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  • 1.
    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)
  • 2.
    Ballet, Caroline
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Correia, Mario S. P.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Conway, Louis P.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Locher, Theresa L.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Lehmann, Laura C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Garg, Neeraj
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Chemical Biology for Biomarker Discovery. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Vujasinovic, Miroslav
    Karolinska Univ Hosp, Dept Digest Dis, Stockholm, Sweden.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lohr, J. -Matthias
    Karolinska Inst, Dept Clin Sci Intervent & Technol CLINTEC, Stockholm, Sweden;Karolinska Univ Hosp, Dept Digest Dis, Stockholm, Sweden.
    Globisch, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Chemical Biology for Biomarker Discovery. Uppsala University, Science for Life Laboratory, SciLifeLab.
    New enzymatic and mass spectrometric methodology for the selective investigation of gut microbiota-derived metabolites2018In: Chemical Science, ISSN 2041-6520, E-ISSN 2041-6539, Vol. 9, no 29, p. 6233-6239Article in journal (Refereed)
    Abstract [en]

    Gut microbiota significantly impact human physiology through metabolic interaction. Selective investigation of the co-metabolism of bacteria and their human host is a challenging task and methods for their analysis are limited. One class of metabolites associated with this co-metabolism are O-sulfated compounds. Herein, we describe the development of a new enzymatic assay for the selective mass spectrometric investigation of this phase II modification class. Analysis of human urine and fecal samples resulted in the detection of 206 sulfated metabolites, which is three times more than reported in the Human Metabolome Database. We confirmed the chemical structure of 36 sulfated metabolites including unknown and commonly reported microbiota-derived sulfated metabolites using synthesized internal standards and mass spectrometric fragmentation experiments. Our findings demonstrate that enzymatic sample pre-treatment combined with state-of-the-art metabolomics analysis represents a new and efficient strategy for the discovery of unknown microbiota-derived metabolites in human samples. Our described approach can be adapted for the targeted investigation of other metabolite classes as well as the discovery of biomarkers for diseases affected by microbiota.

  • 3.
    Baltekin, Özden
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Astrego Diagnost AB, Uppsala, Sweden.
    KN Direct phenotypic antimicrobial susceptibility testing under 30 minutes: Dynamics of single cell response revealed in automated microscopy with microfluidics2018In: Journal of Veterinary Pharmacology and Therapeutics, ISSN 0140-7783, E-ISSN 1365-2885, Vol. 41, no S1, p. 17-17Article in journal (Other academic)
  • 4.
    Baltekin, Özden
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Boucharin, Alexis
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tano, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Infection medicine.
    Andersson, Dan I
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Antibiotic susceptibility testing in less than 30 min using direct single-cell imaging2017In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 34, p. 9170-9175Article in journal (Refereed)
    Abstract [en]

    The emergence and spread of antibiotic-resistant bacteria are aggravated by incorrect prescription and use of antibiotics. A core problem is that there is no sufficiently fast diagnostic test to guide correct antibiotic prescription at the point of care. Here, we investigate if it is possible to develop a point-of-care susceptibility test for urinary tract infection, a disease that 100 million women suffer from annually and that exhibits widespread antibiotic resistance. We capture bacterial cells directly from samples with low bacterial counts (10(4) cfu/mL) using a custom-designed microfluidic chip and monitor their individual growth rates using microscopy. By averaging the growth rate response to an antibiotic over many individual cells, we can push the detection time to the biological response time of the bacteria. We find that it is possible to detect changes in growth rate in response to each of nine antibiotics that are used to treat urinary tract infections in minutes. In a test of 49 clinical uropathogenic Escherichia coli (UPEC) isolates, all were correctly classified as susceptible or resistant to ciprofloxacin in less than 10 min. The total time for antibiotic susceptibility testing, from loading of sample to diagnostic readout, is less than 30 min, which allows the development of a point-of-care test that can guide correct treatment of urinary tract infection.

  • 5.
    Balzarotti, Francisco
    et al.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Eilers, Yvan
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Gwosch, Klaus C.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Gynnå, Arvid H.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Westphal, Volker
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany..
    Stefani, Fernando D.
    Consejo Nacl Invest Cient & Tecn, Ctr Invest Bionanociencias CIBION, Buenos Aires, DF, Argentina.;Univ Buenos Aires, Fac Ciencias Exactas & Nat, Dept Fis, Buenos Aires, DF, Argentina..
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hell, Stefan W.
    Max Planck Inst Biophys Chem, Dept NanoBiophoton, Gottingen, Germany.;Max Planck Inst Med Res, Dept Opt Nanoscopy, Heidelberg, Germany.;German Canc Res Ctr, Opt Nanoscopy Div, Heidelberg, Germany..
    Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes2017In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 355, no 6325, p. 606-612Article in journal (Refereed)
    Abstract [en]

    We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolutionmicroscopy, MINFLUXattained similar to 1-nanometer precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30S ribosomal subunits in living Escherichia coli. As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.

  • 6.
    Belikov, Sergey
    et al.
    Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden.;Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, SE-10691 Stockholm, Sweden..
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Wrange, Orjan
    Karolinska Inst, Dept Cell & Mol Biol, SE-17177 Stockholm, Sweden..
    Quantification of transcription factor-DNA binding affinity in a living cell2016In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 44, no 7, p. 3045-3058Article in journal (Refereed)
    Abstract [en]

    The apparent dissociation constant (K-d) for specific binding of glucocorticoid receptor (GR) and androgen receptor (AR) to DNA was determined in vivo in Xenopus oocytes. The total nuclear receptor concentration was quantified as specifically retained [H-3]-hormone in manually isolated oocyte nuclei. DNA was introduced by nuclear microinjection of single stranded phagemid DNA, chromatin is then formed during second strand synthesis. The fraction of DNA sites occupied by the expressed receptor was determined by dimethylsulphate in vivo footprinting and used for calculation of the receptor-DNA binding affinity. The forkhead transcription factor FoxA1 enhanced the DNA binding by GR with an apparent K-d of similar to 1 mu M and dramatically stimulated DNA binding by AR with an apparent K-d of similar to 0.13 mu M at a composite androgen responsive DNA element containing one FoxA1 binding site and one palindromic hormone receptor binding site known to bind one receptor homodimer. FoxA1 exerted a weak constitutive- and strongly cooperative DNA binding together with AR but had a less prominent effect with GR, the difference reflecting the licensing function of FoxA1 at this androgen responsive DNA element.

  • 7.
    Belliveau, Nathan M.
    et al.
    CALTECH, Div Biol & Biol Engn, Pasadena, USA.
    Barnes, Stephanie L.
    CALTECH, Div Biol & Biol Engn, Pasadena, USA.
    Ireland, William T.
    CALTECH, Dept Phys, Pasadena, USA.
    Jones, Daniel L.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sweredoski, Michael J.
    CALTECH, Beckman Inst, Proteome Explorat Lab, Pasadena, USA.
    Moradian, Annie
    CALTECH, Beckman Inst, Proteome Explorat Lab, Pasadena, USA.
    Hess, Sonja
    CALTECH, Beckman Inst, Proteome Explorat Lab, Pasadena, CA 91125 USA;MedImmune, Antibody Discovery & Prot Engn, Gaithersburg, USA.
    Kinney, Justin B.
    Cold Spring Harbor Lab, Simons Ctr Quantitat Biol, POB 100, Cold Spring Harbor, USA.
    Phillips, Rob
    CALTECH, Div Biol & Biol Engn, Pasadena, USA;CALTECH, Dept Phys, Pasadena, CA 91125 USA;CALTECH, Dept Appl Phys, Pasadena, CA 91125 USA.
    Systematic approach for dissecting the molecular mechanisms of transcriptional regulation in bacteria2018In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 115, no 21, p. E4796-E4805Article in journal (Refereed)
    Abstract [en]

    Gene regulation is one of the most ubiquitous processes in biology. However, while the catalog of bacterial genomes continues to expand rapidly, we remain ignorant about how almost all of the genes in these genomes are regulated. At present, characterizing the molecular mechanisms by which individual regulatory sequences operate requires focused efforts using low-throughput methods. Here, we take a first step toward multipromoter dissection and show how a combination of massively parallel reporter assays, mass spectrometry, and information-theoretic modeling can be used to dissect multiple bacterial promoters in a systematic way. We show this approach on both well-studied and previously uncharacterized promoters in the enteric bacterium Escherichia coli. In all cases, we recover nucleotide-resolution models of promoter mechanism. For some promoters, including previously unannotated ones, the approach allowed us to further extract quantitative biophysical models describing input-output relationships. Given the generality of the approach presented here, it opens up the possibility of quantitatively dissecting the mechanisms of promoter function in E. coli and a wide range of other bacteria.

  • 8.
    Berg, Otto G.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Mahmutovic, Anel
    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.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    The helical structure of DNA facilitates binding2016In: Journal of Physics A: Mathematical and Theoretical, ISSN 1751-8113, E-ISSN 1751-8121, Vol. 9, no 36, article id 364002Article in journal (Other academic)
    Abstract [en]

    The helical structure of DNA imposes constraints on the rate of diffusion-limited protein binding. Here we solve the reaction-diffusion equations for DNA-like geometries and extend with simulations when necessary. We find that the helical structure can make binding to the DNA more than twice as fast compared to a case where DNA would be reactive only along one side. We also find that this rate advantage remains when the contributions from steric constraints and rotational diffusion of the DNA-binding protein are included. Furthermore, we find that the association rate is insensitive to changes in the steric constraints on the DNA in the helix geometry, while it is much more dependent on the steric constraints on the DNA-binding protein. We conclude that the helical structure of DNA facilitates the nonspecific binding of transcription factors and structural DNA-binding proteins in general.

  • 9.
    Doudna, Jennifer
    et al.
    Univ Calif Berkeley, Berkeley, CA 94720 USA..
    Bar-Ziv, Roy
    Weizmann Inst Sci, IL-76100 Rehovot, Israel..
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Noireaux, Vincent
    Univ Minnesota, Minneapolis, MN 55455 USA..
    Berro, Julien
    Yale Univ, New Haven, CT 06520 USA..
    Saiz, Leonor
    Univ Calif Davis, Davis, CA 95616 USA..
    Vavylonis, Dimitrios
    Lehigh Univ, Bethlehem, PA 18015 USA..
    Faulon, Jean-Loup
    INRA, Jouy En Josas, France..
    Fordyce, Polly
    Stanford Univ, Stanford, CA 94305 USA..
    How Will Kinetics and Thermodynamics Inform Our Future Efforts to Understand and Build Biological Systems?2017In: CELL SYSTEMS, ISSN 2405-4712, Vol. 4, no 2, p. 144-146Article in journal (Refereed)
  • 10.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hypothesis: Homologous Recombination Depends on Parallel Search2016In: CELL SYSTEMS, ISSN 2405-4712, Vol. 3, no 4, p. 325-327Article in journal (Refereed)
    Abstract [en]

    It is not known how a cell manages to find a specific DNA sequence sufficiently fast to repair a broken chromosome through homologous recombination. I propose a solution based on freely diffusing molecules that are programmed with sequences corresponding to those flanking the break site. In such a process, the target search would be parallelized.

  • 11.
    Elias-Wolff, Federico
    et al.
    Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden; Stockholm Univ, Dept Mat & Environm Chem, Stockholm, Sweden.
    Lindén, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Lyubartsev, Alexander P.
    Stockholm Univ, Dept Mat & Environm Chem, Stockholm, Sweden.
    Brandt, Erik G.
    Stockholm Univ, Dept Mat & Environm Chem, Stockholm, Sweden.
    Computing Curvature Sensitivity of Biomolecules in Membranes by Simulated Buckling2018In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 14, no 3, p. 1643-1655Article in journal (Refereed)
    Abstract [en]

    Membrane curvature sensing, where the binding free energies of membrane-associated molecules depend on the local membrane curvature, is a key factor to modulate and maintain the shape and organization of cell membranes. However, the microscopic mechanisms are not well understood, partly due to absence of efficient simulation methods. Here, we describe a method to compute the curvature dependence of the binding free energy of a membrane associated probe molecule that interacts with a buckled membrane, which has been created by lateral compression of a flat bilayer patch. This buckling approach samples a wide range of curvatures in a single simulation, and anisotropic effects can be extracted from the orientation statistics. We develop an efficient and robust algorithm to extract the motion of the probe along the buckled membrane surface, and evaluate its numerical properties by extensive sampling of three coarse-grained model systems: local lipid density in a curved environment for single-component bilayers, curvature preferences of individual lipids in two-component membranes, and curvature sensing by a homotrimeric transmembrane protein. The method can be used to complement experimental data from curvature partition assays and provides additional insight into mesoscopic theories and molecular mechanisms for curvature sensing.

  • 12.
    Ghosh, Anirban
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Wäneskog, Marcus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elkhalifa, Dina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Larsson, Disa
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Contact-dependent growth inhibition induces high levels of antibiotic-tolerant persister cells in clonal bacterial populations2018In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 37, no 9, article id UNSP e98026Article in journal (Refereed)
    Abstract [en]

    Bacterial populations can use bet-hedging strategies to cope with rapidly changing environments. One example is non-growing cells in clonal bacterial populations that are able to persist antibiotic treatment. Previous studies suggest that persisters arise in bacterial populations either stochastically through variation in levels of global signalling molecules between individual cells, or in response to various stresses. Here, we show that toxins used in contact-dependent growth inhibition (CDI) create persisters upon direct contact with cells lacking sufficient levels of CdiI immunity protein, which would otherwise bind to and neutralize toxin activity. CDI-mediated persisters form through a feedforward cycle where the toxic activity of the CdiA toxin increases cellular (p)ppGpp levels, which results in Lon-mediated degradation of the immunity protein and more free toxin. Thus, CDI systems mediate a population density-dependent bet-hedging strategy, where the fraction of non-growing cells is increased only when there are many cells of the same genotype. This may be one of the mechanisms of how CDI systems increase the fitness of their hosts.

  • 13.
    Jones, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Leroy, Prune
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Unoson, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Curic, Vladimir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lawson, Michael J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala Univ, Dept Cell & Mol Biol, Sci Life Lab, Uppsala, Sweden..
    Kinetics of dCas9 target search in Escherichia coli2017In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 357, no 6358, p. 1420-1423Article in journal (Refereed)
    Abstract [en]

    How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.

  • 14.
    Jones, Daniel
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Unoson, Cecilia
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Leroy, Prune
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Curic, Vladimir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Kinetics of dCas9 Target Search in Escherichia Coli2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 314A-314AArticle in journal (Other academic)
  • 15.
    Kipper, Kalle
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Eremina, Nadja
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Tubasum, Sumera
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mao, Guanzhong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lehmann, Laura Christina
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. 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. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Structure-guided approach to site-specific fluorophore labeling of the lac repressor LacI2018In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 13, no 6, article id e0198416Article in journal (Refereed)
    Abstract [en]

    The lactose operon repressor protein LacI has long served as a paradigm of the bacterial transcription factors. However, the mechanisms whereby LacI rapidly locates its cognate binding site on the bacterial chromosome are still elusive. Single-molecule fluorescence imaging approaches are well suited for the study of these mechanisms but rely on a functionally compatible fluorescence labeling of LacI. Particularly attractive for protein fluorescence labeling are synthetic fluorophores due to their small size and favorable photophysical characteristics. Synthetic fluorophores are often conjugated to natively occurring cysteine residues using maleimide chemistry. For a site-specific and functionally compatible labeling with maleimide fluorophores, the target protein often needs to be redesigned to remove unwanted native cysteines and to introduce cysteines at locations better suited for fluorophore attachment. Biochemical screens can then be employed to probe for the functional activity of the redesigned protein both before and after dye labeling. Here, we report a mutagenesis- based redesign of LacI to enable a functionally compatible labeling with maleimide fluorophores. To provide an easily accessible labeling site in LacI, we introduced a single cysteine residue at position 28 in the DNA-binding headpiece of LacI and replaced two native cysteines with alanines where derivatization with bulky substituents is known to compromise the protein's activity. We find that the redesigned LacI retains a robust activity in vitro and in vivo, provided that the third native cysteine at position 281 is retained in LacI. In a total internal reflection microscopy assay, we observed individual Cy3-labeled LacI molecules bound to immobilized DNA harboring the cognate O-1 operator sequence, indicating that the dye-labeled LacI is functionally active. We have thus been able to generate a functional fluorescently labeled LacI that can be used to unravel mechanistic details of LacI target search at the single molecule level.

  • 16.
    Kipper, Kalle
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundius, Ebba G.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ćurić, Vladimir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Nikic, Ivana
    European Mol Biol Lab, Cell Biol & Biophys Unit, Struct & Computat Biol Unit, D-69117 Heidelberg, Germany..
    Wiessler, Manfred
    Deutsch Krebsforschungszentrum, Biol Chem, D-69120 Heidelberg, Germany..
    Lemke, Edward A.
    European Mol Biol Lab, Cell Biol & Biophys Unit, Struct & Computat Biol Unit, D-69117 Heidelberg, Germany..
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Application of Noncanonical Amino Acids for Protein Labeling in a Genomically Recoded Escherichia coli2017In: ACS Photonics, E-ISSN 2330-4022, Vol. 6, no 2, p. 233-255Article in journal (Refereed)
    Abstract [en]

    Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.

  • 17.
    Lawson, Michael J.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Camsund, Daniel
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Larsson, Jimmy
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Baltekin, Özden
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Fange, David
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Elf, Johan
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    In situ genotyping of a pooled strain library after characterizing complex phenotypes2017In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 13, no 10, article id 947Article in journal (Refereed)
    Abstract [en]

    In this work, we present a proof-of-principle experiment that extends advanced live cell microscopy to the scale of pool-generated strain libraries. We achieve this by identifying the genotypes for individual cells in situ after a detailed characterization of the phenotype. The principle is demonstrated by single-molecule fluorescence time-lapse imaging of Escherichia coli strains harboring barcoded plasmids that express a sgRNA which suppresses different genes in the E.coli genome through dCas9 interference. In general, the method solves the problem of characterizing complex dynamic phenotypes for diverse genetic libraries of cell strains. For example, it allows screens of how changes in regulatory or coding sequences impact the temporal expression, location, or function of a gene product, or how the altered expression of a set of genes impacts the intracellular dynamics of a labeled reporter.

  • 18.
    Lehmann, Laura C.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hewitt, Graeme
    Francis Crick Inst, 1 Midland Rd, London NW1 1AT, England..
    Aibara, Shintaro
    Stockholm Univ, Dept Biochem & Biophys, Sci Life Lab, S-17165 Solna, Sweden..
    Leitner, Alexander
    Swiss Fed Inst Technol, Inst Mol Syst Biol, Dept Biol, CH-8093 Zurich, Switzerland..
    Marklund, Emil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Maslen, Sarah L.
    MRC Lab Mol Biol, Francis Crick Ave,Cambridge Biomed Campus, Cambridge CB2 0QH, England..
    Maturi, Varun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Chen, Yang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    van der Spoel, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Skehel, J. Mark
    MRC Lab Mol Biol, Francis Crick Ave,Cambridge Biomed Campus, Cambridge CB2 0QH, England..
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Boulton, Simon J.
    Francis Crick Inst, 1 Midland Rd, London NW1 1AT, England..
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mechanistic Insights into Autoinhibition of the Oncogenic Chromatin Remodeler ALC12017In: Molecular Cell, ISSN 1097-2765, E-ISSN 1097-4164, Vol. 68, no 5, p. 847-859.e7Article in journal (Refereed)
    Abstract [en]

    Human ALC1 is an oncogene-encoded chromatin-remodeling enzyme required for DNA repair that possesses a poly(ADP-ribose) (PAR)-binding macro domain. Its engagement with PARylated PARP1 activates ALC1 at sites of DNA damage, but the underlying-mechanism remains unclear. Here, we establish a dual role for the macro domain in autoinhibition of ALC1 ATPase activity and coupling to nucleosome mobilization. In the absence of DNA damage, an inactive conformation of the ATPase is maintained by juxtaposition of the macro domain against predominantly the C-terminal ATPase lobe through conserved electrostatic interactions. Mutations within this interface displace the macro domain, constitutively activate the ALC1 ATPase independent of PARylated PARP1, and alter the dynamics of ALC1 recruitment at DNA damage sites. Upon DNA damage, binding of PARylated PARP1 by the macro domain induces a conformational change that relieves autoinhibitory interactions with the ATPase motor, which selectively activates ALC1 remodeling upon recruitment to sites of DNA damage.

  • 19.
    Levendosky, Robert F.
    et al.
    Johns Hopkins Univ, TC Jenkins Dept Biophys, Baltimore, MD 21218 USA..
    Sabantsev, Anton
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bowman, Gregory D.
    Johns Hopkins Univ, TC Jenkins Dept Biophys, Baltimore, MD 21218 USA..
    The Chd1 chromatin remodeler shifts hexasomes unidirectionally2016In: eLIFE, E-ISSN 2050-084X, Vol. 5, article id e21356Article in journal (Refereed)
    Abstract [en]

    Despite their canonical two-fold symmetry, nucleosomes in biological contexts are often asymmetric: functionalized with post-translational modifications (PTMs), substituted with histone variants, and even lacking H2A/H2B dimers. Here we show that the Widom 601 nucleosome positioning sequence can produce hexasomes in a specific orientation on DNA, providing a useful tool for interrogating chromatin enzymes and allowing for the generation of nucleosomes with precisely defined asymmetry. Using this methodology, we demonstrate that the Chd1 chromatin remodeler from Saccharomyces cerevisiae requires H2A/H2B on the entry side for sliding, and thus, unlike the back-and-forth sliding observed for nucleosomes, Chd1 shifts hexasomes unidirectionally. Chd1 takes part in chromatin reorganization surrounding transcribing RNA polymerase II (Pol II), and using asymmetric nucleosomes we show that ubiquitin-conjugated H2B on the entry side stimulates nucleosome sliding by Chd1. We speculate that biased nucleosome and hexasome sliding due to asymmetry contributes to the packing of arrays observed in vivo.

  • 20.
    Liljeruhm, Josefine
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Funk, Saskia K.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Tietscher, Sandra
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Edlund, Anders D.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Jamal, Sabri
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Yuen, Pikkei
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Dyrhage, Karl
    Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Gynnå, Arvid H.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Ivermark, Katarina
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Lövgren, Jessica
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Törnblom, Viktor
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Virtanen, Anders
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Lundin, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala Univ, iGEM Uppsala, Uppsala, Sweden.
    Wistand-Yuen, Erik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Forster, Anthony C.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology2018In: Journal of Biological Engineering, ISSN 1754-1611, E-ISSN 1754-1611, Vol. 12, article id 8Article in journal (Refereed)
    Abstract [en]

    Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them.

    Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors.

    Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts.

  • 21.
    Lind, Peter A.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Umea Univ, Dept Mol Biol, S-90187 Umea, Sweden..
    Arvidsson, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Variation in Mutational Robustness between Different Proteins and the Predictability of Fitness Effects2017In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 34, no 2, p. 408-418Article in journal (Refereed)
    Abstract [en]

    Random mutations in genes from disparate protein classes may have different distributions of fitness effects (DFEs) depending on different structural, functional, and evolutionary constraints. We measured the fitness effects of 156 single mutations in the genes encoding AraC (transcription factor), AraD (enzyme), and AraE (transporter) used for bacterial growth on L-arabinose. Despite their different molecular functions these genes all had bimodal DFEs with most mutations either being neutral or strongly deleterious, providing a general expectation for the DFE. This contrasts with the unimodal DFEs previously obtained for ribosomal protein genes where most mutations were slightly deleterious. Based on theoretical considerations, we suggest that the 33-fold higher average mutational robustness of ribosomal proteins is due to stronger selection for reduced costs of translational and transcriptional errors. Whereas the large majority of synonymous mutations were deleterious for ribosomal proteins genes, no fitness effects could be detected for the AraCDE genes. Four mutations in AraC and AraE increased fitness, suggesting that slightly advantageous mutations make up a significant fraction of the DFE, but that they often escape detection due to the limited sensitivity of commonly used fitness assays. We show that the fitness effects of amino acid substitutions can be predicted based on evolutionary conservation, but those weakly deleterious mutations are less reliably detected. This suggests that large-effect mutations and the fraction of highly deleterious mutations can be computationally predicted, but that experiments are required to characterize the DFE close to neutrality, where many mutations ultimately fixed in a population will occur.

  • 22.
    Lindén, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    An early peak in ion channel research2018In: Nature Physics, ISSN 1745-2473, E-ISSN 1745-2481, Vol. 14, no 2, p. 105-105Article in journal (Other academic)
  • 23.
    Lindén, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Curic, Vladimir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Amselem, Elias
    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.
    Pointwise error estimates in localization microscopy2017In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, article id 15115Article in journal (Refereed)
    Abstract [en]

    Pointwise localization of individual fluorophores is a critical step in super-resolution localization microscopy and single particle tracking. Although the methods are limited by the localization errors of individual fluorophores, the pointwise localization precision has so far been estimated using theoretical best case approximations that disregard, for example, motion blur, defocus effects and variations in fluorescence intensity. Here, we show that pointwise localization precision can be accurately estimated directly from imaging data using the Bayesian posterior density constrained by simple microscope properties. We further demonstrate that the estimated localization precision can be used to improve downstream quantitative analysis, such as estimation of diffusion constants and detection of changes in molecular motion patterns. Finally, the quality of actual point localizations in live cell super-resolution microscopy can be improved beyond the information theoretic lower bound for localization errors in individual images, by modelling the movement of fluorophores and accounting for their pointwise localization uncertainty.

  • 24.
    Lindén, Martin
    et al.
    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.
    Variational Algorithms for Analyzing Noisy Multistate Diffusion Trajectories2018In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 115, no 2, p. 276-282Article in journal (Refereed)
    Abstract [en]

    Single-particle tracking offers a noninvasive high-resolution probe of biomolecular reactions inside living cells. However, efficient data analysis methods that correctly account for various noise sources are needed to realize the full quantitative potential of the method. We report algorithms for hidden Markov-based analysis of single-particle tracking data, which incorporate most sources of experimental noise, including heterogeneous localization errors and missing positions. Compared to previous implementations, the algorithms offer significant speedups, support for a wider range of inference methods, and a simple user interface. This will enable more advanced and exploratory quantitative analysis of single-particle tracking data.

  • 25.
    Lindén, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Ćurić, Vladimir
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Boucharin, Alexis
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Fange, David
    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.
    Simulated single molecule microscopy with SMeagol2016In: Bioinformatics, ISSN 1367-4803, E-ISSN 1367-4811, Vol. 32, no 15, p. 2394-2395Article in journal (Refereed)
    Abstract [en]

    Summary: SMeagol is a software tool to simulate highly realistic microscopy data based on spatial systems biology models, in order to facilitate development, validation and optimization of advanced analysis methods for live cell single molecule microscopy data.

    Availability and implementation: SMeagol runs on Matlab R2014 and later, and uses compiled binaries in C for reaction–diffusion simulations. Documentation, source code and binaries for Mac OS, Windows and Ubuntu Linux can be downloaded from http://smeagol.sourceforge.net.

  • 26.
    Marklund, Emil
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Amselem, Elias
    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.
    Measuring the Orientation of Single Proteins Interacting with DNA using Fluorescence Polarization Microscopy2017In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 112, no 3, p. 169A-169AArticle in journal (Other academic)
  • 27.
    Martyna, Agnieszka
    et al.
    Univ Kent, Sch Biosci, Canterbury CT2 7NJ, Kent, England..
    Gomez-Llobregat, Jordi
    Stockholm Univ, Dept Biochem & Biophys, Ctr Biomembrane Res, SE-10691 Stockholm, Sweden..
    Lindén, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Stockholm Univ, Dept Biochem & Biophys, Ctr Biomembrane Res, SE-10691 Stockholm, Sweden..
    Rossman, Jeremy S.
    Univ Kent, Sch Biosci, Canterbury CT2 7NJ, Kent, England..
    Curvature Sensing by a Viral Scission Protein2016In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 25, p. 3493-3496Article in journal (Refereed)
    Abstract [en]

    Membrane scission is the final step in all budding processes wherein a membrane neck is sufficiently constricted so as to allow for fission and the release of the budded particle. For influenza viruses, membrane scission is mediated by an amphipathic helix (AH) domain in the viral M2 protein. While it is known that the M2AH alters membrane curvature, it is not known how the protein is localized to the center neck of budding virions where it would be able to cause membrane scission. Here, we use molecular dynamics simulations on buckled lipid bilayers to show that the M2AH senses membrane curvature and preferentially localizes to regions of high membrane curvature, comparable to that seen at the center neck of budding influenza viruses. These results were then validated using in vitro binding assays to show that the M2AH senses membrane curvature by detecting lipid packing defects in the membrane. Our results show that the M2AH senses membrane curvature and suggest that the AH domain may localize the protein at the viral neck where it can then mediate membrane scission and the release of budding viruses.

  • 28.
    Pandzic, Tatjana
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Rendo, Verónica
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lim, Jinyeong
    Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, Goyangsi, Republic of Korea.
    Larsson, Chatarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Larsson, Jimmy
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Stoimenov, Ivaylo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kundu, Snehangshu
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Ali, Muhammad Akhtar
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology.
    Hellström, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    He, Liqun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lindroth, Anders M.
    Department of Cancer Biomedical Science, National Cancer Center Graduate School of Cancer Science and Policy, Goyangsi, Republic of Korea.
    Sjöblom, Tobias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Experimental and Clinical Oncology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Somatic PRDM2 c.4467delA mutations in colorectal cancers control histone methylation and tumor growth2017In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 8, no 58, p. 98646-98659Article in journal (Refereed)
    Abstract [en]

    The chromatin modifier PRDM2/RIZ1 is inactivated by mutation in several forms of cancer and is a putative tumor suppressor gene. Frameshift mutations in the C-terminal region of PRDM2, affecting (A)8 or (A)9 repeats within exon 8, are found in one third of colorectal cancers with microsatellite instability, but the contribution of these mutations to colorectal tumorigenesis is unknown. To model somatic mutations in microsatellite unstable tumors, we devised a general approach to perform genome editing while stabilizing the mutated nucleotide repeat. We then engineered isogenic cell systems where the PRDM2 c.4467delA mutation in human HCT116 colorectal cancer cells was corrected to wild-type by genome editing. Restored PRDM2 increased global histone 3 lysine 9 dimethylation and reduced migration, anchorage-independent growth and tumor growth in vivo. Gene set enrichment analysis revealed regulation of several hallmark cancer pathways, particularly of epithelial-to-mesenchymal transition (EMT), with VIM being the most significantly regulated gene. These observations provide direct evidence that PRDM2 c.4467delA is a driver mutation in colorectal cancer and confirms PRDM2 as a cancer gene, pointing to regulation of EMT as a central aspect of its tumor suppressive action.

  • 29.
    Saupe, Falk
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Reichel, Matthias
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Huijbers, Elisabeth J. M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Femel, Julia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Markgren, Per-Olof
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Andersson, C. Evalena
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Deindl, Sebastian
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Hellman, Lars T.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Olsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala Univ, Dept Med Biochem & Microbiol, Husargatan 3,Box 582, SE-75123 Uppsala, Sweden..
    Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously2017In: The FASEB Journal, ISSN 0892-6638, E-ISSN 1530-6860, Vol. 31, no 3, p. 1204-1214Article in journal (Refereed)
    Abstract [en]

    With the aim to improve the efficacy of therapeutic vaccines that target self-antigens, we have developed a novel fusion protein vaccine on the basis of the C-terminal multimerizing end of the variable lymphocyte receptor B (VLRB), the Ig equivalent in jawless fishes. Recombinant vaccines were produced in Escherichia coli by fusing the VLRB sequence to 4 different cancer-associated target molecules. The anti-self-immune response generated in mice that were vaccinated with VLRB vaccines was compared with the response in mice that received vaccines that contained bacterial thioredoxin (TRX), previously identified as an efficient carrier. The anti-self-Abswere analyzed with respect to titers, binding properties, and duration of response. VLRB-vaccinatedmice displayed a 2-to 10-fold increase in anti-self-Ab titers and a substantial decrease in Abs against the foreign part of the fusion protein compared with the response in TRX-vaccinated mice (P < 0.01). VLRB-generated Ab response had duration similar to the corresponding TRX-generatedAbs, but displayed a higher diversity in binding characteristics. Of importance, VLRB vaccines could sustain an immune response against several targets simultaneously. VLRB vaccines fulfill several key criteria for an efficient therapeutic vaccine that targets self-antigens as a result of its small size, its multimerizing capacity, and nonexposed foreign sequences in the fusion protein.- Saupe, F., Reichel, M., Huijbers, E. J. M., Femel, J., Markgren, P.- O., Andersson, C. E., Deindl, S., Danielson, U. H., Hellman, L. T., Olsson, A.- K. Development of a novel therapeutic vaccine carrier that sustains high antibody titers against several targets simultaneously.

  • 30.
    Travin, Dmitrii Y.
    et al.
    Lomonosov Moscow State Univ, Dept Bioengn & Bioinformat, Moscow, Russia; Skolkovo Inst Sci & Technol, Ctr Data Intens Biomed & Biotechnol, Skolkovo, Russia.
    Metelev, Mikhail
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Skolkovo Inst Sci & Technol, Ctr Data Intens Biomed & Biotechnol, Skolkovo, Russia; Russian Acad Sci, Inst Gene Biol, Moscow, Russia.
    Serebryakova, Marina
    Skolkovo Inst Sci & Technol, Ctr Data Intens Biomed & Biotechnol, Skolkovo, Russia; Lomonosov Moscow State Univ, Dept Chem, Moscow, Russia; Lomonosov Moscow State Univ, AN Belozersky Inst Physicochem Biol, Moscow, Russia.
    Komarova, Ekaterina S.
    Lomonosov Moscow State Univ, Dept Bioengn & Bioinformat, Moscow, Russia; Skolkovo Inst Sci & Technol, Ctr Data Intens Biomed & Biotechnol, Skolkovo, Russia.
    Osterman, Ilya A.
    Lomonosov Moscow State Univ, Dept Chem, Moscow, Russia; Lomonosov Moscow State Univ, AN Belozersky Inst Physicochem Biol, Moscow, Russia; Skolkovo Inst Sci & Technol, Ctr Translat Biomed, Skolkovo, Russia.
    Ghilarov, Dmitry
    Skolkovo Inst Sci & Technol, Ctr Data Intens Biomed & Biotechnol, Skolkovo, Russia; Russian Acad Sci, Inst Gene Biol, Moscow, Russia; Jagiellonian Univ, Malopolska Ctr Biotechnol, Krakow, Poland.
    Severinov, Konstantin
    Skolkovo Inst Sci & Technol, Ctr Data Intens Biomed & Biotechnol, Skolkovo, Russia; Russian Acad Sci, Inst Gene Biol, Moscow, Russia; Rutgers State Univ, Waksman Inst Microbiol, Piscataway, NJ USA.
    Biosynthesis of Translation Inhibitor Klebsazolicin Proceeds through Heterocyclization and N-Terminal Amidine Formation Catalyzed by a Single YcaO Enzyme2018In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, no 16, p. 5625-5633Article in journal (Refereed)
    Abstract [en]

    Klebsazolicin (KLB) is a recently discovered Klebsiella pneumonia peptide antibiotic targeting the exit tunnel of bacterial ribosome. KLB contains an N-terminal amidine ring and four azole heterocycles installed into a ribosomally synthesized precursor by dedicated maturation machinery. Using an in vitro system for KLB production, we show that the YcaO-domain KlpD maturation enzyme is a bifunctional cyclodehydratase required for the formation of both the core heterocycles and the N-terminal amidine ring. We further demonstrate that the amidine ring is formed concomitantly with proteolytic cleavage of azole-containing pro-KLB by a cellular protease TldD/E. Members of the YcaO family are diverse enzymes known to activate peptide carbonyls during natural product biosynthesis leading to the formation of azoline, macroamidine, and thioamide moieties. The ability of KlpD to simultaneously perform two distinct types of modifications is unprecedented for known YcaO proteins. The versatility of KlpD opens up possibilities for rational introduction of modifications into various peptide backbones.

  • 31.
    Volkov, Ivan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Aguirre, Javier
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Lindén, Martin
    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.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    In Vivo Measurements of Protein Synthesis Kinetics using Single-Molecule Tracking of E.Coli tRNAS2016In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 110, no 3, p. 351A-351AArticle in journal (Other academic)
  • 32.
    Volkov, Ivan
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Lindén, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Aguirre, Javier
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Ieong, Ka-Weng
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Metelev, Mikhail
    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.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    tRNA tracking for direct measurements of protein synthesis kinetics in live cells2018In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, no 6, p. 618-626Article in journal (Refereed)
    Abstract [en]

    Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy.Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNA(Phe) that are in perfect agreement with previous indirect estimates, and once fMet-tRNA(fMet) has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.

  • 33.
    Walldén, Mats
    et al.
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fange, David
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundius, Ebba Gregorsson
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Baltekin, Özden
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Elf, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    The Synchronization of Replication and Division Cycles in Individual E. coli Cells2016In: Cell, ISSN 0092-8674, E-ISSN 1097-4172, Vol. 166, no 3, p. 729-739Article in journal (Refereed)
    Abstract [en]

    Isogenic E. coli cells growing in a constant environment display significant variability in growth rates, division sizes, and generation times. The guiding principle appears to be that each cell, during one generation, adds a size increment that is uncorrelated to its birth size. Here, we investigate the mechanisms underlying this "adder'' behavior by mapping the chromosome replication cycle to the division cycle of individual cells using fluorescence microscopy. We have found that initiation of chromosome replication is triggered at a fixed volume per chromosome independent of a cell's birth volume and growth rate. Each initiation event is coupled to a division event after a growth-rate-dependent time. We formalize our findings in a model showing that cell-to-cell variation in division timing and cell size is mainly driven by variations in growth rate. The model also explains why fast-growing cells display adder behavior and correctly predict deviations from the adder behavior at slow growth.

  • 34.
    Wistrand-Yuen, Erik
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Knopp, Michael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hjort, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Koskiniemi, Sanna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Evolution of high-level resistance during low-level antibiotic exposure2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, no 1, article id 1599Article in journal (Refereed)
    Abstract [en]

    It has become increasingly clear that low levels of antibiotics present in many environments can select for resistant bacteria, yet the evolutionary pathways for resistance development during exposure to low amounts of antibiotics remain poorly defined. Here we show that Salmonella enterica exposed to sub-MIC levels of streptomycin evolved high-level resistance via novel mechanisms that are different from those observed during lethal selections. During lethal selection only rpsL mutations are found, whereas at sub-MIC selection resistance is generated by several small-effect resistance mutations that combined confer high-level resistance via three different mechanisms: (i) alteration of the ribosomal RNA target (gidB mutations), (ii) reduction in aminoglycoside uptake (cyoB, nuoG, and trkH mutations), and (iii) induction of the aminoglycoside-modifying enzyme AadA (znuA mutations). These results demonstrate how the strength of the selective pressure influences evolutionary trajectories and that even weak selective pressures can cause evolution of high-level resistance.

1 - 34 of 34
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