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  • 351.
    Amirkhanov, N V
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Pradeepkumar, P I
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Chattopadhyaya, J
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Kinetic Analysis of the RNA Cleavage of the oxetane modified Antisense-RNA Hybrid Duplex by RNase H.2002In: J. Chem. Soc. Perkin 2, Vol. 5, p. 976-984Article in journal (Refereed)
  • 352.
    Amirkhanov, NV
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Zamaratski, E
    Chattopadhyaya, J
    The recognition and cleavage of RNA in the antisense oligo-RNA hybrid duplexes by RNase H2001In: TETRAHEDRON LETTERS, ISSN 0040-4039, Vol. 42, no 3, p. 489-491Article in journal (Refereed)
    Abstract [en]

    The different extent of the target RNA cleavage at t(99.9%) by RNase H in the AON-RNA duplexes, at the RNA saturation condition by antisense oligo, is due to different recognition and catalytic properties of RNase H toward the hybrids owing to different s

  • 353.
    Amlinger, Lina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    The type I-E CRISPR-Cas system: Biology and applications of an adaptive immune system in bacteria2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    CRISPR-Cas systems are adaptive immune systems in bacteria and archaea, consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) array and CRISPR associated (Cas) proteins. In this work, the type I-E CRISPR-Cas system of Escherichia coli was studied.

    CRISPR-Cas immunity is divided into three stages. In the first stage, adaptation, Cas1 and Cas2 store memory of invaders in the CRISPR array as short intervening sequences, called spacers. During the expression stage, the array is transcribed, and subsequently processed into small CRISPR RNAs (crRNA), each consisting of one spacer and one repeat. The crRNAs are bound by the Cascade multi-protein complex. During the interference step, Cascade searches for DNA molecules complementary to the crRNA spacer. When a match is found, the target DNA is degraded by the recruited Cas3 nuclease.

    Host factors required for integration of new spacers into the CRISPR array were first investigated. Deleting recD, involved in DNA repair, abolished memory formation by reducing the concentration of the Cas1-Cas2 expression plasmid, leading to decreased amounts of Cas1 to levels likely insufficient for spacer integration. Deletion of RecD has an indirect effect on adaptation. To facilitate detection of adaptation, a sensitive fluorescent reporter was developed where an out-of-frame yfp reporter gene is moved into frame when a new spacer is integrated, enabling fluorescent detection of adaptation. Integration can be detected in single cells by a variety of fluorescence-based methods. A second aspect of this thesis aimed at investigating spacer elements affecting target interference. Spacers with predicted secondary structures in the crRNA impaired the ability of the CRISPR-Cas system to prevent transformation of targeted plasmids. Lastly, in absence of Cas3, Cascade was successfully used to inhibit transcription of specific genes by preventing RNA polymerase access to the promoter.

    The CRISPR-Cas field has seen rapid development since the first demonstration of immunity almost ten years ago. However, much research remains to fully understand these interesting adaptive immune systems and the research presented here increases our understanding of the type I-E CRISPR-Cas system. 

    List of papers
    1. Deletion of recD indirectly reduce adaptation in the type I-E CRISPR-Cas system
    Open this publication in new window or tab >>Deletion of recD indirectly reduce adaptation in the type I-E CRISPR-Cas system
    (English)Manuscript (preprint) (Other academic)
    National Category
    Microbiology
    Identifiers
    urn:nbn:se:uu:diva-312230 (URN)
    Available from: 2017-01-08 Created: 2017-01-08 Last updated: 2017-01-09
    2. Quantification of CRISPR-Cas spacer integration using a fluorescent reporter
    Open this publication in new window or tab >>Quantification of CRISPR-Cas spacer integration using a fluorescent reporter
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Microbiology
    Identifiers
    urn:nbn:se:uu:diva-312231 (URN)
    Available from: 2017-01-08 Created: 2017-01-08 Last updated: 2017-01-09
    3. Effect of spacer sequence on efficiency of Type I-E CRISPR-Cas systems
    Open this publication in new window or tab >>Effect of spacer sequence on efficiency of Type I-E CRISPR-Cas systems
    (English)Manuscript (preprint) (Other academic)
    National Category
    Microbiology
    Identifiers
    urn:nbn:se:uu:diva-312233 (URN)
    Available from: 2017-01-08 Created: 2017-01-08 Last updated: 2017-01-09
    4. Efficient programmable gene silencing by Cascade
    Open this publication in new window or tab >>Efficient programmable gene silencing by Cascade
    Show others...
    2015 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 43, no 1, p. 237-246Article in journal (Refereed) Published
    Abstract [en]

    Methods that permit controlled changes in the expression of genes are important tools for biological and medical research, and for biotechnological applications. Conventional methods are directed at individually changing each gene, its regulatory elements or its mRNA's translation rate. We demonstrate that the CRISPR-associated DNA-binding Cascade complex can be used for efficient, long-lasting and programmable gene silencing. When Cascade is targeted to a promoter sequence the transcription of the downstream gene is inhibited, resulting in dramatically reduced expression. The specificity of Cascade binding is provided by the integral crRNA component, which is easily designed to target virtually any stretch of DNA. Cascade targeted to the ORF sequence of the gene can also silence expression, albeit at lower efficiency. The system can be used to silence plasmid and chromosome targets, simultaneously target several genes and is active in different bacterial species and strains. The findings described here are an addition to the expanding range of CRISPR-based technologies and may be adapted to additional organisms and cell systems.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-249042 (URN)10.1093/nar/gku1257 (DOI)000350207100026 ()25435544 (PubMedID)
    Available from: 2015-04-23 Created: 2015-04-10 Last updated: 2018-02-28Bibliographically approved
  • 354.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, E. Gerhart H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Quantification of CRISPR-Cas spacer integration using a fluorescent reporterManuscript (preprint) (Other academic)
  • 355.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed)
    Abstract [en]

    CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

  • 356.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Hoekzema, Mirthe
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Wagner, Gerhart E. H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Fluorescent CRISPR Adaptation Reporter for rapid quantification of spacer acquisition2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 10392Article in journal (Refereed)
    Abstract [en]

    CRISPR-Cas systems are adaptive prokaryotic immune systems protecting against horizontally transferred DNA or RNA such as viruses and other mobile genetic elements. Memory of past invaders is stored as spacers in CRISPR loci in a process called adaptation. Here we developed a novel assay where spacer integration results in fluorescence, enabling detection of memory formation in single cells and quantification of as few as 0.05% cells with expanded CRISPR arrays in a bacterial population. Using this fluorescent CRISPR Adaptation Reporter (f-CAR), we quantified adaptation of the two CRISPR arrays of the type I-E CRISPR-Cas system in Escherichia coli, and confirmed that more integration events are targeted to CRISPR-II than to CRISPR-I. The f-CAR conveniently analyzes and compares many samples, allowing new insights into adaptation. For instance, we show that in an E. coli culture the majority of acquisition events occur in late exponential phase.

  • 357.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Larsson, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Deletion of recD indirectly reduce adaptation in the type I-E CRISPR-Cas systemManuscript (preprint) (Other academic)
  • 358.
    Amlinger, Lina
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Saunders, Sita J.
    Lundgren, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Backofen, Rolf
    Effect of spacer sequence on efficiency of Type I-E CRISPR-Cas systemsManuscript (preprint) (Other academic)
  • 359.
    Ammenberg, P.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Centre for Image Analysis. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Evolution, Limnology. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computerized Image Analysis.
    Flink, P
    Lindell, T.
    Strömbeck, N.
    Bio-optical Modelling Combined with Remote Sensing to Assess Water Quality2002In: International Journal of Remote Sensing, ISSN 0143-1161, Vol. 23, no 8, p. 1621-1638Article in journal (Refereed)
  • 360.
    Ammunet, Tea
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Evolution and diversification of secreted protein effectors in the order Legionellales2018Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    The evolution of a large, diverse group of intracellular bacteria was previously very difficult to study. Recent advancements in both metagenomic methods and bioinformatics has made it possible. This thesis investigates the evolution of the order Legionellales. The study concentrates on a group of proteins essential for pathogenesis and host manipulation in the order, called effector proteins. The role of effectors in host adaptation, evolutionary history and the diversification of the order were investigated using a multitude of bioinformatics methods. First, the abundance and distribution of the known effector proteins in the orderwas found to cover newly discovered clades. There was a clear distinction between the proteins present in Legionellales and the outgoup, indicating the important role of the effectors in the order. Further, the effectors with known functions found in the new clades, particularly in Berkiella, revealed potential modes of host manipulation of this group. Secondly, the evolution of the effector gene content in the order shed light on theevolution of the order, as well as on the potential evolutionary differences between Legionellaceae and Coxiellaceae. In general, most of the effectors were gained early in the last common ancestor of Legionellales and Legionellaceae, as further indication of their role in the diversification of the order. New effector genes were acquired in the Legionellaceae even up to recent speciation events, whereas Coxiellacea have lost more protein coding genes with time. These differences may be due to horizontal gene transfer in the case of gene gains in Legionellaceae and loss of selection in the case of gene losses in Coxiellaceae. Third, the early evolution of core gained effector proteins for the order was studied.Two of the eight investigated core effectors seem to have a connection to eukaryotes, the rest to other bacteria, indicating both inter-domain and within bacteria horizontal gene transfer. In particular, one effector protein with eukaryotic motif gained at the last common ancestor of Legionellales, was found in all the clades and is therefore an important evolutionary link that may have allowed Legionellales to utilize eukaryotic hosts.

  • 361.
    Amnesten, Josefine
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Ekologiska effekter vid återintroduktion av visent i södra Sveriges lövskog2014Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [sv]

    Förr betades södra Sveriges skogar av megaherbivorer, varav många arter idag är utdöda. På grund av deras utdöende och mycket på grund av de senaste 200 årens förändringar och Sveriges jord- och skogsbruk, som bland annat innebär att skogsbete inte längre används i lika hög grad som förr, så breder nu barrskogarna ut sig i söder. Resultaten har blivit att de öppna lövskogar som hyser ädellövträd som ek är på väg att växa igen. Marken utsätts inte längre för samma markstörning och den skuggtåliga granen som gynnas av skogsbruket etablerar sig. Det stora gamla ädellövträden blir allt färre när deras livsmiljö försvinner och med dem försvinner en mängd evertebrater och fåglar som är beroende av träden i öppna lövskogar. När arter försvinner kan man aldrig få dem tillbaka och det kan vara arter som påverkar hela ekosystem och på lång sikt även oss människor.

    Ett alternativ för att behålla de öppna lövskogarna är att återintroducera visenter i södra Sverige. Visenter har funnits i Skandinavien, men utrotades för runt 8000 år sen. De betar gräs och örter på samma sätt som tamboskap och kan utföra den markstörning som behövs för att gynna denna biotop. I delar av Europa levde vilda visenter kvar fram till första världskrigets slut 1919 och har sedan 1950-talet återintroducerats i flera länder. Hit hör bland annat Polen, Ukraina, Ryssland, Vitryssland och Litauen. Syftet har då varit att bevara visenten som art, men framgången har varit varierande. Man har haft problem med lokalbefolkningens attityd då de förstör grödor samt att de har låg överlevnad till följd av inavel. Det krävs en del planering för att det skulle kunna bli verklighet i Sverige och man måste lokalisera tillräcklig stora områden där de inte kommer i allt för stor konflikt med lokalbefolkningen. Förutom fördelen i form naturvård kan man både öka acceptans hos befolkningen och få ekonomiska fördelar på att i framtiden använda visenten inom jakt och turism. Å andra sidan innebär själva introduktionen en kostnad och man kan behöva betala ut ersättning för förstörda åkrar. En annan nackdel är trafikfaran och rädsla för djuren. Man måste här göra en avvägning mellan kostnad och naturnytta. Redan idag läggs stora summor av skattebetalarnas pengar på att skydda och restaurera hotade naturmiljöer och kanske är visenter i förhållande till det en billig och naturlig lösning som kan sköta sig relativt bra själv.

  • 362.
    Amrein, Beat A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Duarte, Fernanda
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Janfalk Carlsson, Åsa
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Naworyta, Agata
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Kamerlin, Shina C. L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Expanding the catalytic triad in epoxide hydrolases and related enzymes2015In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed)
    Abstract [en]

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

  • 363.
    Amrein, Beat Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Extending the Reach of Computational Approaches to Model Enzyme Catalysis2017Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Recent years have seen tremendous developments in methods for computational modeling of (bio-) molecular systems. Ever larger reactive systems are being studied with high accuracy approaches, and high-level QM/MM calculations are being routinely performed. However, applying high-accuracy methods to large biological systems is computationally expensive and becomes problematic when conformational sampling is needed. To address this challenge, classical force field based approaches such as free energy perturbation (FEP) and empirical valence bond calculations (EVB) have been employed in this work. Specifically:

    1. Force-field independent metal parameters have been developed for a range of alkaline earth and transition metal ions, which successfully reproduce experimental solvation free energies, metal-oxygen distances, and coordination numbers. These are valuable for the computational study of biological systems.

    2. Experimental studies have shown that the epoxide hydrolase from Solanum tuberosum (StEH1) is not only an enantioselective enzyme, but for smaller substrates, displays enantioconvergent behavior. For StEH1, two detailed studies, involving combined experimental and computational efforts have been performed: We first used trans-stilbene oxide to establish the basic reaction mechanism of this enzyme. Importantly, a highly conserved and earlier ignored histidine was identified to be important for catalysis. Following from this, EVB and experiment have been used to investigate the enantioconvergence of the StEH1-catalyzed hydrolysis of styrene oxide. This combined approach involved wildtype StEH1 and an engineered enzyme variant, and established a molecular understanding of enantioconvergent behavior of StEH1.

    3. A novel framework was developed for the Computer-Aided Directed Evolution of Enzymes (CADEE), in order to be able to quickly prepare, simulate, and analyze hundreds of enzyme variants. CADEE’s easy applicability is demonstrated in the form of an educational example.

    In conclusion, classical approaches are a computationally economical means to achieve extensive conformational sampling. Using the EVB approach has enabled me to obtain a molecular understanding of complex enzymatic systems. I have also increased the reach of the EVB approach, through the implementation of CADEE, which enables efficient and highly parallel in silico testing of hundreds-to-thousands of individual enzyme variants.

    List of papers
    1. Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
    Open this publication in new window or tab >>Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
    Show others...
    2014 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 16, p. 4351-4362Article in journal (Refereed) Published
    Abstract [en]

    The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

    National Category
    Physical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-225523 (URN)10.1021/jp501737x (DOI)000335113600010 ()
    Funder
    Swedish National Infrastructure for Computing (SNIC), 2013/26-1
    Available from: 2014-06-23 Created: 2014-06-04 Last updated: 2018-12-03Bibliographically approved
    2. Expanding the catalytic triad in epoxide hydrolases and related enzymes
    Open this publication in new window or tab >>Expanding the catalytic triad in epoxide hydrolases and related enzymes
    Show others...
    2015 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed) Published
    Abstract [en]

    Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-260232 (URN)10.1021/acscatal.5b01639 (DOI)000362391500006 ()
    Funder
    EU, FP7, Seventh Framework Programme, 306474Swedish Research Council, 621-2011-6055, 621-2010-5145Swedish National Infrastructure for Computing (SNIC), 2015/16-12
    Available from: 2015-08-18 Created: 2015-08-18 Last updated: 2017-12-04Bibliographically approved
    3. Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
    Open this publication in new window or tab >>Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
    Show others...
    2016 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed) Published
    Abstract [en]

    Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-282015 (URN)10.1039/C6OB00060F (DOI)000378933400042 ()27049844 (PubMedID)
    Funder
    Swedish National Infrastructure for Computing (SNIC), 25/2-10EU, European Research Council, 306474;283570Swedish Research Council, 621-2011-6055Carl Tryggers foundation , CTS13:104
    Available from: 2016-04-01 Created: 2016-04-01 Last updated: 2017-11-30Bibliographically approved
    4. CADEE: Computer-Aided Directed Evolution of Enzymes
    Open this publication in new window or tab >>CADEE: Computer-Aided Directed Evolution of Enzymes
    Show others...
    2017 (English)In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 4, no 1, p. 50-64Article in journal (Refereed) Published
    Abstract [en]

    The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

    Keywords
    computational directed evolution, computational enzyme design, distributed computing, empirical valence bond, triosephosphate isomerase
    National Category
    Structural Biology Bioinformatics (Computational Biology) Theoretical Chemistry
    Identifiers
    urn:nbn:se:uu:diva-314218 (URN)10.1107/S2052252516018017 (DOI)000392925800007 ()
    Funder
    EU, FP7, Seventh Framework Programme, 306474Knut and Alice Wallenberg FoundationThe Royal Swedish Academy of SciencesSwedish Research Council, 2015-04928Swedish National Infrastructure for Computing (SNIC), 2015/16-12
    Available from: 2017-01-31 Created: 2017-01-31 Last updated: 2018-01-13Bibliographically approved
  • 364.
    Amrein, Beat Anton
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Steffen-Munsberg, Fabian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Szeler, Ireneusz
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Purg, Miha
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kulkarni, Yashraj
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamerlin, Shina Caroline Lynn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    CADEE: Computer-Aided Directed Evolution of Enzymes2017In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 4, no 1, p. 50-64Article in journal (Refereed)
    Abstract [en]

    The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

  • 365.
    Amselem, Elias
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Kipper, Kalle
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Real- Time Single Protein Tracking with Polarization Readout using a Confocal Microscope2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 295A-295AArticle in journal (Other academic)
  • 366. An, Junghwa
    et al.
    Bechet, Arnaud
    Berggren, Åsa
    Brown, Sarah K.
    Bruford, Michael W.
    Cai, Qingui
    Cassel-Lundhagen, Anna
    Cezilly, Frank
    Chen, Song-Lin
    Cheng, Wei
    Choi, Sung-Kyoung
    Ding, X.Y.
    Fan, Yong
    Feldheim, Kevin A.
    Feng, Z.Y.
    Friesen, Vicki L.
    Gaillard, Maria
    Galaraza, Juan A.
    Gallo, Leonardo
    Ganeshaiah, K. N.
    Geraci, Julia
    Gibbons, John G.
    Grant, William S.
    Grauvogel, Zac
    Gustafsson, Stefan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Functional Genomics.
    Guyon, Jeffrey R.
    Han, L.
    Heath, Daniel D.
    Hemmilä, Sofia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Functional Genomics.
    Hogan, Derek
    Hou, B. W.
    Jakse, Jernej
    Javornik, Branka
    Kanuch, Peter
    Kim, Kyung-Kil
    Kim, Kyung-Seok
    Kim, Sang-Gyu
    Kim, Sang-In
    Kim, Woo-Jin
    Kim, Yi-Kyung
    Klich, Maren A.
    Kreiser, Brian R.
    Kwan, Ye-Seul
    Lam, Athena W.
    Lasater, Kelly
    Lascoux, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Evolutionary Functional Genomics.
    Lee, Hang
    Lee, Yun-Sun
    Li, D. L.
    Li, Shao-Jing
    Li, W. Y.
    Liao, Xiaolin
    Liber, Zlatko
    Lin, Lin
    Liu, Shaoying
    Luo, Xin-Hui
    Ma, Y. H.
    Ma, Yajun
    Marchelli, Paula
    Min, Mi-Sook
    Moccia, Maria Domenica
    Mohana, Kumara P.
    Moore, Marcelle
    Morris-Pocock, James A.
    Park, Han-Chan
    Pfunder, Monika
    Ivan, Radosavljevic
    Ravikanth, G.
    Roderick, George K.
    Rokas, Antonis
    Sacks, Benjamin N.
    Saski, Christopher A.
    Satovic, Zlatko
    Schoville, Sean D.
    Sebastiani, Federico
    Sha, Zhen-Xia
    Shin, Eun-Ha
    Soliani, Carolina
    Sreejayan, N.
    Sun, Zhengxin
    Tao, Yong
    Taylor, Scott A.
    Templin, William D.
    Shaanker, R. Uma
    Vasudeva, R.
    Vendramin, Giovanni G.
    Walter, Ryan P.
    Wang, Gui-Zhong
    Wang, Ke-Jian
    Wang, Y. Q.
    Wattier, Rémi A.
    Wei, Fuwen
    Widmer, Alex
    Woltmann, Stefan
    Won, Yong-Jin
    Wu, Jing
    Xie, M. L.
    Xu, Genbo
    Xu, Xiao-Jun
    Ye, Hai-Hui
    Zhan, Xiangjiang
    Zhang, F.
    Zhong, J.
    Permanent Genetic Resources added to Molecular Ecology Resources Database 1 October 2009-30 November 20092010In: Molecular Ecology Resources, ISSN 1755-098X, Vol. 10, no 2, p. 404-408Article in journal (Refereed)
    Abstract [en]

    This article documents the addition of 411 microsatellite marker loci and 15 pairs of Single Nucleotide Polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Acanthopagrus schlegeli, Anopheles lesteri, Aspergillus clavatus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus oryzae, Aspergillus terreus, Branchiostoma japonicum, Branchiostoma belcheri, Colias behrii, Coryphopterus personatus, Cynogolssus semilaevis, Cynoglossus semilaevis, Dendrobium officinale, Dendrobium officinale, Dysoxylum malabaricum, Metrioptera roeselii, Myrmeciza exsul, Ochotona thibetana, Neosartorya fischeri, Nothofagus pumilio, Onychodactylus fischeri, Phoenicopterus roseus, Salvia officinalis L., Scylla paramamosain, Silene latifo, Sula sula, and Vulpes vulpes. These loci were cross-tested on the following species: Aspergillus giganteus, Colias pelidne, Colias interior, Colias meadii, Colias eurytheme, Coryphopterus lipernes, Coryphopterus glaucofrenum, Coryphopterus eidolon, Gnatholepis thompsoni, Elacatinus evelynae, Dendrobium loddigesii Dendrobium devonianum, Dysoxylum binectariferum, Nothofagus antarctica, Nothofagus dombeyii, Nothofagus nervosa, Nothofagus obliqua, Sula nebouxii, and Sula variegata. This article also documents the addition of 39 sequencing primer pairs and 15 allele specific primers or probes for Paralithodes camtschaticus.

  • 367.
    Anandhi, Aavudai
    et al.
    Florida A&M Univ, Coll Agr & Food Sci, Biol Syst Engn, Tallahassee, FL 32307 USA;Florida A&M Univ, Coll Agr & Food Sci, Ctr Water Resources, Tallahassee, FL 32307 USA.
    Pierson, Don
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Ecology and Genetics, Limnology.
    Frei, Allan
    CUNY, Hunter Coll, Dept Geog, New York, NY 10065 USA;CUNY, CUNY Inst Sustainable Cities, New York, NY 10065 USA.
    Evaluation of Climate Model Performance for Water Supply Studies: Case Study for New York City2019In: Journal of water resources planning and management, ISSN 0733-9496, E-ISSN 1943-5452, Vol. 145, no 8, article id 06019006Article in journal (Refereed)
    Abstract [en]

    Evaluating the suitability of data from global climate models (GCMs) for use as input in water supply models is an important step in the larger task of evaluating the effects of climate change on water resources management such as that of water supply operations. The purpose of this paper is to present the process by which GCMs were evaluated and incorporated into the New York City (NYC) water supply's planning activities and to provide conclusions regarding the overall effectiveness of the ranking procedure used in the evaluation. A suite of GCMs participating in Phase 3 of the Coupled Model Intercomparison Project (CMIP3) were evaluated for use in climate change projections in the watersheds of the NYC water supply that provide 90% of the water consumed by NYC. GCM data were aggregated using the seven land-grid points surrounding NYC watersheds, and these data with a daily timestep were evaluated seasonally using probability-based skill scores for various combinations of five meteorological variables (precipitation, average, maximum and minimum temperatures, and wind speed). These are the key variables for the NYC water supply because they affect the timing and magnitude of water, energy, sediment, and nutrient fluxes into the reservoirs as well as in simulating watershed hydrology and reservoir hydrodynamics. We attempted to choose a subset of GCMs based on the average of several skill metrics that compared baseline (20C3M) GCM results to observations. Skill metrics for the study indicate that the skill in simulating the frequency distributions of measured data is highest for temperature and lowest for wind. However, our attempts to identify the best model or subgroup of models were not successful because we found that no single model performs best when considering all of the variables and seasons.

  • 368.
    Andaloussi, Mounir
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Henriksson, Lena M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Wieckowska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Lindh, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Larsson, Anna M.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Suresh, Surisetti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Iyer, Harini
    Srinivasa, Bachally R.
    Bergfors, Terese
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Unge, Torsten
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Mowbray, Sherry L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Jones, T. Alwyn
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase2011In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 14, p. 4964-4976Article in journal (Refereed)
    Abstract [en]

    The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. alpha-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 mu M on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 mu g/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

  • 369.
    Andaloussi, Mounir
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Lindh, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Björkelid, Christofer
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Suresh, Surisetti
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Wieckowska, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Iyer, Harini
    Karlén, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Larhed, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry.
    Substitution of the phosphonic acid and hydroxamic acid functionalities of the DXR inhibitor FR900098: An attempt to improve the activity against Mycobacterium tuberculosis2011In: Bioorganic & Medicinal Chemistry Letters, ISSN 0960-894X, E-ISSN 1090-2120, Vol. 21, no 18, p. 5403-5407Article in journal (Refereed)
    Abstract [en]

    Two series of FR900098/fosmidomycin analogs were synthesized and evaluated for MtDXR inhibition and Mycobacterium tuberculosis whole-cell activity. The design rationale of these compounds involved the exchange of either the phosphonic acid or the hydroxamic acid part for alternative acidic and metal-coordinating functionalities. The best inhibitors provided IC(50) values in the micromolar range, with a best value of 41 mu M.

  • 370. Anders, Alfjorden
    et al.
    Astvaldsson, Asgeir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eva, Jansson
    Svärd, Staffan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Experimental challenge of Atlantic salmon (Salmo salar) with the diplomonad parasite Spironucleus salmonicida to characterize the infection cycleManuscript (preprint) (Other academic)
    Abstract [en]

    Experimental infections were performed of Atlantic salmon (Salmo salar) from the Baltic Sea region with the Diplomonad fish parasite Spironucleus salmonicida in order to define the infection cycle, specifically the time-line and putative routes of transmission. An oral infection protocol using axenic parasites was developed, as were new diagnostic tools using PCR and specific antibodies. We also produced firefly luciferase expressing S. salmonicida parasites that could be identified in the infected fish using in vivo and ex vivo imaging. The new tools made it possible to follow the S. salmonicida infection cycle in detail. Three different stages of the infection were identified: one initial intestinal stage, followed by a blood stage and a final tissue stage. Parasites intubated into the intestine attached to the intestinal surface and were identified in the blood after 1-3 weeks. Skin lesions and infections of the muscles, internal organs and eyes were seen 4-10 weeks after initiation of infection. Several morphologically different forms of S. salmonicida cells were detected in ex vivo cell-cultures of biopsies from skin lesions. By this infection trial we have been able to show that S. salmonicida may use several alternative routes of transmission. One alternative is the fecal-oral route, similar to other Diplomonad parasites but the parasites can also be excreted directly into the surrounding water from the mucous layer of the skin or from an ulcerated skin lesion. This information can be used to prevent the transmission of the parasite in fish farms.

  • 371. Anderson, Bruce
    et al.
    Alexandersson, Ronny
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Johnson, Steven D
    Evolution and coexistence of pollination ecotypes in an African Gladiolus (Iridaceae)2010In: Evolution, ISSN 0014-3820, E-ISSN 1558-5646, Vol. 64, no 4, p. 960-972Article in journal (Refereed)
    Abstract [en]

    Pollinator-mediated selection has been suggested as a key driver of speciation in plants. We examined the potential role of hawkmoth pollinators in driving allopatric divergence and maintaining sympatric coexistence of morphotypes in the African iris Gladiolus longicollis. Floral tube length in this species varies from 35 mm to 130 mm across its geographic range and reflects the prevailing tongue lengths of local hawkmoth assemblages. The distribution of floral tube lengths is bimodal with two relatively discrete categories—long (about 90 mm) or short (about 50 mm)—that match the bimodal distribution of hawkmoth tongue lengths in eastern South Africa. At a contact site between these two floral morphs, we found few individuals of intermediate length, suggesting limited gene flow between morphs despite their interfertility. A difference in flowering phenology appears to be the main isolating barrier between morphs at this site. Long- and short-tubed morphs differed markedly in the chemical composition of their floral fragrance, a trait that could be used as a cue for morph-specific foraging by hawkmoths. Positive directional selection on tube length was found to occur in both morphs.

  • 372.
    Anderson, Cajsa Lisa
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Department of Evolution, Genomics and Systematics. Systematisk botanik.
    All we need now is fossils; a new phylogenetic dating method (PATHd8) allowing thousands of taxa and multiple fossil constraints.2006In: Ancient life and modern approaches: Abstracts of the second International Paleontological Congress, 2006, p. 45-Conference paper (Refereed)
    Abstract [en]

    Estimation of divergence times in phylogenetic trees using sequence data

    becomes increasingly popular, but so far dating studies have given widely different results,

    and especially datings of the lower nodes within the angiosperms and metazoans, have given

    much older ages than those obtained from the fossil record. It has been concluded in different

    studies that more taxa, and more fossils are needed for more reliable age estimates. For this

    reason, a dating method that can handle very large data sets with multiple fossil constraints is

    necessary.

    Chronograms obtained by e.g. penalized likelihood and Bayesian methods, often

    adds a large "ghost range" to the fossil record, and produces chronograms with a more or less

    smooth appearance, even if the corresponding phylograms have apparently very

    heterogeneous rates. Compared to the other methods, our recently developed method,

    PATHd8, gives the results with the best agreement with the fossil record, which coincides

    with the least smooth appearance of the chronograms. When other programs often run into

    computational problems when analysing trees with hundreds of leaves, PATHd8 has no

    problems analysing thousands of taxa instantaneously. An arbitrary number of fossil age

    constraints can be specified, either as fixed-, minimum or maximum age.

    With our new method, the biggest problem in dating studies is that we need

    more fossils, and these fossils must be well dated and assigned to the correct branches of the

    phylogeny. Therefore, to accomplish divergence time estimates, which hopefully approximate

    the real ages, biologists now need to cooperate with palaeontologists.

  • 373.
    Anderson, Cajsa Lisa
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Department of Evolution, Genomics and Systematics.
    Dating Divergence Times in Phylogenies2007Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis concerns different aspects of dating divergence times in phylogenetic trees, using molecular data and multiple fossil age constraints.

    Datings of phylogenetically basal eudicots, monocots and modern birds (Neoaves) are presented. Large phylograms and multiple fossil constraints were used in all these studies. Eudicots and monocots are suggested to be part of a rapid divergence of angiosperms in the Early Cretaceous, with most families present at the Cretaceous/Tertiary boundary. Stem lineages of Neoaves were present in the Late Cretaceous, but the main divergence of extant families took place around the Cre-taceous/Tertiary boundary.

    A novel method and computer software for dating large phylogenetic trees, PATHd8, is presented. PATHd8 is a nonparametric smoothing method that smoothes one pair of sister groups at a time, by taking the mean of the added branch lengths from a terminal taxon to a node. Because of the local smoothing, the algorithm is simple, hence providing stable and very fast analyses, allowing for thousands of taxa and an arbitrary number of age constraints.

    The importance of fossil constraints and their placement are discussed, and concluded to be the most important factor for obtaining reasonable age estimates.

    Different dating methods are compared, and it is concluded that differences in age estimates are obtained from penalized likelihood, PATHd8, and the Bayesian autocorrelation method implemented in the multidivtime program. In the Bayesian method, prior assumptions about evolutionary rate at the root, rate variance and the level of rate smoothing between internal edges, are suggested to influence the results.

    List of papers
    1. Dating phylogenetically basal eudicots using rbcL sequences and multiple fossil constraints.
    Open this publication in new window or tab >>Dating phylogenetically basal eudicots using rbcL sequences and multiple fossil constraints.
    2005 In: American Journal of Botany, Vol. 92, no 10, p. 1737–1748-Article in journal (Refereed) Published
    Identifiers
    urn:nbn:se:uu:diva-96058 (URN)
    Available from: 2007-09-03 Created: 2007-09-03Bibliographically approved
    2. Diversification of Neoaves through time: Integration of molecular sequence data and fossils
    Open this publication in new window or tab >>Diversification of Neoaves through time: Integration of molecular sequence data and fossils
    Show others...
    2006 (English)In: Biology Letters, ISSN 1744-9561, E-ISSN 1744-957X, Vol. 2, no 4, p. 543-547Article in journal (Refereed) Published
    Abstract [en]

    Patterns of diversification and timing of evolution within Neoaves, which includes almost 95% of all bird species, are virtually unknown. On the other hand, molecular data consistently indicate a Cretaceous origin of many neoavian lineages and the fossil record seems to support an Early Tertiary diversification. Here, we present the first well-resolved molecular phylogeny for Neoaves, together with divergence time estimates calibrated with a large number of stratigraphically and phylogenetically well-documented fossils. Our study defines several well-supported clades within Neoaves. The calibration results suggest that Neoaves, after an initial split from Galloanseres in Mid-Cretaceous, diversified around or soon after the K/T boundary. Our results thus do not contradict palaeontological data and show that there is no solid molecular evidence for an extensive pre-Tertiary radiation of Neoaves.

    Keywords
    Divergence times, Fossils, Molecular clock, Neoaves, Nuclear DNA, Phylogeny
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-96059 (URN)10.1098/rsbl.2006.0523 (DOI)000242686500018 ()17148284 (PubMedID)
    Available from: 2007-09-03 Created: 2007-09-03 Last updated: 2017-12-14Bibliographically approved
    3. Hangin’ on to our rocks ’n clocks: a reply to Brown et al.
    Open this publication in new window or tab >>Hangin’ on to our rocks ’n clocks: a reply to Brown et al.
    2007 (English)In: Biology Letters, ISSN 1744-9561, E-ISSN 1744-957X, Vol. 3, no 3, p. 260-261Article in journal (Refereed) Published
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-96060 (URN)10.1098/rsbl.2007.0103. (DOI)
    Available from: 2007-09-03 Created: 2007-09-03 Last updated: 2017-12-14Bibliographically approved
    4. Estimating divergence times in large phylogenetic trees
    Open this publication in new window or tab >>Estimating divergence times in large phylogenetic trees
    Show others...
    2007 (English)In: Systematic Biology, ISSN 1063-5157, E-ISSN 1076-836X, Vol. 56, no 5, p. 741-752Article in journal (Refereed) Published
    Abstract [en]

    A new method, PATHd8, for estimating ultrametric trees from trees with edge (branch) lengths proportional to the number of substitutions is proposed. The method allows for an arbitrary number of reference nodes for time calibration, each defined either as absolute age, minimum age, or maximum age, and the tree need not be fully resolved. The method is based on estimating node ages by mean path lengths from the node to the leaves but correcting for deviations from a molecular clock suggested by reference nodes. As opposed to most existing methods allowing substitution rate variation, the new method smoothes substitution rates locally, rather than simultaneously over the whole tree, thus allowing for analysis of very large trees. The performance of PATHd8 is compared with other frequently used methods for estimating divergence times. In analyses of three separate data sets, PATHd8 gives similar divergence times to other methods, the largest difference being between crown group ages, where unconstrained nodes get younger ages when analyzed with PATHd8. Overall, chronograms obtained from other methods appear smoother, whereas PATHd8 preserves more of the heterogeneity seen in the original edge lengths. Divergence times are most evenly spread over the chronograms obtained from the Bayesian implementation and the clock-based Langley-Fitch method, and these two methods produce very similar ages for most nodes. Evaluations of PATHd8 using simulated data suggest that PATHd8 is slightly less precise compared with penalized likelihood, but it gives more sensible answers for extreme data sets. A clear advantage with PATHd8 is that it is more or less instantaneous even with trees having several thousand leaves, whereas other programs often run into problems when analyzing trees with hundreds of leaves. PATHd8 is implemented in freely available software.

    Keywords
    Divergence times, estimation, molecular clock, phylogenetic trees, substitution rates
    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-96061 (URN)10.1080/10635150701613783 (DOI)000250959000004 ()17886144 (PubMedID)
    Available from: 2007-09-03 Created: 2007-09-03 Last updated: 2017-12-14Bibliographically approved
    5. Monocots
    Open this publication in new window or tab >>Monocots
    2009 (English)In: Timetree of life / [ed] S. Blair Hedges and Sudhir Kumar, Oxford University Press, 2009Chapter in book (Other academic)
    Place, publisher, year, edition, pages
    Oxford University Press, 2009
    Series
    Oxford biology
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-96062 (URN)978-0-19-953503-3 (ISBN)
    Available from: 2007-09-03 Created: 2007-09-03 Last updated: 2013-06-18Bibliographically approved
    6. Dating phylogenies: an evaluation of three methods based on the lycopod family Selaginellaceae.
    Open this publication in new window or tab >>Dating phylogenies: an evaluation of three methods based on the lycopod family Selaginellaceae.
    Manuscript (Other academic)
    Identifiers
    urn:nbn:se:uu:diva-96063 (URN)
    Available from: 2007-09-03 Created: 2007-09-03 Last updated: 2010-01-13Bibliographically approved
  • 374.
    Anderson, Cajsa Lisa
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Department of Evolution, Genomics and Systematics.
    Dating phylogenies: an evaluation of three methods based on the lycopod family Selaginellaceae.Manuscript (Other academic)
  • 375.
    Anderson, Cajsa Lisa
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Department of Evolution, Genomics and Systematics. Systematisk botanik.
    PATHd8 - a new phylogenetic dating method allowing thousands of taxa and multiple fossil constraints2006In: 7th european paleobotany-palynology: program and abstracts, 2006, p. 6-Conference paper (Refereed)
    Abstract [en]

    Estimation of divergence times in phylogenetic trees using DNA sequence data becomes increasingly popular, but so far dating studies have given widely different results, and especially datings of the lower nodes within the angiosperms and metazoans, have given much older ages than those obtained from the fossil record. It has been concluded in different studies that more taxa, and more fossils are needed for more reliable age estimates. For this reason, a dating method that can handle very large data sets with multiple fossil constraints is necessary.

    Chronograms obtained by the currently most used methods often adds a large "ghost range" to the fossil record. Compared to the other methods, our recently developed method, PATHd8, gives the most reasonable results, with the best agreement with the fossil record, in all studies performed so far.

    The only way to improve the datings further, and hopefully obtain divergence time estimates which approximate the real ages, is to include more fossils. The combination of allowing an arbitrary number of fossil age constraints with the capability to analyse thousands of taxa instantaneously, makes PATHd8 a strong alternative to other methods. All we need now to accomplish better studies, is cooperation between biologists and paleontologists.

  • 376.
    Anderson, Cajsa Lisa
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Faculty of Science and Technology, Department of Evolution, Genomics and Systematics.