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  • 1. Nykvist, Marie
    et al.
    Gillman, Anna
    Söderström Lindström, Hanna
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Yrkes- och miljömedicin. Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Tang, Chaojun
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Fedorova, Ganna
    Lundkvist, Åke
    Latorre-Margalef, Neus
    Wille, Michelle
    Järhult, Josef D.
    In vivo mallard experiments indicate that zanamivir has less potential for environmental influenza A virus resistance development than oseltamivir2017Ingår i: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 98, s. 2937-2949Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Neuraminidase inhibitors are a cornerstone of influenza pandemic preparedness before vaccines can be mass-produced and thus a neuraminidase inhibitor-resistant pandemic is a serious threat to public health. Earlier work has demonstrated the potential for development and persistence of oseltamivir resistance in influenza A viruses exposed to environmentally relevant water concentrations of the drug when infecting mallards, the natural influenza reservoir that serves as the genetic base for human pandemics. As zanamivir is the major second-line neuraminidase inhibitor treatment, this study aimed to assess the potential for development and persistence of zanamivir resistance in an in vivo mallard model; especially important as zanamivir will probably be increasingly used. Our results indicate less potential for development and persistence of resistance due to zanamivir than oseltamivir in an environmental setting. This conclusion is based on: (1) the lower increase in zanamivir IC50 conferred by the mutations caused by zanamivir exposure (2-17-fold); (2) the higher zanamivir water concentration needed to induce resistance (at least 10 µg l-1); (3) the lack of zanamivir resistance persistence without drug pressure; and (4) the multiple resistance-related substitutions seen during zanamivir exposure (V116A, A138V, R152K, T157I and D199G) suggesting lack of one straight-forward evolutionary path to resistance. Our study also adds further evidence regarding the stability of the oseltamivir-induced substitution H275Y without drug pressure, and demonstrates the ability of a H275Y-carrying virus to acquire secondary mutations, further boosting oseltamivir resistance when exposed to zanamivir. Similar studies using influenza A viruses of the N2-phylogenetic group of neuraminidases are recommended.

  • 2.
    Tang, Chaojun
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gandla, Madhavi Latha
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Jönsson, Leif J.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Comparison of solid and liquid fractions of pretreated Norway spruce as reductants in LPMO-supported saccharification of cellulose2022Ingår i: Frontiers in Bioengineering and Biotechnology, E-ISSN 2296-4185, Vol. 10, artikel-id 1071159Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    The role of lignin in enzymatic saccharification of cellulose involving lytic polysaccharide monooxygenase (LPMO) was investigated in experiments with the solid and liquid fractions of pretreated Norway spruce from a biorefinery demonstration plant using hydrothermal pretreatment and impregnation with sulfur dioxide. Pretreated biomass before and after enzymatic saccharification was characterized using HPAEC, HPLC, Py-GC/MS, 2D-HSQC NMR, FTIR, and SEM. Chemical characterization indicated that relatively harsh pretreatment conditions resulted in that the solid phase contained no or very little hemicellulose but considerable amounts of pseudo-lignin, and that the liquid phase contained a relatively high concentration (∼5 g/L) of lignin-derived phenolics. As judged from reactions continuously supplied with either air or nitrogen gas, lignin and lignin fragments from both the solid and the liquid phases efficiently served as reductants in LPMO-supported saccharification. When air was used to promote LPMO activity, the enzymatic conversion of cellulose after 72 h was 25% higher in reactions with pretreated solids and buffer, and 14% higher in reactions with pretreatment liquid and microcrystalline cellulose. Research in this area is useful for designing efficient saccharification steps in biochemical conversion of lignocellulosic biomass.

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  • 3.
    Tang, Chaojun
    et al.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Gandla, Madhavi Latha
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Jönsson, Leif J.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    LPMO-supported saccharification of biomass: effects of continuous aeration of reaction mixtures with variable fractions of water-insoluble solids and cellulolytic enzymes2023Ingår i: Biotechnology for Biofuels and Bioproducts, E-ISSN 2731-3654, Vol. 16, nr 1, artikel-id 156Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Background: High substrate concentrations and high sugar yields are important aspects of enzymatic saccharification of lignocellulosic substrates. The benefit of supporting the catalytic action of lytic polysaccharide monooxygenase (LPMO) through continuous aeration of slurries of pretreated softwood was weighed against problems associated with increasing substrate content (quantitated as WIS, water-insoluble solids, in the range 12.5–17.5%), and was compared to the beneficial effect on the saccharification reaction achieved by increasing the enzyme preparation (Cellic CTec3) loadings. Aerated reactions were compared to reactions supplied with N2 to assess the contribution of LPMO to the saccharification reactions. Analysis using 13C NMR spectroscopy, XRD, Simons’ staining, BET analysis, and SEM analysis was used to gain further insights into the effects of the cellulolytic enzymes on the substrate under different reaction conditions.

    Results: Although glucose production after 72 h was higher at 17.5% WIS than at 12.5% WIS, glucan conversion decreased with 24% (air) and 17% (N2). Compared to reactions with N2, the average increases in glucose production for aerated reactions were 91% (12.5% WIS), 70% (15.0% WIS), and 67% (17.5% WIS). Improvements in glucan conversion through aeration were larger (55–86%) than the negative effects of increasing WIS content. For reactions with 12.5% WIS, increased enzyme dosage with 50% improved glucan conversion with 25–30% for air and N2, whereas improvements with double enzyme dosage were 30% (N2) and 39% (air). Structural analyses of the solid fractions revealed that the enzymatic reaction, particularly with aeration, created increased surface area (BET analysis), increased disorder (SEM analysis), decreased crystallinity (XRD), and increased dye adsorption based on the cellulose content (Simons' staining).

    Conclusions: The gains in glucan conversion with aeration were larger than the decreases observed due to increased substrate content, resulting in higher glucan conversion when using aeration at the highest WIS value than when using N2 at the lowest WIS value. The increase in glucan conversion with double enzyme preparation dosage was smaller than the increase achieved with aeration. The results demonstrate the potential in using proper aeration to exploit the inherent capacity of LPMO in enzymatic saccharification of lignocellulosic substrates and provide detailed information about the characteristics of the substrate after interaction with cellulolytic enzymes.

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  • 4. Tepper, Viktoria
    et al.
    Nykvist, Marie
    Gillman, Anna
    Skog, Erik
    Wille, Michelle
    Söderström Lindström, Hanna
    Umeå universitet, Medicinska fakulteten, Institutionen för folkhälsa och klinisk medicin, Avdelningen för hållbar hälsa.
    Tang, Chaojun
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lindberg, Richard H.
    Umeå universitet, Teknisk-naturvetenskapliga fakulteten, Kemiska institutionen.
    Lundkvist, Åke
    Järhult, Josef D.
    Influenza A/H4N2 mallard infection experiments further indicate zanamivir as less prone to induce environmental resistance development than oseltamivir2020Ingår i: Journal of General Virology, ISSN 0022-1317, E-ISSN 1465-2099, Vol. 101, nr 8, s. 816-824Artikel i tidskrift (Refereegranskat)
    Abstract [en]

    Neuraminidase inhibitors (NAIs) are the gold standard treatment for influenza A virus (IAV). Oseltamivir is mostly used, followed by zanamivir (ZA). NAIs are not readily degraded in conventional wastewater treatment plants and can be detected in aquatic environments. Waterfowl are natural IAV hosts and replicating IAVs could thus be exposed to NAIs in the environment and develop resistance. Avian IAVs form the genetic basis for new human IAVs, and a resistant IAV with pandemic potential poses a serious public health threat, as NAIs constitute a pandemic preparedness cornerstone. Resistance development in waterfowl IAVs exposed to NAIs in the water environment has previously been investigated in an in vivo mallard model and resistance development was demonstrated in several avian IAVs after the exposure of infected ducks to oseltamivir, and in an H1N1 IAV after exposure to ZA. The N1 and N2 types of IAVs have different characteristics and resistance mutations, and so the present study investigated the exposure of an N2-type IAV (H4N2) in infected mallards to 1, 10 and 100 µg l−1 of ZA in the water environment. Two neuraminidase substitutions emerged, H274N (ZA IC50 increased 5.5-fold) and E119G (ZA IC50 increased 110-fold) at 10 and 100 µg l−1 of ZA, respectively. Reversion towards wild-type was observed for both substitutions in experiments with removed drug pressure, indicating reduced fitness of both resistant viruses. These results corroborate previous findings that the development of resistance to ZA in the environment seems less likely to occur than the development of resistance to oseltamivir, adding information that is useful in planning for prudent drug use and pandemic preparedness.

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