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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    Pestalotiopsis revisited
    Maharachchikumbura, S.S.N. ; Hyde, K.D. ; Groenewald, J.Z. ; Xu, J. ; Crous, P.W. - \ 2014
    Studies in Mycology 79 (2014). - ISSN 0166-0616 - p. 121 - 186.
    ribosomal dna-sequences - sp-nov - morphological characters - conidial structure - camellia-sinensis - natural-products - twig blight - primer sets - leaf-spot - disease
    Species of Pestalotiopsis occur commonly as plant pathogens, and represent a fungal group known to produce a wide range of chemically novel, diverse metabolites. In the present study, we investigated 91 Pestalotiopsis isolates from the CBS-KNAW Fungal Biodiversity Centre (CBS) culture collection. The phylogeny of the Amphisphaeriaceae was constructed based on analysis of 28S nrRNA gene (LSU) sequence data, and taxonomic changes are proposed to reflect more natural groupings. We combined morphological and DNA data, and segregated two novel genera from Pestalotiopsis, namely Neopestalotiopsis and Pseudopestalotiopsis. The three genera are easily distinguishable on the basis of their conidiogenous cells and colour of their median conidial cells. We coupled morphological and combined sequence data of internal transcribed spacer (ITS), partial ß-tubulin (TUB) and partial translation elongation factor 1-alpha (TEF) gene regions, which revealed 30 clades in Neopestalotiopsis and 43 clades in Pestalotiopsis. Based on these data, 11 new species are introduced in Neopestalotiopsis, 24 in Pestalotiopsis, and two in Pseudopestalotiopsis. Several new combinations are proposed to emend monophyly of Neopestalotiopsis, Pestalotiopsis and Pseudopestalotiopsis.
    Coral aquaculture to support drug discovery
    Leal, M.C. ; Calado, R. ; Sheridan, C. ; Alimonti, A. ; Osinga, R. - \ 2013
    Trends in Biotechnology 31 (2013)10. - ISSN 0167-7799 - p. 555 - 561.
    reef restoration - natural-products - sinularia-flexibilis - cultivation - growth - mucus - bacteria - diseases - ecology - sponges
    Marine natural products (NP) are unanimously acknowledged as the blue gold in the urgent quest for new pharmaceuticals. Although corals are among the marine organisms with the greatest diversity of secondary metabolites, growing evidence suggest that their symbiotic bacteria produce most of these bioactive metabolites. The ex hospite culture of coral symbiotic microbiota is extremely challenging and only limited examples of successful culture exist today. By contrast, in toto aquaculture of corals is a commonly applied technology to produce corals for aquaria. Here, we suggest that coral aquaculture could as well be a viable and economically feasible option to produce the biomass required to execute the first steps of the NP-based drug discovery pipeline.
    Solvent exchange module for LC-NMR hyphenation using machine vision-controlled droplet evaporation
    Schoonen, J.W. ; Vulto, P. ; Roo, N. de; Duynhoven, J.P.M. van; Linden, H. van der; Hankemeier, T. - \ 2013
    Analytical Chemistry 85 (2013)12. - ISSN 0003-2700 - p. 5734 - 5739.
    solid-phase extraction - nuclear-magnetic-resonance - red wine/grape juice - ms-spe-nmr - natural-products - liquid-chromatography - flow probe - black tea - identification - system
    We report the use of pendant droplet evaporation for exchange of eluents for 1H nuclear magnetic resonance (1H NMR) purposes. Analytes are fed and retained in 500 nL droplets, which are concentrated by evaporation and subsequently redissolved in deuterated solvent. Droplet size is monitored by machine vision (MV), and heating rates are adjusted concordingly to maintain a stable droplet volume. Evaporation control is independent of solvent properties, and the setup handles feed rates up to 7 µL min–1. The interface is capable of exchanging up to 90% of solvent for deuterated solvent, with a good recovery and repeatability for tomato extracts (Solanum lycopersicum). The system was capable of handling both polar and nonpolar analytes in one run. Volatiles such as formate, acetate, and lactate and the thermosensitive compound epigallocatechin gallate were recovered without significant losses. Ethanol and propionate were recovered with significant losses due to the formation of a minimum boiling azeotrope. The current setup is ideally suited for on- and off-line hyphenation of liquid chromatography and NMR, as it is comprehensive, fully automated, and easy to operate.
    Phyllosticta capitalensis, a widespread endophyte of plants
    Wikee, S. ; Lombard, L. ; Crous, P.W. ; Nakashima, C. ; Motohashi, K. ; Chukeatirote, E. ; Alias, S.A. ; McKenzie, E.H.C. ; Hyde, K.D. - \ 2013
    Fungal Diversity 60 (2013)1. - ISSN 1560-2745 - p. 91 - 105.
    citrus black spot - guignardia-citricarpa - fungal endophytes - natural-products - latent pathogens - musa-acuminata - woody-plants - diversity - thailand - banana
    Phyllosticta capitalensis is an endophyte and weak plant pathogen with a worldwide distribution presently known from 70 plant families. This study isolated P. capitalensis from different host plants in northern Thailand, and determined their different life modes. Thirty strains of P. capitalensis were isolated as endophytes from 20 hosts. An additional 30 strains of P. capitalensis from other hosts and geographic locations were also obtained from established culture collections. Phylogenetic analysis using ITS, ACT and TEF gene data confirmed the identity of all isolates. Pathogenicity tests with five strains of P. capitalensis originating from different hosts were completed on their respective host plants. In all cases there was no infection of healthy leaves, indicating that this endophyte does not cause disease on healthy, unstressed host plants. That P. capitalensis is often isolated as an endophyte has important implications in fungal biology and plant health. Due to its endophytic nature, P. capitalensis is commonly found associated with lesions of plants, and often incorrectly identified as a species of quarantine importance, which again has implications for trade in agricultural and forestry production.
    The Gac regulon of Pseudomonas fluorescens SBW25
    Cheng, X. ; Bruijn, I. de; Voort, M. van der; Loper, J.E. ; Raaijmakers, J.M. - \ 2013
    Environmental Microbiology Reports 5 (2013)4. - ISSN 1758-2229 - p. 608 - 619.
    iii secretion system - signal-transduction system - mediated iron uptake - biological-control - protein secretion - natural-products - gene-expression - vi secretion - fumarase c - small rnas
    Transcriptome analysis of Pseudomonas fluorescens SBW25 showed that 702 genes were differentially regulated in a gacS::Tn5 mutant, with 300 and 402 genes up- and downregulated respectively. Similar to the Gac regulon of other Pseudomonas species, genes involved in motility, biofilm formation, siderophore biosynthesis and oxidative stress were differentially regulated in the gacS mutant of SBW25. Our analysis also revealed, for the first time, that transcription of 19 rhizosphere-induced genes and of genes involved in type II secretion, (exo)polysaccharide and pectate lyase biosynthesis, twitching motility and an orphan non-ribosomal peptide synthetase (NRPS) were significantly affected in the gacS mutant. Furthermore, the gacS mutant inhibited growth of oomycete, fungal and bacterial pathogens significantly more than wild type SBW25. Since RP-HPLC analysis did not reveal any potential candidate metabolites, we focused on the Gac-regulated orphan NRPS gene cluster that was predicted to encode an eight-amino-acid ornicorrugatin-like peptide. Site-directed mutagenesis indicated that the encoded peptide is not involved in the enhanced antimicrobial activity of the gacS mutant but may function as a siderophore. Collectively, this genome-wide analysis revealed that a mutation in the GacS/A two-component regulatory system causes major transcriptional changes in SBW25 and significantly enhances its antimicrobial activities by yet unknown mechanisms.
    Sponge Hybridomas: Applications and Implications
    Pomponi, S.A. ; Jevitt, A. ; Patel, J. ; Diaz, M.C. - \ 2013
    Integrative and Comparative Biology 53 (2013)3. - ISSN 1540-7063 - p. 524 - 530.
    cell-cultures - natural-products - porifera - populations - diversity - systems
    Many sponge-derived natural products with applications to human health have been discovered over the past three decades. In vitro production has been proposed as one biological alternative to ensure adequate supply of marine natural products for preclinical and clinical development of drugs. Although primary cell cultures have been established for many marine phyla, no cell lines with an extended life span have been established for marine invertebrates. Hybridoma technology has been used for production of monoclonal antibodies for application to human health. We hypothesized that a sponge cell line could be formed by fusing sponge cells of one species with those of another, or by fusing sponge cells with rapidly dividing, marine-derived, non-sponge cells. Using standard methods for formation of hybridomas, with appropriate modifications for temperature and salinity, cells from individuals of the same sponge species, as well as cells from individuals of two different sponge species were successfully fused. Research in progress is focused on optimizing fusion to produce a cell line and to stimulate expression of natural products with therapeutic relevance. Experimental hybridomas may also be used as models to test hypotheses related to naturally occurring sponge chimeras and hybridomas.
    Mass spectral molecular networking of living microbial colonies
    Watrous, J. ; Roach, P. ; Alexandrov, T. ; Heath, B.S. ; Yang, J.Y. ; Kersten, R.D. ; Voort, M. van der; Pogliano, K. ; Gross, H. ; Raaijmakers, J. ; Moore, B.S. ; Laskin, J. ; Bandeira, N. ; Dorrestein, P.C. - \ 2012
    Proceedings of the National Academy of Sciences of the United States of America 109 (2012)26. - ISSN 0027-8424 - p. E1743 - E1752.
    desorption electrospray-ionization - assisted laser desorption/ionization - bacillus-subtilis - metabolic exchange - ambient conditions - natural-products - assembly-line - spectrometry - identification - antibiotics
    Integrating the governing chemistry with the genomics and phenotypes of microbial colonies has been a “holy grail” in microbiology. This work describes a highly sensitive, broadly applicable, and cost-effective approach that allows metabolic profiling of live microbial colonies directly from a Petri dish without any sample preparation. Nanospray desorption electrospray ionization mass spectrometry (MS), combined with alignment of MS data and molecular networking, enabled monitoring of metabolite production from live microbial colonies from diverse bacterial genera, including Bacillus subtilis, Streptomyces coelicolor, Mycobacterium smegmatis, and Pseudomonas aeruginosa. This work demonstrates that, by using these tools to visualize small molecular changes within bacterial interactions, insights can be gained into bacterial developmental processes as a result of the improved organization of MS/MS data. To validate this experimental platform, metabolic profiling was performed on Pseudomonas sp. SH-C52, which protects sugar beet plants from infections by specific soil-borne fungi [R. Mendes et al. (2011) Science 332:1097–1100]. The antifungal effect of strain SH-C52 was attributed to thanamycin, a predicted lipopeptide encoded by a nonribosomal peptide synthetase gene cluster. Our technology, in combination with our recently developed peptidogenomics strategy, enabled the detection and partial characterization of thanamycin and showed that it is a monochlorinated lipopeptide that belongs to the syringomycin family of antifungal agents. In conclusion, the platform presented here provides a significant advancement in our ability to understand the spatiotemporal dynamics of metabolite production in live microbial colonies and communities
    antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences
    Medema, M.H. ; Blin, K. ; Cimermancic, P. ; Jager, V.C.L. de; Zakrzewski, P. ; Fischbach, M.A. ; Weber, T. ; Takano, E. ; Breitling, R. - \ 2011
    Nucleic acids research 39 (2011)Suppl. 2. - ISSN 0305-1048 - p. W339 - W346.
    polyketide synthases - natural-products - prediction - evolution - domains - specificity
    Bacterial and fungal secondary metabolism is a rich source of novel bioactive compounds with potential pharmaceutical applications as antibiotics, anti-tumor drugs or cholesterol-lowering drugs. To find new drug candidates, microbiologists are increasingly relying on sequencing genomes of a wide variety of microbes. However, rapidly and reliably pinpointing all the potential gene clusters for secondary metabolites in dozens of newly sequenced genomes has been extremely challenging, due to their biochemical heterogeneity, the presence of unknown enzymes and the dispersed nature of the necessary specialized bioinformatics tools and resources. Here, we present antiSMASH (antibiotics & Secondary Metabolite Analysis Shell), the first comprehensive pipeline capable of identifying biosynthetic loci covering the whole range of known secondary metabolite compound classes (polyketides, non-ribosomal peptides, terpenes, aminoglycosides, aminocoumarins, indolocarbazoles, lantibiotics, bacteriocins, nucleosides, beta-lactams, butyrolactones, siderophores, melanins and others). It aligns the identified regions at the gene cluster level to their nearest relatives from a database containing all other known gene clusters, and integrates or cross-links all previously available secondary-metabolite specific gene analysis methods in one interactive view. antiSMASH is available at
    Differential gene expression in nearly isogenic lines with QTL for partial resistance to Puccinia hordei in barley
    Chen, X. ; Niks, R.E. ; Hedley, P.E. ; Morris, J. ; Druka, A. ; Marcel, T.C. ; Vels, S.A. ; Waugh, R. - \ 2010
    BMC Genomics 11 (2010). - ISSN 1471-2164 - 13 p.
    heterologous rust fungi - disease resistance - quantitative resistance - pathogen interactions - rhizoctonia-solani - natural-products - plant immunity - powdery mildew - sheath blight - cell-wall
    Background - The barley-Puccinia hordei (barley leaf rust) pathosystem is a model for investigating partial disease resistance in crop plants and genetic mapping of phenotypic resistance has identified several quantitative trait loci (QTL) for partial resistance. Reciprocal QTL-specific near-isogenic lines (QTL-NILs) have been developed that combine two QTL, Rphq2 and Rphq3, the largest effects detected in a recombinant-inbred-line (RIL) population derived from a cross between the super-susceptible line L94 and partially-resistant line Vada. The molecular mechanism underpinning partial resistance in these QTL-NILs is unknown. Results - An Agilent custom microarray consisting of 15,000 probes derived from barley consensus EST sequences was used to investigate genome-wide and QTL-specific differential expression of genes 18 hours post-inoculation (hpi) with Puccinia hordei. A total of 1,410 genes were identified as being significantly differentially expressed across the genome, of which 55 were accounted for by the genetic differences defined by QTL-NILs at Rphq2 and Rphq3. These genes were predominantly located at the QTL regions and are, therefore, positional candidates. One gene, encoding the transcriptional repressor Ethylene-Responsive Element Binding Factor 4 (HvERF4) was located outside the QTL at 71 cM on chromosome 1H, within a previously detected eQTL hotspot for defence response. The results indicate that Rphq2 or Rphq3 contains a trans-eQTL that modulates expression of HvERF4. We speculate that HvERF4 functions as an intermediate that conveys the response signal from a gene(s) contained within Rphq2 or Rphq3 to a host of down-stream defense responsive genes. Our results also reveal that barley lines with extreme or intermediate partial resistance phenotypes exhibit a profound similarity in their spectrum of Ph-responsive genes and that hormone-related signalling pathways are actively involved in response to Puccinia hordei. Conclusions - Differential gene expression between QTL-NILs identifies genes predominantly located within the target region(s) providing both transcriptional and positional candidate genes for the QTL. Genetically mapping the differentially expressed genes relative to the QTL has the potential to discover trans-eQTL mediated regulatory relays initiated from genes within the QTL regions
    Recent developments in the rapid analysis of plants and tracking their bioactive constituents
    Beek, T.A. van; Tetala, K.K.R. ; Koleva, I. ; Dapkevicius, A. ; Exarchou, V. ; Jeurissen, S.M.F. ; Claassen, F.W. ; Klift, E.J.C. van der - \ 2009
    Phytochemistry Reviews 8 (2009)2. - ISSN 1568-7767 - p. 387 - 399.
    desorption electrospray-ionization - performance liquid-chromatography - 2-dimensional gas-chromatography - mass-spectrometric detection - radical scavenging compounds - nuclear-magnetic-resonance - thin-layer-chromatography - natural-products - biochemical detec
    Natural products chemistry has witnessed many new developments in the last 5 years like extractions with subcritical water and ionic liquids, LC/HRMS and LC/SPE/cryo-NMR, UHPLC, TLC/MS, MS-based preparative HPLC, comprehensive chromatography (GC × GC, LC × LC), high-throughput screening, introduction of monolithic columns, miniaturisation, and automated structure identification. Nevertheless identifying bioactive constituents in complex plant extracts remains a tedious process. The classical approach of bioassay guided fractionation is time-consuming while off-line screening of extracts does not provide information on individual compounds and sometimes suffers from false positives or negatives. One way out of this is by coupling chromatography with chemical or biochemical assays, so called high resolution screening. An example is the development of HPLC on-line assays for antioxidants. By the post-column addition of a relatively stable coloured radical like DPPH¿ or ABTS¿+, radical scavengers are detected as negative peaks because in a reaction coil they reduce the model radical to its reduced, non-coloured form. When combined with LC/DAD/MS and LC/SPE/NMR, reliable identification of active constituents becomes possible without the necessity of ever isolating them in a classical sense. Also for finding leads for new drugs, combining HPLC with biochemical assays is interesting but technically more difficult. Most enzymes do not work at the organic modifier concentrations commonly encountered in RP-HPLC and the reaction time is often longer requiring dilution and lengthy coils respectively. Therefore, new techniques have to be implemented to gain the required sensitivity for on-line enzyme assays. For stable analytes, high temperature LC offers a solution to the organic modifier problem. When enzymes are highly expensive, like those used in the screening for Cytochrome P450 inhibitors, miniaturisation to chip format may offer a way out. Microreactors (chips) are not only useful for miniaturising larger assays but also offer completely new prospects in phytochemical analysis. One such application is in the sample clean-up of acids and bases like alkaloids. In a lay-out of three parallel channels of 100 ¿m width with the middle one containing organic phase and the two outer ones water of high pH (feed phase) and low pH (trapping phase) such a chip replaces two classical LLE steps but is much faster and requires less solvents and less manpower input.
    A procedure for the metagenomics exploration of disease-suppressive soils
    Elsas, J.D. van; Speksnijder, A. ; Overbeek, L.S. van - \ 2008
    Journal of Microbiological Methods 75 (2008)3. - ISSN 0167-7012 - p. 515 - 522.
    microbial community structure - gene-cluster - uncultured microorganisms - environmental libraries - natural-products - diversity - dna - bacteria - expression - access
    The microbiota of, in particular, disease-suppressive soils contains a wealth of antibiotic biosynthetic loci that are inaccessible by traditional cultivation-based techniques. Hence, we developed a methodology based on soil microbial DNA, which allowed the metagenomics-based unlocking of the relevant genes. Here, a streamlined soil metagenomics protocol is presented. The protocol consists of an optimized method to extract bacterial cells from a Rhizoctonia solani AG3 suppressive loamy sand soil followed by DNA extraction and purification, and the preparation of a clone library in an efficient host/vector system. Methods for the functional and genetic screening of the library for antibiotic production loci are also described. Using the suppressive soil, we thus produced, screened and tested an approximate 15,000-membered metagenomic library of fosmids in an Escherichia coli host. Functional screens, based on dual culturing of clone arrays with R. solani AG3 and Bacillus subtilis 168, were largely negative. Genetic screens, based on hybridizations with soil-generated probes for polyketide biosynthesis, non-ribosomal protein synthesis and gacA, revealed several inserts, of around 40-kb in size, with potential antibiotic production capacity. We present the full sequences of three selected clones. We further examine the challenges that still impinge on the metagenomic exploration of disease-suppressive soil.
    The metagenomics of disease-suppressive soils - Experiences from the METACONTROL project (accepted)
    Elsas, J.D. van; Jansson, J. ; Sjöling, S. ; Bailey, M. ; Nalin, R. ; Vogel, T. ; Costa, R. ; Overbeek, L.S. van - \ 2008
    Trends in Biotechnology 26 (2008)11. - ISSN 0167-7799 - p. 591 - 601.
    wide host-range - community structure - uncultured microorganisms - functional diversity - escherichia-coli - environmental libraries - microbial communities - natural-products - gene-expression - dna
    Soil teems with microbial genetic information that can be exploited for biotechnological innovation. Because only a fraction of the soil microbiota is cultivable, our ability to unlock this genetic complement has been hampered. Recently developed molecular tools, which make it possible to utilize genomic DNA from soil, can bypass cultivation and provide information on the collective soil metagenome with the aim to explore genes that encode functions of key interest to biotechnology. The metagenome of disease-suppressive soils is of particular interest given the expected prevalence of antibiotic biosynthetic clusters. However, owing to the complexity of soil microbial communities, deciphering this key genetic information is challenging. Here, we examine crucial issues and challenges that so far have hindered the metagenomic exploration of soil by drawing on experience from a trans-European project on disease-suppressive soils denoted METACONTROL.
    Antioxidant activity assays on-line with liquid chromatography
    Niederländer, H.A.G. ; Beek, T.A. van; Barsatute, A. ; Koleva, I. - \ 2008
    Journal of Chromatography. A, Including electrophoresis and other separation methods 1210 (2008)2. - ISSN 0021-9673 - p. 121 - 134.
    radical scavenging compounds - coulometric array detection - flow-injection system - dpph screening method - dad-spe-nmr - phenolic-compounds - electrochemical detection - natural-products - plant-extracts - chemiluminescence detection
    Screening for antioxidants requires simple in vitro model systems to investigate antioxidant activity. High resolution screening (HRS), combining a separation technique like HPLC with fast post-column (bio)chemical detection can rapidly pinpoint active compounds in complex mixtures. In this paper both electrochemical and chemistry-based assays are reviewed and discussed. The focus is on the mechanisms involved and differences between the assays, rather than on the matrix or analytes. With 45 applications high resolution antioxidant screening has now become an almost routine tool for the rapid identification of antioxidants in plant extracts, foods and beverages. The methods based on true reactive oxygen species (ROS) provide the most realistic measure of antioxidant activity. Unfortunately these methods are difficult to set up and control and have not been applied since they were reported. The methods based on electrochemical detection are more practical, but have still received only limited attention for practical screening purposes. The methods based on a single relatively stable reagent such as DPPH and ABTS+ have become most popular, because of their simple set-up and ease of control. The methods have been combined with on-line DAD, MS and NMR detection for rapid identification of active constituents.
    Stereochemical preference of yeast epoxide hydrolase for the O-axial C3 epimers of 1-oxaspiro[2.5] octanes
    Weijers, C.A.G.M. ; Koenst, P. ; Franssen, M.C.R. ; Sudhölter, E.J.R. - \ 2007
    Organic & Biomolecular Chemistry 5 (2007)19. - ISSN 1477-0520 - p. 3106 - 3114.
    angiogenesis inhibitors - kinetic resolution - natural-products - methionine aminopeptidase - conformational-analysis - rhodotorula-glutinis - variable-temperature - fumagillin analogs - stereoselectivity - hydrolysis
    The 1-oxaspiro[2.5]octane moiety is a common motif in many biologically active spiroepoxide compounds. Stereochemistry plays an important role in the action of these spiroepoxides, since the O-axial C3 epimers are predominantly responsible for biological activity. In view of this, the reactivity of the yeast epoxide hydrolase (YEH) from Rhodotorula glutinis towards both O-axial and O-equatorial C3 epimers of various 1-oxaspiro[2.5]octanes was investigated. O-axial C3 Epimers were hydrolyzed faster than the O-equatorial C3 epimers. The stereochemical preference was greatly dependent on the type of substitution on the cyclohexane ring. The preference of YEH for O-axial C3 epimers, found throughout this study, illustrates the effectiveness of YEH in enzymatic detoxification of spiroepoxides
    Finding the needles in the meta-genome haystack
    Kowalchuk, G.A. ; Speksnijder, A.G.C.L. ; Zhang, K. ; Goodman, R.M. ; Veen, J.A. van - \ 2007
    Microbial Ecology 53 (2007)3. - ISSN 0095-3628 - p. 475 - 485.
    whole-genome amplification - ribosomal-rna genes - soil dna libraries - wide host-range - enrichment cultures - uncultured microorganisms - microbial diversity - escherichia-coli - natural-products - messenger-rna
    In the collective genomes (the metagenome) of the microorganisms inhabiting the Earth's diverse environments is written the history of life on this planet. New molecular tools developed and used for the past 15 years by microbial ecologists are facilitating the extraction, cloning, screening, and sequencing of these genomes. This approach allows microbial ecologists to access and study the full range of microbial diversity, regardless of our ability to culture organisms, and provides an unprecedented access to the breadth of natural products that these genomes encode. However, there is no way that the mere collection of sequences, no matter how expansive, can provide full coverage of the complex world of microbial metagenomes within the foreseeable future. Furthermore, although it is possible to fish out highly informative and useful genes from the sea of gene diversity in the environment, this can be a highly tedious and inefficient procedure. Microbial ecologists must be clever in their pursuit of ecologically relevant, valuable, and niche-defining genomic information within the vast haystack of microbial diversity. In this report, we seek to describe advances and prospects that will help microbial ecologists glean more knowledge from investigations into metagenomes. These include technological advances in sequencing and cloning methodologies, as well as improvements in annotation and comparative sequence analysis. More significant, however, will be ways to focus in on various subsets of the metagenome that may be of particular relevance, either by limiting the target community under study or improving the focus or speed of screening procedures. Lastly, given the cost and infrastructure necessary for large metagenome projects, and the almost inexhaustible amount of data they can produce, trends toward broader use of metagenome data across the research community coupled with the needed investment in bioinformatics infrastructure devoted to metagenomics will no doubt further increase the value of metagenomic studies in various environments.
    Marine sponges as pharmacy
    Sipkema, D. ; Franssen, M.C.R. ; Osinga, R. ; Tramper, J. ; Wijffels, R.H. - \ 2005
    Marine Biotechnology 7 (2005)3. - ISSN 1436-2228 - p. 142 - 162.
    protein-kinase-c - inositol 1,4,5-trisphosphate receptor - immunodeficiency-virus type-1 - phospholipase a(2) inhibitor - natural-products - antimalarial activity - acanthella-cavernosa - imidazole alkaloids - alpha-glucosidase - crambe-crambe
    Marine sponges have been considered as a gold mine during the past 50 years, with respect to the diversity of their secondary metabolites. The biological effects of new metabolites from sponges have been reported in hundreds of scientific papers, and they are reviewed here. Sponges have the potential to provide future drugs against important diseases, such as cancer, a range of viral diseases, malaria, and inflammations. Although the molecular mode of action of most metabolites is still unclear, for a substantial number of compounds the mechanisms by which they interfere with the pathogenesis of a wide range of diseases have been reported. This knowledge is one of the key factors necessary to transform bioactive compounds into medicines. Sponges produce a plethora of chemical compounds with widely varying carbon skeletons, which have been found to interfere with pathogenesis at many different points. The fact that a particular disease can be fought at different points increases the chance of developing selective drugs for specific targets
    Large-scale production of pharmaceuticals by marine sponges: Sea, cell, or synthesis?
    Sipkema, D. ; Osinga, R. ; Schatton, W. ; Mendola, D. ; Tramper, J. ; Wijffels, R.H. - \ 2005
    Biotechnology and Bioengineering 90 (2005)2. - ISSN 0006-3592 - p. 201 - 222.
    enantioselective total-synthesis - antitumor polyether macrolides - natural-products - suberites-domuncula - perfusion system - growth dynamics - crambe-crambe - dysidea-avara - culture - primmorphs
    Marine sponges are known to produce an overwhelming array of secondary metabolites with pharmaceutical potential. The technical and economical potential of using marine sponges for large-scale production of these compounds was assessed for two cases: the anticancer molecule halichondrin B from a Lissodendoryx sp., and avarol from Dysidea avara for its antipsoriasis activity. An economic and technical analysis was done for three potential production methods: mariculture, ex situ culture (in tanks), and cell culture. We concluded that avarol produced by mariculture or ex situ culture could become a viable alternative to currently used pharmaceuticals for the treatment of psoriasis. Production of halichondrin B from sponge biomass was found to not be a feasible process, mainly due to the extremely low concentration of the compound in the sponge. Technical feasibility was also analyzed for five alternatives: chemical synthesis, wild harvest, primmorph culture, genetic modification and semi-synthesis. It was concluded that the latter two approaches could prove to be valuable methods for the production of pharmaceuticals, based on chemical structures of secondary metabolites present in trace amounts in marine sponges. © 2005 Wiley Periodicals, Inc.
    Realizing the promises of marine biotechnology
    Luiten, E.E.M. ; Akkerman, I. ; Koulman, A. ; Kamermans, P. ; Reith, H. ; Barbosa, M.J. ; Sipkema, D. ; Wijffels, R.H. - \ 2003
    Biomolecular Engineering 20 (2003). - ISSN 1389-0344 - p. 429 - 439.
    High-quality research in the field of marine biotechnology is one of the key-factors for successful innovation in exploiting the vast diversity of marine life. However, fascinating scientific research with promising results and claims on promising potential applications (e.g. for pharmaceuticals, nutritional supplements, (feed-)products for aquaculture and bioremediation solutions) is not the only factor to realise the commercial applications of marine biotechnology. What else is needed to exploit the promising potential of marine biotechnology and to create new industrial possibilities? In the study project 'Ocean Farming-Sustainable exploitation of marine organisms', we explore the possibilities of marine organisms to fulfill needs, such as safe and healthy food, industrial (raw) materials and renewable energy in a sustainable way. One of the three design groups is envisioning the future of strong land-based 'marine' market chains. Marine biotechnology is one of the foci of attention in this design group. This article provides a model of future-oriented thinking in which a variety of experts actively participate. (C) 2003 Elsevier B.V. All rights reserved.
    LC-UV-solid-phase extraction-NMR-MS combined with a cryogenic flow probe and its application to the identification of compounds present in Greek oregano
    Exarchou, V. ; Godejohann, M. ; Beek, T.A. van; Gerothanassis, I.P. ; Vervoort, J.J.M. - \ 2003
    Analytical Chemistry 75 (2003)22. - ISSN 0003-2700 - p. 6288 - 6294.
    magnetic-resonance-spectroscopy - performance liquid-chromatography - high-resolution nmr - tandem mass-spectrometry - to-noise ratio - hplc-nmr - solvent-suppression - natural-products - plant-extracts - constituents
    Structure elucidation of natural products usually relies on a combination of NMR spectroscopy with mass spectrometry whereby NMR trails MS in terms of the minimum sample amount required. In the present study, the usefulness of on-line solid-phase extraction (SPE) in LC-NMR for peak storage after the LC separation prior to NMR analysis is demonstrated. The SPE unit allows the use of normal protonated solvents for the LC separation and fully deuterated solvents for flushing the trapped compounds to the NAIR probe. Thus, solvent suppression is no longer necessary. Multiple trapping of the same analyte from repeated LC injections was utilized to solve the problem of low concentration and to obtain 2D heteronuclear NMR spectra. In addition, a combination of the SPE unit with a recently developed cryoflow NMR probe and an MS was evaluated. This on-line LC-UV-SPE-NMR-MS system was used for the automated analysis of a Greek oregano extract. Combining the data provided by the UV, MS, and NMR spectra, the flavonoids taxifolin, aromadendrin, eriodictyol, naringenin, and apigenin, the phenolic acid rosmarinic acid, and the monoterpene carvacrol were identified. This automated technique is very useful for natural product analysis, and the large sensitivity improvement leads to significantly reduced NAIR acquisition times.
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