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- Hedman, Johannes, et al.
(författare)
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Validation guidelines for PCR workflows in bioterrorism preparedness, food safety and forensics
- 2018
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Ingår i: Accreditation and Quality Assurance. - : Springer Science and Business Media LLC. - 0949-1775 .- 1432-0517. ; 23:3, s. 133-144
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Tidskriftsartikel (refereegranskat)abstract
- The polymerase chain reaction (PCR) is the backbone of contemporary DNA/RNA analysis, ideally enabling detection of one or just a few target molecules. However, when analysing food or forensic samples the analytical procedure is often challenged by low amounts of poor quality template molecules and complex matrices. Applying optimised and validated methods in all steps of the analysis workflow, i.e. sampling, sample treatment, DNA/RNA extraction and PCR (including reverse transcription for RNA analysis), is thus necessary to ensure the reliability of analysis. In this paper, we describe how in-house validation can be performed for the different modules of the diagnostic PCR process, providing practical examples as tools for laboratories in their planning of validation studies. The focus is analysis of heterogeneous samples with interfering matrices, with relevance in food testing, forensic DNA analysis, bioterrorism preparedness and veterinary medicine. Our objective is to enable rational in-house validation for reliable and swift quality assurance when results are urgent, for example in the event of a crisis such as a foodborne outbreak or a crime requiring the analysis of a large number of diverse samples. To that end, we explain the performance characteristics associated with method validation from a PCR and biological sample matrix perspective and suggest which characteristics to investigate depending on the type of method to be validated. Also, we include a modular approach to validation within the PCR workflow, aiming at efficient validation and a flexible use of methods.
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- Salomonsson, Lina, et al.
(författare)
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Deuterium isotope effect of proton pumping in cytochrome c oxidase
- 2008
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1777:4, s. 343-350
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Tidskriftsartikel (refereegranskat)abstract
- In mitochondria and many aerobic bacteria cytochrome c oxidase is the terminal enzyme of the respiratory chain where it catalyses the reduction of oxygen to water. The free energy released in this process is used to translocate (pump) protons across the membrane such that each electron transfer to the catalytic site is accompanied by proton pumping. To investigate the mechanism of electron–proton coupling in cytochrome c oxidase we have studied the pH-dependence of the kinetic deuterium isotope effect of specific reaction steps associated with proton transfer in wild-type and structural variants of cytochrome c oxidases in which amino-acid residues in proton-transfer pathways have been modified. In addition, we have solved the structure of one of these mutant enzymes, where a key component of the proton-transfer machinery, Glu286, was modified to an Asp. The results indicate that the P3 → F3 transition rate is determined by a direct proton-transfer event to the catalytic site. In contrast, the rate of the F3 → O4 transition, which involves simultaneous electron transfer to the catalytic site and is characteristic of any transition during CytcO turnover, is determined by two events with similar rates and different kinetic isotope effects. These reaction steps involve transfer of protons, that are pumped, via a segment of the protein including Glu286 and Arg481.
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- Salomonsson, Lina, 1977-
(författare)
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Proton, Electron, and O2 transfer in Cytochrome c Oxidase
- 2006
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In mitochondria and aerobic bacteria energy conservation involves electron transfer through the membrane-bound protein of the respiratory chain to oxygen. Cytochrome c oxidase (CcO) catalyzes the reduction of oxygen to water. Part of the energy released in this reaction is used to pump protons across the membrane. The protons that are pumped, and those needed for reduction to water, are taken from the inside of the membrane. This helps to maintain an electrochemical potential over the membrane, which is necessary for the survival of the cell and is used for example to produce ATP.This work has been focused on the proton, electron and oxygen transfer in CcO from Rhodobacter sphaeroides. Results presented here show that there is a need for a specific O2-channel, due to structural rigidity of the area close to the catalytic site. In other parts of the enzyme regions that undergo redox-dependent structural changes were identified, and we proposed areas involved in controlling proton pumping and in forming the proton exit route.The results from investigating vectorial proton translocation defined the order and timing of proton uptake and release during a pumping-cycle. Indications from these results also suggested a mechanism by which CcO couples electron transfer and proton pumping, assuring that electrons are not transferred without simultaneous proton pumping.
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- Salomonsson, Lina, et al.
(författare)
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Proton transfer in the quinol dependent nitric oxide reductase from geobacillus stearothermophilus during reduction of oxygen
- 2012
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Ingår i: Biochimica et Biophysica Acta - Bioenergetics. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1817:10, s. 1914-1920
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial nitric oxide reductases (NOR) are integral membrane proteins that catalyse the reduction of nitric oxide to nitrous oxide, often as a step in the process of denitrification. Most functional data has been obtained with NORs that receive their electrons from a soluble cytochrome c in the periplasm and are hence termed cNOR. Very recently, the structure of a different type of NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246]. In this study, we have investigated the reaction between this gNOR and oxygen. Our results show that, like some cNORs, the C. stearothermophilus gNOR is capable of 02 reduction with a turnover of similar to 3 electrons s(-1) at 40 degrees C. Furthermore, using the so-called flow-flash technique, we show that the fully reduced (with three available electrons) gNOR reacts with oxygen in a reaction with a time constant of 1.8 ms that oxidises the low-spin heme b. This reaction is coupled to proton uptake from solution and presumably forms a ferryl intermediate at the active site. The pH dependence of the reaction is markedly different from a corresponding reaction in cNOR from Paracoccus denitrificans, indicating that possibly the proton uptake mechanism and/or pathway differs between gNOR and cNOR. This study furthermore forms the basis for investigation of the proton transfer pathway in gNOR using both variants with putative proton transfer elements modified and measurements of the vectorial nature of the proton transfer. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
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