| 1. |
- Högberg, Liselotte Diaz, et al.
(författare)
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Antibiotic use worldwide
- 2014
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Ingår i: The Lancet - Infectious diseases. - 1473-3099 .- 1474-4457. ; 14:12, s. 1179-1180
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Tidskriftsartikel (refereegranskat)
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| 2. |
- Ke, Rongqin, et al.
(författare)
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Colorimetric Nucleic Acid Testing Assay for RNA Virus Detection Based on Circle-to-Circle Amplification of Padlock Probes
- 2011
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Ingår i: Journal of Clinical Microbiology. - 0095-1137 .- 1098-660X. ; 49:12, s. 4279-4285
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Tidskriftsartikel (refereegranskat)abstract
- We developed a molecular diagnostic method for detection of RNA virus based on padlock probes and colorimetric readout. The feasibility of our approach was demonstrated by using detection of Crimean-Congo hemorrhagic fever (CCHF) virus as a model. Compared with conventional PCR-based methods, our approach does not require advanced equipment, involves easier assay design, and has a sensitivity of 103 viral copies/ml. By using a cocktail of padlock probes, synthetic templates representing different viral strain variants could be detected. We analyzed 34 CCHF patient samples, and all patients were correctly diagnosed when the results were compared to those of the current real-time PCR method. This is the first time that highly specific padlock probes have been applied to detection of a highly variable target sequence typical of RNA viruses.
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| 3. |
- Pavlov, Michael Y., et al.
(författare)
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Activation of initiation factor 2 by ligands and mutations for rapid docking of ribosomal subunits
- 2011
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 30:2, s. 289-301
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Tidskriftsartikel (refereegranskat)abstract
- We previously identified mutations in the GTPase initiation factor 2 (IF2), located outside its tRNA-binding domain, compensating strongly (A-type) or weakly (B-type) for initiator tRNA formylation deficiency. We show here that rapid docking of 30S with 50S subunits in initiation of translation depends on switching 30S subunit-bound IF2 from its inactive to active form. Activation of wild-type IF2 requires GTP and formylated initiator tRNA (fMet-tRNA(i)). In contrast, extensive activation of A-type IF2 occurs with only GTP or with GDP and fMet-tRNA(i), implying a passive role for initiator tRNA as activator of IF2 in subunit docking. The theory of conditional switching of GTPases quantitatively accounts for all our experimental data. We find that GTP, GDP, fMet-tRNA(i) and A-type mutations multiplicatively increase the equilibrium ratio, K, between active and inactive forms of IF2 from a value of 4 × 10(-4) for wild-type apo-IF2 by factors of 300, 8, 80 and 20, respectively. Functional characterization of the A-type mutations provides keys to structural interpretation of conditional switching of IF2 and other multidomain GTPases.
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| 4. |
- Zorzet, Anna, et al.
(författare)
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Compensatory mutations in agrC partly restore fitness in vitro to peptide deformylase inhibitor-resistant Staphylococcus aureus
- 2012
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Ingår i: Journal of Antimicrobial Chemotherapy. - : Oxford University Press (OUP). - 0305-7453 .- 1460-2091. ; 67:8, s. 1835-1842
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVESTo determine how the fitness cost of deformylase inhibitor resistance conferred by fmt mutations can be genetically compensated.METHODSResistant mutants were isolated and characterized with regard to their growth rates in vitro and in neutropenic mice, MIC and DNA sequence. Faster-growing compensated mutants were isolated by serial passage in culture medium, and for a subset of the resistant and compensated mutants whole-genome sequencing was performed.RESULTSStaphylococcus aureus mutants resistant to the peptide deformylase inhibitor actinonin had mutations in the fmt gene that conferred high-level actinonin resistance and reduced bacterial growth rate. Compensated mutants that remained fully resistant to actinonin and showed increased growth rates appeared within 30-60 generations of growth. Whole-genome sequencing and localized DNA sequencing of mutated candidate genes showed that alterations in the gene agrC were present in the majority of compensated strains. Resistant and compensated mutants grew at similar rates as the wild-type in a mouse thigh infection model.CONCLUSIONSResistance to deformylase inhibitors due to fmt mutations reduces bacterial growth rates, but these costs can be reduced by mutations in the agrC gene. Mutants defective in fmt (with or without compensatory agrC mutations) grew well in an animal model, implying that they can also cause infection in a host.
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| 5. |
- Zorzet, Anna, 1977-, et al.
(författare)
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Error-prone initiation factor 2 mutations reduce the fitness cost of antibiotic resistance
- 2010
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Ingår i: Molecular Microbiology. - : Wiley-Blackwell. - 0950-382X .- 1365-2958. ; 75:5, s. 1299-1313
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Tidskriftsartikel (refereegranskat)abstract
- Mutations in the fmt gene (encoding formyl methionine transferase) that eliminate formylation of initiator tRNA (Met-tRNA(i)) confer resistance to the novel antibiotic class of peptide deformylase inhibitors (PDFIs) while concomitantly reducing bacterial fitness. Here we show in Salmonella typhimurium that novel mutations in initiation factor 2 (IF2) located outside the initiator tRNA binding domain can partly restore fitness of fmt mutants without loss of antibiotic resistance. Analysis of initiation of protein synthesis in vitro showed that with non-formylated Met-tRNA(i) IF2 mutants initiated much faster than wild-type IF2, whereas with formylated fMet-tRNA(i) the initiation rates were similar. Moreover, the increase in initiation rates with Met-tRNA(i) conferred by IF2 mutations in vitro correlated well with the increase in growth rate conferred by the same mutations in vivo, suggesting that the mutations in IF2 compensate formylation deficiency by increasing the rate of in vivo initiation with Met-tRNA(i). IF2 mutants had also a high propensity for erroneous initiation with elongator tRNAs in vitro, which could account for their reduced fitness in vivo in a formylation-proficient strain. More generally, our results suggest that bacterial protein synthesis is mRNA-limited and that compensatory mutations in IF2 could increase the persistence of PDFI-resistant bacteria in clinical settings.
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| 6. |
- Zorzet, Anna, 1977-
(författare)
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Mechanisms of Adaptation to Deformylase Inhibitors
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotic resistance is a growing problem on a global scale. Increasing numbers of bacteria resistant toward one or multiple antibiotics could return us to the high mortality rates for infectious diseases of the pre-antibiotic era. The need for development of new classes of antibiotics is great as is increased understanding of the mechanisms underlying the development of antibiotic resistance. We have investigated the emergence of resistance to peptide deformylase inhibitors, a new class of antibiotics that target bacterial protein synthesis. The fitness of resistant mutants as well as their propensity to acquire secondary compensatory mutations was assessed in order to gain some insight into the potential clinical risk of resistance development. Most of this work was done in the bacterium Salmonella typhimurium, due to the availability of excellent genetic tools to study these phenomena. In addition, we have studied the bacterium Staphylococcus aureus as peptide deformylase inhibitors have been shown to have the greatest effect on Gram-positive organisms. In the course of this work we also examined the mechanistic aspects of translation initiation. Using a cell-free in vitro translation system we studied the effects of various components on translation initiation. These results have been combined with results obtained from resistant and compensated bacterial strains in vivo to gain new insights into the mechanisms of translation initiation.
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| 7. |
- Zorzet, Anna
(författare)
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Overcoming scientific and structural bottlenecks in antibacterial discovery and development
- 2014
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Ingår i: Upsala Journal of Medical Sciences. - : Uppsala Medical Society. - 0300-9734 .- 2000-1967. ; 119:2, s. 170-175
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Forskningsöversikt (refereegranskat)abstract
- Antibiotic resistance is becoming an increasing threat, with too few novel antibiotics coming to market to replace those lost due to resistance development. Efforts by the pharmaceutical industry to screen for and design novel antibacterials have not been successful, with several companies minimizing or closing down their antibacterial research units, leading to a loss of skills and know-how. At the same time, antibiotic innovation in academia is not filling the void due to misaligned incentive structures and lack of vital knowledge of drug discovery. The scientific and structural difficulties in discovering new antibiotics have only begun to be appreciated in the latest years. Part of the problem has been a paradigm shift within both industry and academia to focus on 'rational' drug development with an emphasis on single targets and high-throughput screening of large chemical libraries, which may not be suited to target bacteria. The very particular aspects of 'targeting an organism inside another organism' have not been given enough attention. In this paper, researcher interviews have complemented literature studies to delve deeper into the specifics of the different scientific and structural barriers, and some potential solutions are offered.
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