoter from cassava (Manihot esculenta Crantz)
    Koehorst-van Putten, H.J.J. ; Wolters, A.M.A. ; Pereira-Bertram, I.J. ; Berg, H. ; Krol, A.R. van der; Visser, R.G.F. - \ 2012
    Planta 236 (2012)6. - ISSN 0032-0935 - p. 1955 - 1965.
    bound-starch-synthase - storage roots - transformation - expression - gene - agrobacterium - elements - sequences - database - program
    In order to obtain a tuberous root-specific promoter to be used in the transformation of cassava, a 1,728 bp sequence containing the cassava granule-bound starch synthase (GBSSI) promoter was isolated. The sequence proved to contain light- and sugar-responsive cis elements. Part of this sequence (1,167 bp) was cloned into binary vectors to drive expression of the firefly luciferase gene. Cassava cultivar Adira 4 was transformed with this construct or a control construct in which the luciferase gene was cloned behind the 35S promoter. Luciferase activity was measured in leaves, stems, roots and tuberous roots. As expected, the 35S promoter induced luciferase activity in all organs at similar levels, whereas the GBSSI promoter showed very low expression in leaves, stems and roots, but very high expression in tuberous roots. These results show that the cassava GBSSI promoter is an excellent candidate to achieve tuberous root-specific expression in cassava.
    Pig welfare and what you can tell from the carcase
    Lambooij, E. - \ 2012
    Veterinary Record 171 (2012)24. - ISSN 0042-4900 - p. 619 - 620.
    different halothane genotypes - pre-slaughter conditions - meat quality - muscle metabolism - anesthetized pigs - domestic pigs - behavior - transport - gene
    QUALITY-oriented meat production has grown during the past decade and its aim is to improve the harmonisation of product characteristics and consumer demands. Consumer concerns about quality are not limited solely to intrinsic characteristics, for example, meat quality, but often include extrinsic aspects, such as environment and animal welfare in relation to production. Legislation in various EU-member states is often based on animal health and welfare considerations, protecting personnel working with animals, as well as the health and safety of the animals themselves. This interaction, whether it be during housing, transport or slaughter, should not lead to unnecessary excitement, pain or suffering. The current level of animal welfare protection in the EU is one of the highest in the world. However, the European approach is under pressure because of the differences in animal welfare standards between EU member states. Postmortem measurements in the slaughterhouse provide valuable information for welfare evaluation; however, a good feedback system should be available and a process-oriented animal welfare assurance system developed. It is suggested using the meat inspection as an animal welfare diagnostic tool or in animal welfare surveillance schemes at national and international levels; however, the meat inspection process needs to be standardised, as discussed in a paper on pigs summarised on p 621 in this week's issue of Veterinary Record (Harley and others 2012).
    Identification of key peptidoglycan hydrolases for morphogenesis, autolysis, and peptidoglycan composition of Lactobacillus plantarum WCFS1.
    Rolain, T. ; Bernard, E. ; Courtin, P. ; Bron, P.A. ; Kleerebezem, M. ; Chapot-Chartier, M.P. ; Hols, P. - \ 2012
    Microbial Cell Factories 11 (2012). - ISSN 1475-2859
    lactic-acid bacteria - lactococcus-lactis - n-acetylglucosaminidase - cell-wall - staphylococcus-aureus - bacillus-subtilis - murein hydrolase - gene - genome - electroporation
    Background - Lactobacillus plantarum is commonly used in industrial fermentation processes. Selected strains are also marketed as probiotics for their health beneficial effects. Although the functional role of peptidoglycan-degrading enzymes is increasingly documented to be important for a range of bacterial processes and host-microbe interactions, little is known about their functional roles in lactobacilli. This knowledge holds important potential for developing more robust strains resistant to autolysis under stress conditions as well as peptidoglycan engineering for a better understanding of the contribution of released muramyl-peptides as probiotic immunomodulators. Results - Here, we explored the functional role of the predicted peptidoglycan hydrolase (PGH) complement encoded in the genome of L. plantarum by systematic gene deletion. From twelve predicted PGH-encoding genes, nine could be individually inactivated and their corresponding mutant strains were characterized regarding their cell morphology, growth, and autolysis under various conditions. From this analysis, we identified two PGHs, the predicted N-acetylglucosaminidase Acm2 and NplC/P60 D,L-endopeptidase LytA, as key determinants in the morphology of L. plantarum. Acm2 was demonstrated to be required for the ultimate step of cell separation of daughter cells, whereas LytA appeared to be required for cell shape maintenance and cell-wall integrity. We also showed by autolysis experiments that both PGHs are involved in the global autolytic process with a dominant role for Acm2 in all tested conditions, identifying Acm2 as the major autolysin of L. plantarum WCFS1. In addition, Acm2 and the putative N-acetylmuramidase Lys2 were shown to play redundant roles in both cell separation and autolysis under stress conditions. Finally, the analysis of the peptidoglycan composition of Acm2- and LytA-deficient derivatives revealed their potential hydrolytic activities by the disappearance of specific cleavage products. Conclusion - In this study, we showed that two PGHs of L. plantarum have a predominant physiological role in a range of growth conditions. We demonstrate that the N-acetylglucosaminidase Acm2 is the major autolysin whereas the D,L-endopeptidase LytA is a key morphogenic determinant. In addition, both PGHs have a direct impact on PG structure by generating a higher diversity of cleavage products that could be of importance for interaction with the innate immune system.
    Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans
    Ketelaar, T. ; Meijer, H.J.G. ; Spiekerman, M. ; Weide, R. ; Govers, F. - \ 2012
    Fungal Genetics and Biology 49 (2012)12. - ISSN 1087-1845 - p. 1014 - 1022.
    f-actin - achlya-bisexualis - plant-cells - root hairs - tip growth - filaments - organization - arabidopsis - sequence - gene
    The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 µM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 µM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin cytoskeleton plays a role in preventing the movement of nuclei towards the hyphal tip.
    Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche
    Morin, E. ; Kohler, A. ; Baker, A.R. ; Foulongne-Oriol, M. ; Lombard, V. ; Nagy, L.G. ; Ohm, R.A. ; Patyshakuliyeva, A. ; Brun, A. ; Aerts, A.L. ; Bailey, A.M. ; Billette, C. ; Coutinho, P.M. ; Deakin, G. ; Doddapaneni, H. ; Floudas, D. ; Grimwood, J. ; Hildén, K. ; Kües, U. ; LaButti, K.M. ; Lapidus, A. ; Lindquist, E.A. ; Lucas, S.M. ; Murat, C. ; Riley, R.W. ; Salamov, A.A. ; Schmutz, J. ; Subramanian, V. ; Wösten, H.A.B. ; Xu, J. ; Eastwood, D.C. ; Foster, G.D. ; Sonnenberg, A.S.M. ; Cullen, D. ; Vries, R.P. de; Lundell, T. ; Hibbett, D.S. ; Henrissat, B. ; Burton, K.S. ; Kerrigan, R.W. ; Challen, M.P. ; Grigorievf, I.V. ; Martin, M. - \ 2012
    Proceedings of the National Academy of Sciences of the United States of America 109 (2012)43. - ISSN 0027-8424 - p. 17501 - 17506.
    cultivated mushroom - coprinus-cinereus - schizophyllum-commune - coprinopsis-cinerea - fungi - gene - decomposition - chromosomes - degradation - substances
    Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the “button mushroom” forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and ß-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.
    Analyses of pig genomes provide insight to porcine demography and evolution
    Groenen, M.A.M. ; Megens, H.J.W.C. ; Frantz, L.A.F. ; Bosse, M. ; Crooijmans, R.P.M.A. ; Dibbits, B.W. ; Madsen, O. ; Paudel, Y. - \ 2012
    Nature 491 (2012). - ISSN 0028-0836 - p. 393 - 398.
    sus-scrofa - sequence - gene - receptor - locus - variants - complex - taste
    For 10,000¿years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ~1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model.
    Protective effect of Phellinus linteus polysaccharide extracts against thioacetamide-induced liver fibrosis in rats: a proteomics analysis
    Wang, H. ; Wu, G. ; Park, H.J. ; Jiang, P.P. ; Sit, W.H. ; Griensven, L.J.L.D. van; Wan, J.M.F. - \ 2012
    Chinese Medicine 7 (2012). - ISSN 1749-8546
    hepatocellular-carcinoma - oxidative stress - chain - injury - mice - gene - methyltransferase - differentiation - dehydrogenase - metastasis
    Background: The hepatoprotective potential of Phellinus linteus polysaccharide (PLP) extracts has been described. However, the molecular mechanism of PLP for the inhibition of liver fibrosis is unclear. This study aims to investigate the molecular protein signatures involved in the hepatoprotective mechanisms of PLP via a proteomics approach using a thioacetamide (TAA)-induced liver fibrosis rat model. Methods: Male Sprague-Dawley rats were divided into three groups of six as follows: Normal group; TAA group, in which rats received TAA only; and PLP group, in which rats received PLP and TAA. Liver fibrosis was induced in the rats by repeated intraperitoneal injections of TAA at a dose of 200 mg/kg body weight twice a week for 4 weeks. PLP was given orally at a dose of 50 mg/kg body weight twice a day from the beginning of the TAA treatment until the end of the experiment. The development of liver cirrhosis was verified by histological examination. Liver proteomes were established by two-dimensional gel electrophoresis. Proteins with significantly altered expression levels were identified by matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry and the differentially expressed proteins were validated by immunohistochemical staining and reverse transcription polymerase chain reaction. Results: Histological staining showed a remarkable reduction in liver fibrosis in the rats with PLP treatment. A total of 13 differentially expressed proteins including actin, tubulin alpha-1C chain, preprohaptoglobin, hemopexin, galectin-5, glutathione S-transferase alpha-4 (GSTA4), branched chain keto acid dehydrogenase hterotetrameric E1 subunit alpha (BCKDHA), glutathione S-transferase mu (GSTmu); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); thiosulfate sulfurtransferase (TFT); betaine-homocysteine S-methyltransferase 1 (BHMT1); quinoid dihydropteridine reductase (QDPR); ribonuclease UK114 were observed between the TAA and PLP groups. These proteins are involved in oxidative stress, heme and iron metabolism, cysteine metabolism, and branched-chain amino acid catabolism. Conclusion: The proteomics data indicate that P. linteus may be protective against TAA-induced liver fibrosis via regulation of oxidative stress pathways, heat shock pathways, and metabolic pathways for amino acids and nucleic acids.
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