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  • 1.
    Abbott, Jessica K.
    et al.
    Queen's University.
    Bensch, S.
    Lund University.
    Gosden, Thomas P.
    Lund University.
    Svensson, Erik I.
    Lund University.
    Patterns of differentiation in a colour polymorphism and in neutral markers reveal rapid genetic changes in natural damselfly populations2008In: Molecular Ecology, ISSN 0962-1083, E-ISSN 1365-294X, Vol. 17, no 6, p. 1597-1604Article in journal (Refereed)
    Abstract [en]

    The existence and mode of selection operating on heritable adaptive traits can be inferred by comparing population differentiation in neutral genetic variation between populations (often using F(ST) values) with the corresponding estimates for adaptive traits. Such comparisons indicate if selection acts in a diversifying way between populations, in which case differentiation in selected traits is expected to exceed differentiation in neutral markers [F(ST )(selected) > F(ST )(neutral)], or if negative frequency-dependent selection maintains genetic polymorphisms and pulls populations towards a common stable equilibrium [F(ST) (selected) < F(ST) (neutral)]. Here, we compared F(ST) values for putatively neutral data (obtained using amplified fragment length polymorphism) with estimates of differentiation in morph frequencies in the colour-polymorphic damselfly Ischnura elegans. We found that in the first year (2000), population differentiation in morph frequencies was significantly greater than differentiation in neutral loci, while in 2002 (only 2 years and 2 generations later), population differentiation in morph frequencies had decreased to a level significantly lower than differentiation in neutral loci. Genetic drift as an explanation for population differentiation in morph frequencies could thus be rejected in both years. These results indicate that the type and/or strength of selection on morph frequencies in this system can change substantially between years. We suggest that an approach to a common equilibrium morph frequency across all populations, driven by negative frequency-dependent selection, is the cause of these temporal changes. We conclude that inferences about selection obtained by comparing F(ST) values from neutral and adaptive genetic variation are most useful when spatial and temporal data are available from several populations and time points and when such information is combined with other ecological sources of data.

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  • 2.
    Abd El-Gaber, Amira S.
    et al.
    Menoufia Univ, Fac Sci, Dept Chem, Shibin Al Kawm, Egypt.
    El Gendy, Abdel Nasser G.
    Natl Res Ctr, Med & Aromat Plants Res Dept, 33 El Bohouth St,PO 12622, Giza, Egypt.
    Elkhateeb, Ahmed
    Natl Res Ctr, Phytochem & Plant Systemat Dept, 33 El Bohouth St,PO 12622, Giza, Egypt.
    Saleh, Ibrahim A.
    Natl Res Ctr, Phytochem Dept, 33 El Bohouth St,PO 12622, Giza, Egypt.
    El-Seedi, Hesham
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi. Menoufia Univ, Fac Sci, Dept Chem, Shibin Al Kawm, Egypt;Univ Karachi, ICCBS, Karachi 75270, Pakistan.
    Microwave Extraction of Essential Oil from Anastatica hierochuntica (L): Comparison with Conventional Hydro-Distillation and Steam Distillation2018In: Journal of Essential Oil-Bearing Plants (JEOBP), ISSN 0972-060X, E-ISSN 0976-5026, Vol. 21, no 4, p. 1003-1010Article in journal (Refereed)
    Abstract [en]

    This article stands to introduce microwave assisted extraction (MAE) as a more effective method for extraction of Anastatica hierochuntica (L) essential oils (EOs) compared to traditional hydrodistillation (HD) and steam distillation (SD) methods. Analysis of EOs by gas chromatography-mass spectrometry (GC/MS) showed significant differences in the constituents and percentages of the obtained oils. Using MAE and HD obtained oxygenated monoterpenes 50.79 % whereas SD obtained sesquiterpene hydrocarbons 79.84 % as major contents of the extracted oils. This is the first report of EO composition of the aerials parts of A. heirochunatica. It offered several advantages of MAE technique as a green method with shorter extraction time (60 min) and better yield.

  • 3.
    Abdalaal, Hind
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pundir, Shreya
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ge, Xueliang
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Näsvall, Joakim
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Collateral toxicity limits the evolution of bacterial Release Factor 2 towards total omnipotence2020In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 37, no 10, p. 2918-2930Article in journal (Refereed)
    Abstract [en]

    When new genes evolve through modification of existing genes, there are often trade-offs between the new and original functions, making gene duplication and amplification necessary to buffer deleterious effects on the original function. We have used experimental evolution of a bacterial strain lacking peptide release factor 1 (RF1) in order to study how peptide release factor 2 (RF2) evolves to compensate the loss of RF1. As expected, amplification of the RF2-encoding gene prfB to high copy number was a rapid initial response, followed by the appearance of mutations in RF2 and other components of the translation machinery. Characterization of the evolved RF2 variants by their effects on bacterial growth rate, reporter gene expression, and in vitro translation termination reveals a complex picture of reduced discrimination between the cognate and near cognate stop codons and highlight a functional trade-off that we term “collateral toxicity”. We suggest that this type of trade-off may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary trade-offs between “old” and “new” functions of a gene, as it cannot be overcome by gene copy number changes. Further, we suggest a model for how RF2 autoregulation responds not only to alterations in the demand for RF2 activity, but also for RF1 activity.

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  • 4.
    Abdurakhmanov, Eldar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Discovery and evaluation of direct acting antivirals against hepatitis C virus2015Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Until recently, the standard therapy for hepatitis C treatment has been interferon and ribavirin. Such treatment has only 50% efficacy and is not well tolerated. The emergence of new drugs has increased the treatment efficacy to 90%. Despite such an achievement, the success is limited since the virus mutates rapidly, causing the emergence of drug resistant forms. In addition, most new drugs were developed to treat genotype 1 infections. Thus, development of new potent antivirals is needed and drug discovery against hepatitis C is continued.

    In this thesis, a FRET-based protease assay was used to evaluate new pyrazinone based NS3 protease inhibitors that are structurally different to the newly approved and currently developing drugs. Several compounds in this series showed good potencies in the nanomolar range against NS3 proteases from genotype 1, 3, and the drug resistance variant R155K. We assume that these compounds can be further developed into drug candidates that possess activity against above mentioned enzyme variants.

    By using SPR technology, we analyzed interaction mechanisms and characteristics of allosteric inhibitors targeting NS5B polymerases from genotypes 1 and 3. The compounds exhibited different binding mechanisms and displayed a low affinity against NS5B from genotype 3.

    In order to evaluate the activity and inhibitors of the NS5B polymerase, we established an SPR based assay, which enables the monitoring of polymerization and its inhibition in real time. This assay can readily be implemented for the discovery of inhibitors targeting HCV.

    An SPR based fragment screening approach has also been established. A screen of a fragment library has been performed in order to identify novel scaffolds that can be used as a starting point for development of new allosteric inhibitors against NS5B polymerase. Selected fragments will be further elaborated to generate a new potent allosteric drug candidate.

    Alternative approaches have successfully been developed and implemented to the discovery of potential lead compounds targeting two important HCV drug targets.

    List of papers
    1. Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
    Open this publication in new window or tab >>Discovery of pyrazinone based compounds that potently inhibit the drug resistant enzyme variant R155K of the hepatitis C virus NS3 protease
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    2016 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 24, no 12, p. 2603-2620Article in journal (Refereed) Published
    Abstract [en]

    Herein, we present the design and synthesis of 2(1H)-pyrazinone based HCV NS3 protease inhibitors with variations in the C-terminus. Biochemical evaluation was performed using genotype 1a, both the wildtype and the drug resistant enzyme variant, R155K. Surprisingly, compounds without an acidic sulfonamide retained good inhibition, challenging our previous molecular docking model. Moreover, selected compounds in this series showed nanomolar potency against R155K NS3 protease; which generally confer resistance to all HCV NS3 protease inhibitors approved or in clinical trials. These results further strengthen the potential of this novel substance class, being very different to the approved drugs and clinical candidates, in the development of inhibitors less sensitive to drug resistance.

    Keywords
    Hepatitis C virus; Drug resistance; Pyrazinone; NS3 protease inhibitors; R155K
    National Category
    Organic Chemistry
    Research subject
    Medicinal Chemistry
    Identifiers
    urn:nbn:se:uu:diva-243315 (URN)10.1016/j.bmc.2016.03.066 (DOI)000376727800002 ()27160057 (PubMedID)
    Funder
    Swedish Research Council, D0571301
    Available from: 2015-02-08 Created: 2015-02-08 Last updated: 2022-01-28Bibliographically approved
    2. Pyrazinone based hepatitis C virus NS3 protease inhibitors targeting genotype 1a, 3a and the drug-resistant enzyme variant R155K
    Open this publication in new window or tab >>Pyrazinone based hepatitis C virus NS3 protease inhibitors targeting genotype 1a, 3a and the drug-resistant enzyme variant R155K
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    (English)Manuscript (preprint) (Other academic)
    Abstract
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265295 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2022-01-25
    3. Resolution of the Interaction Mechanisms and Characteristics of Non-nucleoside Inhibitors of Hepatitis C Virus Polymerase - Laying the Foundation for Discovery of Allosteric HCV Drugs
    Open this publication in new window or tab >>Resolution of the Interaction Mechanisms and Characteristics of Non-nucleoside Inhibitors of Hepatitis C Virus Polymerase - Laying the Foundation for Discovery of Allosteric HCV Drugs
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    2013 (English)In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 97, no 3, p. 356-368Article in journal (Other academic) Published
    Abstract [en]

    Development of allosteric inhibitors into efficient drugs is hampered by their indirect mode-of-action and complex structure-kinetic relationships. To enablethe design of efficient allosteric drugs targeting the polymerase of hepatitis C virus(NS5B), the interaction characteristics of three non-nucleoside compounds (filibuvir, VX-222, and tegobuvir) inhibiting HCV replication via NS5B have been analyzed. Since there was no logical correlation between the anti-HCV replicative and enzyme inhibitory effects of the compounds, surface plasmon resonance biosensor technology was used to resolve the mechanistic, kinetic, thermodynamic and chemodynamic features of their interactions with their target and their effect on itsinteraction with RNA. Tegobuvir could not be seen to interact with NS5B at all while filibuvir interacted in a single reversible step (except at low temperatures) and VX-222 in two serial steps, interpreted as an induced fit mechanism. Both filibuvir and VX-222 interfered with the interaction between NS5B and RNA. They competed for binding to the enzyme, suggesting that they had a common inhibition mechanism and identical or overlapping binding sites. The greater anti-HCV replicative activityof VX-222 over filibuvir is hypothesized to be due to a greater allosteric conformational effect, resulting in the formation of a less catalytically competent complex. In addition, the induced fit mechanism of VX-222 gives it a kinetic advantage over filibuvir, exhibited as a longer residence time. These insights have important consequences for the selection and optimization of new allosteric NS5Binhibitors.

    Keywords
    HCV, NS5B, filibuvir, VX-222, tegobuvir, allosteric inhibitor, induced fit, kinetics, chemodynamics, thermodynamics
    National Category
    Biochemistry and Molecular Biology
    Research subject
    Biochemistry; Biochemistry
    Identifiers
    urn:nbn:se:uu:diva-171996 (URN)10.1016/j.antiviral.2012.12.027 (DOI)000317709400018 ()
    Available from: 2012-04-03 Created: 2012-03-31 Last updated: 2017-12-07Bibliographically approved
    4. Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative study
    Open this publication in new window or tab >>Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative study
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265287 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
    5. A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discovery
    Open this publication in new window or tab >>A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discovery
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265290 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
    6. Fragment library screening addressing Hepatitis C protein NS5B from genotypes 1 and 3 using an SPR-based approach
    Open this publication in new window or tab >>Fragment library screening addressing Hepatitis C protein NS5B from genotypes 1 and 3 using an SPR-based approach
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-265292 (URN)
    Available from: 2015-10-26 Created: 2015-10-26 Last updated: 2016-01-13
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  • 5. Abdurakhmanov, Eldar
    et al.
    Danielson, Helena
    A time-resolved surface plasmon resonance based hepatitis C virus NS5B polymerase assay and its application for drug discoveryManuscript (preprint) (Other academic)
  • 6.
    Abdurakhmanov, Eldar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Solbak, Sara
    Danielson, Helena
    Characterization of allosteric inhibitors of hepatitis C virus polymerase – a genotype comparative studyManuscript (preprint) (Other academic)
  • 7.
    Abdurakhmanov, Eldar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Solbak, Sara Oie
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Biophysical Mode-of-Action and Selectivity Analysis of Allosteric Inhibitors of Hepatitis C Virus (HCV) Polymerase2017In: Viruses, E-ISSN 1999-4915, Vol. 9, no 6, article id 151Article in journal (Refereed)
    Abstract [en]

    Allosteric inhibitors of hepatitis C virus (HCV) non-structural protein 5B (NS5B) polymerase are effective for treatment of genotype 1, although their mode of action and potential to inhibit other isolates and genotypes are not well established. We have used biophysical techniques and a novel biosensor-based real-time polymerase assay to investigate the mode-of-action and selectivity of four inhibitors against enzyme from genotypes 1b (BK and Con1) and 3a. Two thumb inhibitors (lomibuvir and filibuvir) interacted with all three NS5B variants, although the affinities for the 3a enzyme were low. Of the two tested palm inhibitors (dasabuvir and nesbuvir), only dasabuvir interacted with the 1b variant, and nesbuvir interacted with NS5B 3a. Lomibuvir, filibuvir and dasabuvir stabilized the structure of the two 1b variants, but not the 3a enzyme. The thumb compounds interfered with the interaction between the enzyme and RNA and blocked the transition from initiation to elongation. The two allosteric inhibitor types have different inhibition mechanisms. Sequence and structure analysis revealed differences in the binding sites for 1b and 3a variants, explaining the poor effect against genotype 3a NS5B. The indirect mode-of-action needs to be considered when designing allosteric compounds. The current approach provides an efficient strategy for identifying and optimizing allosteric inhibitors targeting HCV genotype 3a.

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  • 8. Abel, John H.
    et al.
    Drawert, Brian
    Hellander, Andreas
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computational Science.
    Petzold, Linda R.
    GillesPy: A Python package for stochastic model building and simulation2016In: IEEE Life Sciences Letters, E-ISSN 2332-7685, Vol. 2, p. 35-38Article in journal (Refereed)
  • 9.
    Abosedera, Dalia A.
    et al.
    Univ Sadat City, Environm Studies & Res Inst, Dept Nat Resources, Sadat City, Egypt..
    Emara, S. A.
    Univ Sadat City, Fac Vet Med, Dept Cytol & Histol, Sadat City, Egypt..
    Tamam, Omar A. S.
    Univ Sadat City, Environm Studies & Res Inst, Dept Nat Resources, Sadat City, Egypt..
    Badr, Osama M.
    Univ Sadat City, Anim Biotechnol Dept, Genet Engn & Biotechnol Inst GEBRI, Sadat City, Egypt..
    Khalifa, Shaden A. M.
    Stockholm Univ, Wenner Gren Inst, Dept Mol Biosci, S-10691 Stockholm, Sweden..
    El-Seedi, Hesham
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Farmakognosi. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. BJiangsu Univ, Int Res Ctr Food Nutr & Safety, Zhenjiang 212013, Jiangsu, Peoples R China.;Jiangsu Univ, Jiangsu Educ Dept, Int Joint Res Lab Intelligent Agr & Agriprod Proc, Zhenjiang, Jiangsu, Peoples R China.;Menoufia Univ, Fac Sci, Dept Chem, Menoufia 32511, Egypt..
    Refaey, Mohamed S.
    Univ Sadat City, Fac Pharm, Dept Pharmacognosy, Menoufia 32897, Egypt..
    Metabolomic profile and in vitro evaluation of the cytotoxic activity of Asphodelus microcarpus against human malignant melanoma cells A3752022In: Arabian Journal of Chemistry, ISSN 1878-5352, E-ISSN 1878-5379 , Vol. 15, no 10, article id 104174Article in journal (Refereed)
    Abstract [en]

    Melanoma is a huge worldwide health problem that must be handled more effectively with better therapeutic options. As a result, new treatment drugs are required to treat this condition. The goal of this study was to investigate the cytotoxic activity of the anthraquinone-rich fractions obtained from Asphodelus microcarpus against human melanoma cell A375. On these melanoma cell lines; the cytotoxicity of these fractions had never been studied before. Liquid chromatography linked to mass spectrometry (LC-MS-MS) and Nuclear Magnetic Resonance was used to determine the chemical profiles of these fractions. The cytotoxicity of the fractions studied was determined by measuring cell viability and calculating IC50 values. Both ethyl acetate (EtOAC) and the precipitate fractions (PPT) exhibited selective cytotoxicity on human melanoma A 375 cell line with IC50 values of 83 and 65 mu g/mL, respectively. The antiproliferative properties of EtOAc fraction and PPT were supported by a noticeable decrease in cell numbers during the G2/M cell cycle arrest. Our findings suggest that the anthraquinone content of A. microcarpus tubers is responsible for its anti-proliferative and apoptotic properties and that further in vivo investigations should be conducted to establish the viability of using them to treat human melanomas.

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  • 10.
    Abouzayed, Ayman
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Tano, Hanna
    KTH Royal Inst Technol, AlbaNova Univ Ctr, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, S-10691 Stockholm, Sweden..
    Nagy, Abel
    KTH Royal Inst Technol, AlbaNova Univ Ctr, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, S-10691 Stockholm, Sweden..
    Rinne, Sara S.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics.
    Wadeea, Fadya
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Kumar, Sharmishtaa
    KTH Royal Inst Technol, AlbaNova Univ Ctr, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, S-10691 Stockholm, Sweden..
    Westerlund, Kristina
    KTH Royal Inst Technol, AlbaNova Univ Ctr, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, S-10691 Stockholm, Sweden..
    Tolmachev, Vladimir
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Radiation Science. Research Centrum for Oncotheranostics, Research School of Chemistry and Applied Biomedical Sciences, Tomsk Polytechnic University, Tomsk 634050, Russia.
    Eriksson Karlström, Amelie
    KTH Royal Inst Technol, AlbaNova Univ Ctr, Dept Prot Sci, Sch Engn Sci Chem Biotechnol & Hlth, S-10691 Stockholm, Sweden..
    Orlova, Anna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Theranostics. Uppsala University, Science for Life Laboratory, SciLifeLab. Tomsk Polytech Univ, Res Sch Chem & Appl Biomed Sci, Res Centrum Oncotheranost, Tomsk 634050, Russia..
    Preclinical Evaluation of the GRPR-Targeting Antagonist RM26 Conjugated to the Albumin-Binding Domain for GRPR-Targeting Therapy of Cancer2020In: Pharmaceutics, E-ISSN 1999-4923, Vol. 12, no 10, article id 977Article in journal (Refereed)
    Abstract [en]

    The targeting of gastrin-releasing peptide receptors (GRPR) was recently proposed for targeted therapy, e.g., radiotherapy. Multiple and frequent injections of peptide-based therapeutic agents would be required due to rapid blood clearance. By conjugation of the GRPR antagonist RM26 (D-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH2) to an ABD (albumin-binding domain), we aimed to extend the blood circulation of peptides. The synthesized conjugate DOTA-ABD-RM26 was labelled with indium-111 and evaluated in vitro and in vivo. The labelled conjugate was stable in PBS and retained specificity and its antagonistic function against GRPR. The half-maximal inhibitory concentration (IC50) of In-nat-DOTA-ABD-RM26 in the presence of human serum albumin was 49 +/- 5 nM. [In-111]In-DOTA-ABD-RM26 had a significantly longer residence time in blood and in tumors (without a significant decrease of up to 144 h pi) than the parental RM26 peptide. We conclude that the ABD-RM26 conjugate can be used for GRPR-targeted therapy and delivery of cytotoxic drugs. However, the undesirable elevated activity uptake in kidneys abolishes its use for radionuclide therapy. This proof-of-principle study justified further optimization of the molecular design of the ABD-RM26 conjugate.

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    FULLTEXT01
  • 11.
    Aboye, Teshome L.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Strömstedt, Adam A.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Gunasekera, Sunithi
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Bruhn, Jan G.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    El-Seedi, Hesham
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    Rosengren, K. Johan
    Göransson, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
    A Cactus-Derived Toxin-Like Cystine Knot Peptide with Selective Antimicrobial Activity2015In: ChemBioChem, ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, no 7, p. 1068-1077Article in journal (Refereed)
    Abstract [en]

    Naturally occurring cystine knot peptides show a wide range of biological activity, and as they have inherent stability they represent potential scaffolds for peptide-based drug design and biomolecular engineering. Here we report the discovery, sequencing, chemical synthesis, three-dimensional solution structure determination and bioactivity of the first cystine knot peptide from Cactaceae (cactus) family: Ep-AMP1 from Echinopsis pachanoi. The structure of Ep-AMP1 (35 amino acids) conforms to that of the inhibitor cystine knot (or knottin) family but represents a novel diverse sequence; its activity was more than 500 times higher against bacterial than against eukaryotic cells. Rapid bactericidal action and liposome leakage implicate membrane permeabilisation as the mechanism of action. Sequence homology places Ec-AMP1 in the plant C6-type of antimicrobial peptides, but the three dimensional structure is highly similar to that of a spider neurotoxin.

  • 12.
    Abramson, Jeff
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
    Structural studies on the integral membrane protein, ubiquinol oxidase from Escherichia coli2001Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Heme-copper oxidases are redox-driven proton pumps that couple the reduction of molecular oxygen to water with the vectorial translocation of protons across the membrane. The proton gradient generated by heme-copper oxidases and the other members of the aerobic respiratory chain is ultimately used to drive the synthesis of ATP. There are two main branches of the heme-copper oxidases that are characterized by the electron donating substrate; the cytochrome c oxidases, which use cytochrome c as the electron donor, and the ubiquinol oxidases, which use a lipid-soluble molecule, ubiquinol, as their electron donor. These enzymes share important structural and functional features.

    This thesis presents the procedures that have led to the first crystal structure of a ubiquinol oxidase, cytochrome bo, oxidase from Escherichia coli, at a resolution of 3.5 Å. The overall structure of the enzyme is similar to those of cytochrome c oxidases; however the membrane spanning region of subunit I contains a cluster of polar residues exposed to the interior of the lipid bilayer. No such structural feature is present in cytochrome c oxidases. Mutagenesis studies on residues in this region strongly suggest that this area forms a ubiquinone binding site. A comparison of this region with known ubiquinone binding sites shows remarkable similarities. In light of these findings specific roles for these polar residues is proposed in electron and proton transfer in ubiquinol oxidase.

    A fusion protein of cytochrome bo3-Protein Z was generated in an attempt to increase the hydrophilic surface of the protein, thus extending protein-protein contacts within the crystal lattice structure. Such an approach can be used to facilitate crystallization.

  • 13. Abramsson, Mia L
    et al.
    Sahin, Cagla
    Hopper, Jonathan T S
    Branca, Rui M M
    Danielsson, Jens
    Xu, Mingming
    Chandler, Shane A
    Österlund, Nicklas
    Ilag, Leopold L
    Leppert, Axel
    Costeira-Paulo, Joana
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Lang, Lisa
    Teilum, Kaare
    Robinson, Carol V
    Laganowsky, Arthur
    Benesch, Justin L P
    Oliveberg, Mikael
    Marklund, Erik G
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Allison, Timothy M
    Winther, Jakob R
    Landreh, Michael
    Charge engineering reveals the roles of ionizable side chains in electrospray ionization mass spectrometryManuscript (preprint) (Other academic)
    Abstract [en]

    The role of ionizable side chains in the electrospray ionization mass spectrometry of intact proteins remains hotly debated but has not been conclusively addressed because multiple chargeable sites are present in virtually all proteins. Using engineered soluble proteins, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, whilst co-existing conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. We conclude that the sum of charges is governed solely by Coulombic terms, while their locations affect the stability of the protein in the gas phase.

  • 14.
    Adhikari, Subash
    et al.
    Macquarie Univ, Dept Biomed Sci, Fac Med Hlth & Human Sci, N Ryde, NSW 2109, Australia..
    Nice, Edouard C.
    Macquarie Univ, Dept Biomed Sci, Fac Med Hlth & Human Sci, N Ryde, NSW 2109, Australia.;Monash Univ, Dept Biochem & Mol Biol, Fac Med Nursing & Hlth Sci, Melbourne, Vic 3800, Australia..
    Deutsch, Eric W.
    Inst Syst Biol, 401 Terry Ave North, Seattle, WA 98109 USA..
    Lane, Lydie
    Univ Geneva, SIB Swiss Inst Bioinformat, Fac Med, CMU, Michel Servet 1, CH-1211 Geneva, Switzerland.;Univ Geneva, Dept Microbiol & Mol Med, Fac Med, CMU, Michel Servet 1, CH-1211 Geneva, Switzerland..
    Omenn, Gilbert S.
    Univ Michigan, Dept Computat Med & Bioinformat, Ann Arbor, MI 48109 USA..
    Pennington, Stephen R.
    Univ Coll Dublin, UCD Conway Inst Biomol & Biomed Res, Sch Med, Dublin, Ireland..
    Paik, Young-Ki
    Yonsei Proteome Res Ctr, Sudaemoon Ku, 50 Yonsei ro, Seoul 120749, South Korea..
    Overall, Christopher M.
    Univ British Columbia, Fac Dent, Vancouver, BC, Canada..
    Corrales, Fernando J.
    Ctr Nacl Biotecnol CSIC, Funct Prote Lab, Proteored ISCIII, Madrid 28049, Spain..
    Cristea, Ileana M.
    Princeton Univ, Dept Mol Biol, Princeton, NJ 08544 USA..
    Van Eyk, Jennifer E.
    Smidt Heart Inst, Cedars Sinai Med Ctr, Adv Clin Biosyst Res Inst, Los Angeles, CA 90048 USA..
    Uhlen, Mathias
    KTH Royal Inst Technol, Sch Engn Sci Chem, Sci Life Lab Biotechnol & Hlth, S-17121 Solna, Sweden..
    Lindskog, Cecilia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Clinical and experimental pathology.
    Chan, Daniel W.
    Johns Hopkins Univ, Dept Pathol & Oncol, Sch Med, Baltimore, MD 21224 USA..
    Bairoch, Amos
    Arizona State Univ, Biodesign Inst, Tempe, AZ USA..
    Waddington, James C.
    Justice, Joshua L.
    Arizona State Univ, Biodesign Inst, Tempe, AZ USA..
    LaBaer, Joshua
    Rodriguez, Henry
    NCI, Off Canc Clin Prote Res, NIH, Bethesda, MD 20892 USA..
    He, Fuchu
    Beijing Inst Life, Beijing Proteome Res Ctr, Natl Ctr Prot Sci Beijing, State Key Lab Prote, Beijing 102206, Peoples R China..
    Kostrzewa, Markus
    Bruker Daltonik GmbH, Microbiol & Diagnost, Fahrenheitstr, D-428359 Bremen, Germany..
    Ping, Peipei
    Univ Calif Los Angeles, David Geffen Sch Med, Dept Physiol, Cardiac Prote & Signaling Lab, Los Angeles, CA USA..
    Gundry, Rebekah L.
    Univ Nebraska Med Ctr, Div Cardiovasc Med, Cardiom Program, Ctr Heart & Vasc Res, Omaha, NE 68198 USA.;Univ Nebraska Med Ctr, Dept Cellular & Integrat Physiol, Cardiom Program, Ctr Heart & Vasc Res, Omaha, NE 68198 USA..
    Stewart, Peter
    Royal Prince Alfred Hosp, Dept Chem Pathol, Camperdown, NSW, Australia..
    Srivastava, Sanjeeva
    Indian Inst Technol, Powai 400076, Maharashtra, India..
    Srivastava, Sudhir
    Natl Canc Inst, Natl Inst Hlth, Canc Biomarkers Res Branch, Med Ctr Dr, Suite 5E136, Rockville, MD 20852 USA.;Fed Univ Rio Janeiro, Inst Chem, Lab Prote, Athos da Silveria Ramos,149, BR-21941909 Rio De Janeiro, RJ, Brazil..
    Nogueira, Fabio C. S.
    Fed Univ Rio Janeiro, Inst Chem, Prote Unit, Athos da Silveria Ramos,149, BR-21941909 Rio De Janeiro, RJ, Brazil.;Univ Grenoble Alpes, INSERM, CEA, IRIG BGE,U1038, F-38000 Grenoble, France..
    Domont, Gilberto B.
    Univ Grenoble Alpes, INSERM, CEA, IRIG BGE,U1038, F-38000 Grenoble, France..
    Vandenbrouck, Yves
    Univ Colorado, Dept Med Cardiol, Anschutz Med Campus, Aurora, CO USA.;Univ Colorado, Dept Biochem, Anschutz Med Campus, Aurora, CO USA.;Univ Colorado, Dept Mol Genet, Anschutz Med Campus, Aurora, CO USA..
    Lam, Maggie P. Y.
    Univ Colorado, Dept Med, Div Cardiol, Anschutz Med Campus, Aurora, CO USA.;European Bioinformat Inst, Mol Biol Lab, Wellcome Trust Genome Campus, Cambridge CB10 1SD, England..
    Wennersten, Sara
    Univ New South Wales, Sch Biotechnol & Biomol Sci, Sydney, NSW, Australia..
    Vizcaino, Juan Antonio
    Univ Calif San Diego, Dept Comp Sci & Engn, 9500 Gilman Dr,Mail Code 0404, San Diego, CA 92093 USA..
    Wilkins, Marc
    Lund Univ, Dept Biomed Engn, Lund, Sweden..
    Schwenk, Jochen M.
    Lundberg, Emma
    Bandeira, Nuno
    Univ Texas Hlth Sci Ctr San Antonio, UT Hlth, Dept Biochem & Struct Biol, 7703 Floyd Curl Dr, San Antonio, TX 78229 USA..
    Marko-Varga, Gyorgy
    Univ Rennes, INSERM, EHESP, IREST,UMR S 1085, F-35042 Rennes, France..
    Weintraub, Susan T.
    Leiden Univ, Med Ctr, NL-2333 Leiden, Netherlands..
    Pineau, Charles
    Stanford Sch Med, Dept Genet, Stanford, CA 94305 USA..
    Kusebauch, Ulrike
    Moritz, Robert L.
    Ahn, Seong Beom
    Palmblad, Magnus
    Univ Rennes, INSERM, EHESP, IREST,UMR S 1085, F-35042 Rennes, France..
    Snyder, Michael P.
    Leiden Univ, Med Ctr, NL-2333 Leiden, Netherlands..
    Aebersold, Ruedi
    Stanford Sch Med, Dept Genet, Stanford, CA 94305 USA..
    Baker, Mark S.
    Macquarie Univ, Dept Biomed Sci, Fac Med Hlth & Human Sci, N Ryde, NSW 2109, Australia.;Leiden Univ, Med Ctr, NL-2333 Leiden, Netherlands.;Univ Zurich, Fac Sci, Zurich, Switzerland..
    A high-stringency blueprint of the human proteome2020In: Nature Communications, E-ISSN 2041-1723, Vol. 11, no 1, article id 5301Article in journal (Refereed)
    Abstract [en]

    The Human Proteome Organization (HUPO) launched the Human Proteome Project (HPP) in 2010, creating an international framework for global collaboration, data sharing, quality assurance and enhancing accurate annotation of the genome-encoded proteome. During the subsequent decade, the HPP established collaborations, developed guidelines and metrics, and undertook reanalysis of previously deposited community data, continuously increasing the coverage of the human proteome. On the occasion of the HPP's tenth anniversary, we here report a 90.4% complete high-stringency human proteome blueprint. This knowledge is essential for discerning molecular processes in health and disease, as we demonstrate by highlighting potential roles the human proteome plays in our understanding, diagnosis and treatment of cancers, cardiovascular and infectious diseases. The Human Proteome Project (HPP) was launched in 2010 to enhance accurate annotation of the genome-encoded proteome. Ten years later, the HPP releases its first blueprint of the human proteome, annotating 90% of all known proteins at high-stringency and discussing the implications of proteomics for precision medicine.

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  • 15.
    Adler, Anna
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Fritsch, Marlene
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
    Fromell, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Leneweit, Gero
    Carl Gustav Carus-Institute, Association for the Promotion of Cancer Therapy, Niefern-Öschelbronn, Germany.
    Ekdahl, Kristina N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. Linnaeus Center of Biomaterials Chemistry, Linnaeus University, SE-391 82 Kalmar, Sweden.
    Nilsson, Bo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology.
    Teramura, Yuji
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Vascular Biology. Cellular and Molecular Biotechnology Research Institute (CMB), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central Fifth, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan; Master's/Doctoral Program in Life Science Innovation (T-LSI), University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan.
    Regulation of the innate immune system by fragmented heparin-conjugated lipids on lipid bilayered membranes in vitro2023In: Journal of materials chemistry. B, ISSN 2050-750X, E-ISSN 2050-7518, Vol. 11, no 46, p. 11121-11134Article in journal (Refereed)
    Abstract [en]

    Surface modification with heparin is a powerful biomaterial coating strategy that protects against innate immunity activation since heparin is a part of the proteoglycan heparan sulfate on cell surfaces in the body. We studied the heparinization of cellular and material surfaces via lipid conjugation to a heparin-binding peptide. In the present study, we synthesized fragmented heparin (fHep)-conjugated phospholipids and studied their regulation of the innate immune system on a lipid bilayered surface using liposomes. Liposomes have versatile applications, such as drug-delivery systems, due to their ability to carry a wide range of molecules. Owing to their morphological similarity to cell membranes, they can also be used to mimic a simple cell-membrane to study protein–lipid interactions. We investigated the interaction of complement-regulators, factor H and C4b-binding protein (C4BP), as well as the coagulation inhibitor antithrombin (AT), with fHep-lipids on the liposomal surface. Herein, we studied the ability of fHep-lipids to recruit factor H, C4BP, and AT using a quartz crystal microbalance with dissipation monitoring. With dynamic light scattering, we demonstrated that liposomes could be modified with fHep-lipids and were stable up to 60 days at 4 °C. Using a capillary western blot-based method (Wes), we showed that fHep-liposomes could recruit factor H in a model system using purified proteins and assist in the degradation of the active complement protein C3b to iC3b. Furthermore, we found that fHep-liposomes could recruit factor H and AT from human plasma. Therefore, the use of fHep-lipids could be a potential coating for liposomes and cell surfaces to regulate the immune system on the lipid surface.

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  • 16.
    Adler, Marlen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mechanisms and Dynamics of Carbapenem Resistance in Escherichia coli2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The emergence of extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae worldwide has led to an increased use of carbapenems and may drive the development of carbapenem resistance. Existing mechanisms are mainly due to acquired carbapenemases or the combination of ESBL-production and reduced outer membrane permeability. The focus of this thesis was to study the development of carbapenem resistance in Escherichia coli in the presence and absence of acquired β-lactamases. To this end we used the resistance plasmid pUUH239.2 that caused the first major outbreak of ESBL-producing Enterobacteriaceae in Scandinavia.

    Spontaneous carbapenem resistance was strongly favoured by the presence of the ESBL-encoding plasmid and different mutational spectra and resistance levels arose for different carbapenems. Mainly, loss of function mutations in the regulators of porin expression caused reduced influx of antibiotic into the cell and in combination with amplification of β-lactamase genes on the plasmid this led to high resistance levels. We further used a pharmacokinetic model, mimicking antibiotic concentrations found in patients during treatment, to test whether ertapenem resistant populations could be selected even at these concentrations. We found that resistant mutants only arose for the ESBL-producing strain and that an increased dosage of ertapenem could not prevent selection of these resistant subpopulations. In another study we saw that carbapenem resistance can even develop in the absence of ESBL-production. We found mutants in export pumps and the antibiotic targets to give high level resistance albeit with high fitness costs in the absence of antibiotics. In the last study, we used selective amplification of β-lactamases on the pUUH239.2 plasmid by carbapenems to determine the cost and stability of gene amplifications. Using mathematical modelling we determined the likelihood of evolution of new gene functions in this region. The high cost and instability of the amplified state makes de novo evolution very improbable, but constant selection of the amplified state may balance these factors until rare mutations can establish a new function.

    In my studies I observed the influence of β-lactamases on carbapenem resistance and saw that amplification of these genes would further contribute to resistance. The rapid disappearance of amplified arrays of resistance genes in the absence of antibiotic selection may lead to the underestimation of gene amplification as clinical resistance mechanism. Amplification of β-lactamase genes is an important stepping-stone and might lead to the evolution of new resistance genes.

    List of papers
    1. Influence of acquired β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli
    Open this publication in new window or tab >>Influence of acquired β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli
    2013 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 1, p. 51-59Article in journal (Refereed) Published
    Abstract [en]

    Objectives: To investigate the influence of plasmid-borne β-lactamases on the evolution of spontaneous carbapenem resistance in Escherichia coli and the fitness costs associated with resistance. Methods: Stepwise selection of carbapenem-resistant mutants with or without the extended-spectrum β-lactamase (ESBL)-encoding plasmid pUUH239.2 was performed. Mutation rates and mutational pathways to resistance were determined. In vitro-selected and constructed mutants were characterized regarding the MICs of the carbapenems, porin expression profiles, growth rates and the presence of mutations in the porins ompC/ompF and their regulatory genes. The influence of the plasmid-encoded β-lactamases TEM-1, OXA-1 and CTX-M-15 on resistance development was determined. Results: Results show that E. coli readily developed reduced carbapenem susceptibility and clinical resistance levels by a combination of porin loss and increased β-lactamase expression, especially towards ertapenem. All tested β-lactamases (CTX-M-15, TEM-1 and OXA-1) contributed to reduced carbapenem susceptibility in the absence of porin expression. However, complete loss of porin expression conferred a 20% fitness cost on the bacterial growth rate. Increased β-lactamase expression through spontaneous gene amplification on the plasmid was a major resistance factor. Conclusions: Plasmid-encoded β-lactamases, including non-ESBL enzymes, have a strong influence on the frequency and resistance level of spontaneous carbapenem-resistant mutants. The fitness cost associated with the loss of OmpC/OmpF in E. coli most likely reduces the survivability of porin mutants and could explain why they have not emerged as a clinical problem in this species.

    Keywords
    Fitness cost, Gene amplification, Mutations, Plasmids
    National Category
    Natural Sciences
    Identifiers
    urn:nbn:se:uu:diva-192026 (URN)10.1093/jac/dks368 (DOI)000312646300010 ()
    Available from: 2013-01-24 Created: 2013-01-15 Last updated: 2017-12-06Bibliographically approved
    2. Frequent emergence of porin-deficient subpopulations with reduced carbapenem susceptibility in ESBL-producing Escherichia coli during exposure to ertapenem in an in vitro pharmacokinetic model
    Open this publication in new window or tab >>Frequent emergence of porin-deficient subpopulations with reduced carbapenem susceptibility in ESBL-producing Escherichia coli during exposure to ertapenem in an in vitro pharmacokinetic model
    Show others...
    2013 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 68, no 6, p. 1319-1326Article in journal (Refereed) Published
    Abstract [en]

    OBJECTIVES:

    Ertapenem resistance is increasing in Enterobacteriaceae. The production of extended-spectrum β-lactamases (ESBLs) and reduced expression of outer membrane porins are major mechanisms of resistance in ertapenem-resistant Klebsiella pneumoniae. Less is known of ertapenem resistance in Escherichia coli. The aim of this study was to explore the impact of ESBL production in E. coli on the antibacterial activity of ertapenem.

    METHODS:

    Two E. coli strains, with and without ESBL production, were exposed to ertapenem in vitro for 48 h at concentrations simulating human pharmacokinetics with conventional and higher dosages.

    RESULTS:

    Isolates with non-susceptibility to ertapenem (MICs 0.75-1.5 mg/L) were detected after five of nine time-kill experiments with the ESBL-producing strain. All of these isolates had ompR mutations, which reduce the expression of outer membrane porins OmpF and OmpC. Higher dosage did not prevent selection of porin-deficient subpopulations. No mutants were detected after experiments with the non-ESBL-producing strain. Compared with other experiments, experiments with ompR mutants detected in endpoint samples showed significantly less bacterial killing after the second dose of ertapenem. Impaired antibacterial activity against E. coli with ESBL production and ompR mutation was also demonstrated in time-kill experiments with static antibiotic concentrations.

    CONCLUSIONS:

    The combination of ESBL production and porin loss in E. coli can result in reduced susceptibility to ertapenem. Porin-deficient subpopulations frequently emerged in ESBL-producing E. coli during exposure to ertapenem at concentrations simulating human pharmacokinetics. Inappropriate use of ertapenem should be avoided to minimize the risk of selection of ESBL-producing bacteria with reduced susceptibility to carbapenems.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-197878 (URN)10.1093/jac/dkt044 (DOI)000319468900016 ()23478794 (PubMedID)
    Available from: 2013-04-05 Created: 2013-04-05 Last updated: 2017-12-06Bibliographically approved
    3. Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli
    Open this publication in new window or tab >>Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli
    2016 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, p. 1188-1198Article in journal (Refereed) Published
    Abstract [en]

    The worldwide spread of ESBL-producing Enterobacteriaceae has led to an increased use of carbapenems, the group of beta-lactams with the broadest spectrum of activity. Bacterial resistance to carbapenems is mainly due to acquired carbapenemases or a combination of ESBL production and reduced drug influx via loss of outer-membrane porins. Here, we have studied the development of carbapenem resistance in Escherichia coli in the absence of beta-lactamases. We selected mutants with high-level carbapenem resistance through repeated serial passage in the presence of increasing concentrations of meropenem or ertapenem for similar to 60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance levels to 23 antibiotics were measured. High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure]. Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. beta-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.

    National Category
    Biochemistry and Molecular Biology Microbiology
    Identifiers
    urn:nbn:se:uu:diva-221428 (URN)10.1093/jac/dkv475 (DOI)000376291300008 ()26869688 (PubMedID)
    Funder
    Swedish Research Council Formas, 2013-5476-25194-9EU, European Research Council, 282004
    Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2017-12-05Bibliographically approved
    4. High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms
    Open this publication in new window or tab >>High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms
    Show others...
    2014 (English)In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 31, no 6, p. 1526-1535Article in journal (Refereed) Published
    Abstract [sv]

    An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplication-divergence includes several different sub-models, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kbp of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modelling shows that the fitness costs and instability strongly reduces the likelihood of both sub- and neofunctionalization, but that these effects can be off-set by positive selection for novel beneficial functions.

    National Category
    Microbiology Biochemistry and Molecular Biology Genetics
    Identifiers
    urn:nbn:se:uu:diva-221431 (URN)10.1093/molbev/msu111 (DOI)000337067400019 ()
    Available from: 2014-03-31 Created: 2014-03-31 Last updated: 2017-12-05Bibliographically approved
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  • 17.
    Adler, Marlen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anjum, Mehreen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli2016In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 71, no 5, p. 1188-1198Article in journal (Refereed)
    Abstract [en]

    The worldwide spread of ESBL-producing Enterobacteriaceae has led to an increased use of carbapenems, the group of beta-lactams with the broadest spectrum of activity. Bacterial resistance to carbapenems is mainly due to acquired carbapenemases or a combination of ESBL production and reduced drug influx via loss of outer-membrane porins. Here, we have studied the development of carbapenem resistance in Escherichia coli in the absence of beta-lactamases. We selected mutants with high-level carbapenem resistance through repeated serial passage in the presence of increasing concentrations of meropenem or ertapenem for similar to 60 generations. Isolated clones were whole-genome sequenced, and the order in which the identified mutations arose was determined in the passaged populations. Key mutations were reconstructed, and bacterial growth rates of populations and isolated clones and resistance levels to 23 antibiotics were measured. High-level resistance to carbapenems resulted from a combination of downstream effects of envZ mutation and target mutations in AcrAB-TolC-mediated drug export, together with PBP genes [mrdA (PBP2) after meropenem exposure or ftsI (PBP3) after ertapenem exposure]. Our results show that antibiotic resistance evolution can occur via several parallel pathways and that new mechanisms may appear after the most common pathways (i.e. beta-lactamases and loss of porins) have been eliminated. These findings suggest that strategies to target the most commonly observed resistance mechanisms might be hampered by the appearance of previously unknown parallel pathways to resistance.

  • 18.
    Adler, Marlen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Anjum, Mehreen
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Berg, Otto, G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Andersson, Dan I.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sandegren, Linus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms2014In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 31, no 6, p. 1526-1535Article in journal (Refereed)
    Abstract [sv]

    An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplication-divergence includes several different sub-models, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kbp of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modelling shows that the fitness costs and instability strongly reduces the likelihood of both sub- and neofunctionalization, but that these effects can be off-set by positive selection for novel beneficial functions.

  • 19.
    Afshar, Mostafa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Discovery of peptides in Chinese medicinal plants2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [en]

    In this study, 20 well-known Chinese medicinal plants are included. Traditionally, these medicinal herbs were consumed for different diseases but have one application in common, treatment of rheumatism arthritis. Peptides are pharmacologically attractive substances and the reason behind this work as there is only one study on peptide content of included herbs. Commonly, plant-derived peptides are cysteine-rich peptides. 

    The plants were extracted in series by 60%, 30% and 10% AcN in H2O and FA. Then, the combined extracts were purified by SPE and SEC. Additionally, reduction by DDT, alkylation by IAM, and digestion by trypsin performed. UPLC-QToF was used for separation and identification of potential peptides. 

    The investigation resulted in finding peptides in Coix lacryma-jobi L, Atractylodes lancea, Astragalus membranaceus. Peptides found in these species are mainly in the range 2,5-5 kDa. This finding raises new questions about the function of peptides in these species and the possibility of having pharmacological activity.

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  • 20.
    Afshari Kashanian, Elisa
    Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Detection of celery (Apium graveolens) in food with Real-Time PCR2006Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    Directive EC 2003/89/EC of the European Parliament and of the Council states that certain

    ingredients and products derived there of known to cause allergen reactions must always be

    declared. Furthermore labelling is mandatory irrespective of the amount included. The National

    Food Administration therefore needs methods for monitoring the presence of allergens in food.

    Methods already exist for most of the allergens on the EU-list, but an operational method for

    celery (Apium graveolens) is missing.

    A specific DNA-method was developed, based on TaqMan Real-Time PCR with the celery

    mannitol dehydrogenase gene as target sequence. The analysis was started with homogenisation

    of the sample followed by extraction of DNA. The Real-Time PCR method was shown to be

    specific for celery, producing a 113 bp fragment with two celery varieties and negative results

    with other closely selected species commonly present together with celery in food products (12

    samples). The detection limit was 2-20 pg DNA, which corresponds to 1-7 haploid genome

    copies. When evaluated with model samples of celery in meat, a detection limit of less than

    0,01 % was determined. When used to analyse food products from the market, six out of seven

    products declared to contain celery were correctly identified as positive.

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  • 21.
    Agbaje, Oluwatoosin B. A.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Earth Sciences, Department of Earth Sciences, Palaeobiology. Macquarie Univ, Dept Earth & Environm Sci, Sydney, NSW, Australia.;Macquarie Univ, Dept Biol Sci, Sydney, NSW, Australia..
    Dominguez, J. Gabriel
    Macquarie Univ, Dept Biol Sci, Sydney, NSW, Australia..
    Jacob, Dorrit E.
    Macquarie Univ, Dept Earth & Environm Sci, Sydney, NSW, Australia.;Australian Natl Univ, Res Sch Earth Sci, Canberra, ACT, Australia..
    Organic biopolymers of venus clams: Collagen-related matrix in the bivalve shells with crossed-lamellar ultrastructure2021In: Biochemistry and biophysics reports, ISSN 2405-5808, Vol. 26, article id 100939Article in journal (Refereed)
    Abstract [en]

    Background: Biochemical studies and spectroscopic techniques have shown that chitin-silk fibroins are common in nacroprismatic bivalve shells. However, the nature of organic biopolymers in the less well studied shell architectures, such as crossed lamellar shells, remain unknown. Here, two venus shells, Callista disrupta and Callista kingii, with crossed lamellar ultrastructure have been studied.

    Methods: We employed thermal gravimetric analysis, optical-, confocal- and scanning electron-microscopes, gel-sodium dodecyl sulfate (gel-SDS), FTIR, ultra-performance liquid chromatography and high-performance anion-exchange chromatography system with pulsed amperometric detection to analyse organic macromolecules in the shells.

    Results: Thermal analysis showed a low concentration of organic macromolecules in C. disrupta (1.38 wt%) and in C. kingii (1.71 wt%). A combination of biochemical protocols, including Calcofluor White staining and FTIR spectroscopic assessment, indicate that amino-polysaccharide chitin together with proteins, are present in the organic scaffolding of the shells. Scanning electron microscope of insoluble acid biopolymer extracts as well as FTIR technique show that the hierarchical structural organizations of organic biopolymers consist collagen-related matrix. Our histochemical fixing and staining techniques reveal many discrete proteins and glycoproteins from soluble organic macromolecules on the gel-SDS. We show here 'singlet' and 'doublet' glycosaminoglycan bands that are far above 260 kDa.

    General significance/conclusions: The presence of collagen matrix in Callista shells shows promise for the new source of biomaterials. Most importantly, the structural organization of the proteinaceous motif is predominantly helical structures and not silk-fibroin unlike in nacreous bivalve shells.

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  • 22.
    Aggarwal, Tanya
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Patil, Sourabh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Ceder, Mikaela
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Hayder, Maher
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Fredriksson, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Knockdown of SLC38 Transporter Ortholog-CG13743 Reveals a Metabolic Relevance in Drosophila2020In: Frontiers in Physiology, E-ISSN 1664-042X, Vol. 10, article id 1592Article in journal (Refereed)
    Abstract [en]

    Solute Carrier (SLC) is a cluster of families of membrane bound transporters, of which many members lack defined substrate profile, and many more are poorly characterized. Many play a vital role in regulating metabolic systems, protein synthesis, and post translational modifications. SLC38 is one of the families of SLCs, which are also known as sodium-coupled neutral amino acid transporters (SNATs). In mice, it has 11 members (SNAT1-11) but in Drosophila there are two homologs for the SLC38 family; CG13743 and CG30394. Here, we show characteristics of Drosophila CG13743 which closely resembles SLC38A11 in humans. SLC38A11 still remains an orphan member of the SLC38 family which has not been functionally well studied. We used the UAS-GAL4 system to investigate and control gene expression using RNAi lines for ubiquitous knockdown of the CG13743 gene. It was found to be expressed mainly in salivary gland and brain. Knockdown flies had reduced body weight and consumed less sugar compared with controls. The gene knockdown also affected stored energy pools (lipids and glycogen) and influenced feeding pattern and total activity. In all, this shows novel findings for the characterization of CG13743 in Drosophila and a possible role in maintaining general metabolic pathways and behavior of the fly.

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  • 23.
    Agnadóttir, Védís Mist Eyju
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Investigating the expression and function of RNA-binding protein FUBL-1 in C. elegans2023Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis
    Abstract [en]

    RNA interference (RNAi) is an important mechanism of gene silencing in Caenorhabditis elegans, in which short RNAs direct sequence-specific silencing of gene expression mediated by Argonaute proteins. RNAi in C. elegans can be sorted into exogenous RNAi, where the short RNAs originate from foreign RNA sequences, and endogenous RNAi, where the short RNAs originate from RNA sequences in the genome. One endogenous RNAi pathway is the ERGO-1 pathway, which is active in the germline and in embryos. Mutants deficient in the ERGO-1 pathway show an increased response to exogenous RNAi, which is thought to be due to competition between exogenous and endogenous silencing pathways. 

    FUBL-1 is an RNA-binding protein found in C. elegans, which has three predicted functional isoforms: isoforms a, b and c. Prior research by the Hinas group has indicated that FUBL-1 may play a role in the ERGO-1 pathway of RNAi. FUBL-1 deletion mutants show an increased response to exogenous RNAi similar to ERGO-1 mutants, and also an upregulation of ERGO-1 target genes. They have also found that FUBL-1 is broadly expressed in somatic tissues, but germline expression has not been confirmed. The function of the three different isoforms has not yet been examined.

    The aim of this study was to further investigate the function of FUBL-1 by assessing the function of the different isoforms through RT-qPCR of C. elegans with mutations affecting the different isoforms, to use immunofluorescence staining to see whether FUBL-1 is expressed in the germline, and to identify FUBL-1 RNA targets through CLIP-seq. Preliminary RT-qPCR results indicated that the upregulation of ERGO-1 targets is less in isoform mutants than in FUBL-1 deletion mutants, indicating partial redundancy of the isoforms. Immunofluorescence staining showed that FUBL-1 is expressed in the germline nuclei. Growth protocols have been optimized and crosslinking has been performed on worms, but the full CLIP-seq protocol has not been performed yet.

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  • 24.
    Agrillo, Caroline
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Environmental toxicology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    DNA methylation modifications in human mesenchymal stem cells  induced by exposure to endocrine disrupting plasticiser metabolites MBP, MEP, MBzP, MEHP and MINCH2022Independent thesis Advanced level (degree of Master (Two Years)), 80 credits / 120 HE creditsStudent thesis
    Abstract [en]

    Endocrine disrupting chemicals (EDCs) are exogenous substances which can modify the function of the endocrine system and lead to adverse health effects. Humans experience daily uncontrolled exposure to EDC mixtures. Predicting mixture effects is complicated since the chemicals may produce different effects when combined together. EDCs may produce epigenetic effects such as alterations of the DNA methylation, which could modify the expression of the gene. Previously, 14 chemicals linked to metabolism (mixture G1) were reported to induce DNA methylation changes in an in vitro model. Mixture G1 were based on a Swedish longitudinal study which had identified the chemical burden of >2300 pregnant women. This project aimed to study single chemical driver effects of five individual chemicals from mixture G1, namely the four phthalate metabolites monobutyl phthalate (MBP), monoethyl phthalate (MEP), monobenzyl pthtalate (MBzP), mono-(2-ethylhexyl) phthalate (MEHP) and the non-phthalate plasticiser Bis(7-methyloctyl) Cyclohexane-1,2-dicarboxylate (DINCH) metabolite 2-4-methyl-7-oxyoctyl-oxycarbonyl-cyclohexane carboxylic acid (MINCH). Human mesenchymal stem cells (hMSCs) were exposed to the five compounds individually at the same concentrations as in mixture G1. After exposure, DNA methylation changes in four CpG sites within PGM1, MYOF and HCFC1 genes were analysed. While som chemicals did not show statistically significant effects, one chemical showed significant effects and thus could be a potential driver. The discrepancy between the observed DNA methylation alterations in the analysed genes and the alterations in mixture G1 highlights the need for comparing mixture to single chemical effects to identify drivers within mixes and for increased understanding of mixture effects.

    The full text will be freely available from 2026-01-01 15:14
  • 25.
    Aguirre Rivera, Javier
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Tracking single molecules in uncharted territory: A single-molecule method to study kinetics in live bacteria2020Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The synthesis of proteins, also known as translation, is a fundamental process in every living organism. The steps in the translation of genetic information to functional proteins have been meticulously studied, mostly using in vitro techniques, yielding a detailed model of their mechanisms. However, the use of minimal cell-free systems allows for the possibility to miss interactions from absent components or that reactions are affected by the buffer composition. The work presented in this thesis opens a way to study the kinetics of complex molecular processes, like protein synthesis, directly inside live bacterial cells in real time. We developed and optimized a method to deliver dye-labeled macromolecules inside live cells and generate a kinetic model of the particle’s interactions based on its diffusion inside the cell.

    This method facilitated the study of translation elongation and initiation directly in live cells. Our measurements of reaction times of tRNA in the ribosome, agree with previous reports from in vitro techniques. We further applied the method to examine the effects of three aminoglycoside antibiotics and erythromycin directly in live cells. The aminoglycoside antibiotics slowed-down protein synthesis 2- to 4-fold, while the number of elongation cycles per initiation event decreased significantly. In the case of erythromycin, cells showed a 4-fold slower protein synthesis. Additionally, we measured the kinetics of sequence-specific effects of erythromycin: translational arrest, and peptidyl-tRNA drop-off; these in vivo measurements revealed a complex mechanism of action of the drug, in agreement with models suggested by previous experiments. Additionally, we applied the method to measure the effects, on the kinetics of protein synthesis, caused by modifications in the C-terminal tail of the S13 ribosomal protein. Our measurements showed that specific mutations led to different changes in the occupancy and dwell-time of labeled-tRNA in the ribosome.

    To summarize, the present work will guide the reader through the development of a method to study the kinetics of protein synthesis directly in live bacterial cells, as well as its application to characterize the effects of different antibiotics within the complex environment of a living organism.

    List of papers
    1. tRNA tracking for direct measurements of protein synthesis kinetics in live cells
    Open this publication in new window or tab >>tRNA tracking for direct measurements of protein synthesis kinetics in live cells
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    2018 (English)In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 14, no 6, p. 618-626Article in journal (Refereed) Published
    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.

    Place, publisher, year, edition, pages
    NATURE PUBLISHING GROUP, 2018
    National Category
    Biochemistry and Molecular Biology Cell and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-359663 (URN)10.1038/s41589-018-0063-y (DOI)000435445100019 ()29769736 (PubMedID)
    Funder
    Swedish Research Council, 2015-04111EU, European Research Council, ERC-2013-CoG 616047 SMILEKnut and Alice Wallenberg FoundationWenner-Gren FoundationsCarl Tryggers foundation , CTS 15:243
    Available from: 2018-09-05 Created: 2018-09-05 Last updated: 2020-08-21Bibliographically approved
    2. Real-time measurements of aminoglycoside effects on protein synthesis in live cells
    Open this publication in new window or tab >>Real-time measurements of aminoglycoside effects on protein synthesis in live cells
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    Single-molecule translation protein synthesis fluorescence microscopy bacteria antibiotics aminoglycoside gentamicin paromomycin apramycin
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-417619 (URN)
    Available from: 2020-08-21 Created: 2020-08-21 Last updated: 2020-09-08
    3. Direct measurements of erythromycin’s effect on protein synthesis kinetics in living bacterial cells
    Open this publication in new window or tab >>Direct measurements of erythromycin’s effect on protein synthesis kinetics in living bacterial cells
    2021 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 433, no 10, article id 166942Article in journal (Refereed) Published
    Abstract [en]

    Macrolide antibiotics, such as erythromycin, bind to the nascent peptide exit tunnel (NPET) of the bacterial ribosome and modulate protein synthesis depending on the nascent peptide sequence. Whereas in vitro biochemical and structural methods have been instrumental in dissecting and explaining the molecular details of macrolide-induced peptidyl-tRNA drop-off and ribosome stalling, the dynamic effects of the drugs on ongoing protein synthesis inside live bacterial cells are far less explored. In the present study, we used single-particle tracking of dye-labeled tRNAs to study the kinetics of mRNA translation in the presence of erythromycin, directly inside live Escherichia coli cells. In erythromycin-treated cells, we find that the dwells of elongator tRNA(Phe) on ribosomes extend significantly, but they occur much more seldom. In contrast, the drug barely affects the ribosome binding events of the initiator tRNA(fMet). By overexpressing specific short peptides, we further find context-specific ribosome binding dynamics of tRNA(Phe), underscoring the complexity of erythromycin's effect on protein synthesis in bacterial cells.

    Place, publisher, year, edition, pages
    Elsevier, 2021
    Keywords
    Single-molecule translation protein synthesis fluorescence microscopy bacteria antibiotics erythromycin
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-417623 (URN)10.1016/j.jmb.2021.166942 (DOI)000643684400005 ()33744313 (PubMedID)
    Funder
    Swedish Research Council, 2015-04111Swedish Research Council, 2016-06264Swedish Research Council, 2019-03714Wenner-Gren FoundationsCarl Tryggers foundation , 15:243Carl Tryggers foundation , 17:226
    Available from: 2020-08-21 Created: 2020-08-21 Last updated: 2024-01-15Bibliographically approved
    4. An extended C-terminal tail of the ribosomal protein S13 modulates the speed of ribosomal translocation.
    Open this publication in new window or tab >>An extended C-terminal tail of the ribosomal protein S13 modulates the speed of ribosomal translocation.
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Keywords
    Protein synthesis S13 single-molecule tRNA translocation
    National Category
    Biochemistry and Molecular Biology
    Identifiers
    urn:nbn:se:uu:diva-417624 (URN)
    Available from: 2020-08-21 Created: 2020-08-21 Last updated: 2020-08-21
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  • 26.
    Aguirre Rivera, Javier
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology. Uppsala Universitet.
    Larsson, Jimmy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. 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.
    Seefeldt, A. Carolin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Faculty of Science and Technology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Real-time measurements of aminoglycoside effects on protein synthesis in live cellsManuscript (preprint) (Other academic)
  • 27.
    Aguirre Rivera, Javier
    et al.
    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 Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. 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.
    Volkov, Ivan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Seefeldt, A. Carolin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Faculty of Science and Technology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Johansson, Magnus
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Systems Biology.
    Real-time measurements of aminoglycoside effects on protein synthesis in live cells2021In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 118, no 9, article id e2013315118Article in journal (Refereed)
    Abstract [en]

    The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.

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  • 28.
    Ahi, Ehsan Pashay
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology. University of Helsinki.
    Tsakoumis, Emmanouil
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Brunel, Mathilde
    Department of Molecular Sciences, Swedish University of Agricultural Sciences, Allmas Allé 5, SE-750 07, Uppsala, Sweden.
    Schmitz, Monika
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Transcriptional study reveals a potential leptin-dependent gene regulatory network in zebrafish brain2021In: Fish Physiology & Biochemistry, ISSN 0920-1742, E-ISSN 1573-5168, Vol. 47, no 4, p. 1283-1298Article in journal (Refereed)
    Abstract [en]

    The signal mediated by leptin hormone and its receptor is a major regulator of body weight, food intake and metabolism. In mammals and many teleost fish species, leptin has an anorexigenic role and inhibits food intake by influencing the appetite centres in the hypothalamus. However, the regulatory connections between leptin and downstream genes mediating its appetite-regulating effects are still not fully explored in teleost fish. In this study, we used a loss of function leptin receptor zebrafish mutant and real-time quantitative PCR to assess brain expression patterns of several previously identified anorexigenic genes downstream of leptin signal under different feeding conditions (normal feeding, 7-day fasting, 2 and 6-h refeeding). These downstream factors include members of cart genes, crhb and gnrh2, as well as selected genes co-expressed with them based on a zebrafish co-expression database. Here, we found a potential gene expression network (GRN) comprising the abovementioned genes by a stepwise approach of identifying co-expression modules and predicting their upstream regulators. Among the transcription factors (TFs) predicted as potential upstream regulators of this GRN, we found expression pattern of sp3a to be correlated with transcriptional changes of the downstream gene network. Interestingly, the expression and transcriptional activity of Sp3 orthologous gene in mammals have already been implicated to be under the influence of leptin signal. These findings suggest a potentially conserved regulatory connection between leptin and sp3a, which is predicted to act as a transcriptional driver of a downstream gene network in the zebrafish brain.

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    Ahi et al. 2019
  • 29.
    Ahlström, Anna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Testing the specificity of the pBAD arabinose reporter2017Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [en]

    The project highlights Salmonella enterica subspecies enterica serovar Typhimurium (S. Tm)'s ability to metabolize simple sugars released from dead commensal bacteria, by using the pBAD (araBAD promoter) system as a reporter of L-arabinose availability. Using bioinformatics and homology of conserved L-arabinose transporter genes shared in Escherichia coli K12 (E. coli) and S. Tm, we aimed to create a S. Tm mutant strain unable to obtain L-arabinose from it environment. During the projects course of time it was discovered that L-arabinose transporters are not a shared gene trait between E. coli and S. Tm, and that putative L-arabinose transporter orthologues may exists in the S. Tm genome.

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  • 30.
    Ahlsén, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Structure-activity and resistance studies of HIV-1 protease inhibitors2000Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The present investigation was undertaken in order to identify inhibitors of HIV-1 protease that would be efficient in vivo and against HIV-1 protease carrying mutations known to confer resistance to inhibitors in clinical use. A second interest was to understand details of inhibitory mechanisms and to gain understanding of the molecular details of resistance.

    Linear inhibitors of transition-state type showed to have a resistance pattern similar to protease inhibitors in clinical use, whereas cyclic inhibitors of sulfonamide were somewhat different in their inhibitory profiles. It was found that mutation L90M in some situations could lessen the decrease in overall efficiency suffered by the enzyme when aquiring other mutations. Also presented are results from the characterization of double mutation I84V/L90M, formerly not investigated. Testing of triple and quadruple mutant confirmed the additive features of some mutations. In an attempt to find new leads for inhibitor development, extracts from bee propolis, a natural product, was investigated, and it was found that one extract inhibited wild-type enzyme with an I50-value of 0.2 μg/mL. Even more interesting is the result that propolis extract also inhibited all the investigated mutant enzymes.

  • 31.
    Ahmad, Shabbir
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Hysteretic Behavior, Regioselectivity, and Role of Salt Bridging Residues at the Domain Interface of Potato Epoxide Hydrolase StEH1, Site-Directed Mutagenesis and Kinetic Study2009Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
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  • 32.
    Ahmadova, Nigar
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Mamedov, Fikret
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Formation of tyrosine radicals in photosystem II under far-red illumination2018In: Photosynthesis Research, ISSN 0166-8595, E-ISSN 1573-5079, Vol. 136, no 1, p. 93-106Article in journal (Refereed)
    Abstract [en]

    Photosystem II (PS II) contains two redox-active tyrosine residues on the donor side at symmetrical positions to the primary donor, P680. TyrZ, part of the water-oxidizing complex, is a preferential fast electron donor while TyrD is a slow auxiliary donor to P680 +. We used PS II membranes from spinach which were depleted of the water oxidation complex (Mn-depleted PS II) to study electron donation from both tyrosines by time-resolved EPR spectroscopy under visible and far-red continuous light and laser flash illumination. Our results show that under both illumination regimes, oxidation of TyrD occurs via equilibrium with TyrZ at pH 4.7 and 6.3. At pH 8.5 direct TyrD oxidation by P680 + occurs in the majority of the PS II centers. Under continuous far-red light illumination these reactions were less effective but still possible. Different photochemical steps were considered to explain the far-red light-induced electron donation from tyrosines and localization of the primary electron hole (P680 +) on the ChlD1 in Mn-depleted PS II after the far-red light-induced charge separation at room temperature is suggested.

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  • 33. Ahmed, Aisha S
    et al.
    Li, Jian
    Erlandsson-Harris, Helena
    Stark, André
    Bakalkin, Georgy
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Mahmood, Ahmed
    Suppression of pain and joint destruction by inhibition of the proteasome system in experimental osteoarthritis2012In: Pain, ISSN 0304-3959, E-ISSN 1872-6623, Vol. 153, no 1, p. 18-26Article in journal (Refereed)
    Abstract [en]

    Osteoarthritis is a degenerative joint disease with pain and loss of joint function as major pathological features. Recent studies show that proteasome inhibitors reduce pain in various pathological conditions. We evaluated the effects of MG132, a reversible proteasome inhibitor on pain and joint destruction in a rat model of osteoarthritis. Osteoarthritis was induced by intraarticular injection of monosodium iodoacetate into the rat knee. Knee joint stiffness was scored and nociception was evaluated by mechanical pressure applied to the respective hind paw. Knee joint destruction was assessed by radiological and histological analyses. Expression of matrix metalloproteinase-3 (MMP-3) was analyzed by quantitative reverse transcription polymerase chain reaction in the knee articular cartilage. Expression of substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in the dorsal root ganglia (L4–L6) by quantitative reverse transcription polymerase chain reaction and in the knee joints by immunohistochemistry. Our results indicate that daily treatment of osteoarthritic rats with MG132 significantly increases their mobility while the swelling, pain thresholds, and pathological features of the affected joints were reduced. Furthermore, the upregulated expression of MMP-3, SP, and CGRP in the arthritic rats was normalized by MG132 administration. We conclude that the proteasome inhibitor MG132 reduces pain and joint destruction, probably by involving the peripheral nervous system, and that changes in SP and CGRP expression correlate with alterations in behavioural responses. Our findings suggest that nontoxic proteasome inhibitors may represent a novel pharmacotherapy for osteoarthritis.

  • 34.
    Ahmedova, Sibel Ismetova
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. Mercodia AB.
    Development of a Sandwich ELISA to Quantify the GLP-1 Analogs2022Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    Immunoassay is a technology to quantify the molecules of biological samples. Enzyme-Linked Immunosorbent Assay (ELISA) is one of the most common immunoassays that is used to analyze biological samples. Mercodia is a company that is specialized in developing immunoassays within the area of metabolic disorders such as type 2 diabetes, obesity, and cardiovascular diseases. Mercodia wanted to develop a prototype sandwich ELISA for quantitative measurement of GLP-1 analogs, especially for Exenatide due to the customer requests. Thus, Mercodia in-house antibodies are paired with anti-Exenatide commercial antibodies and initial tests are performed. Various parameters and conditions are tested. Exenatide and Lixisenatide are successfully detected several times at 10 to 1000 pg/mL concentration. However, further optimizations are required in order to be more consistent with the results and to develop a more sensitive assay.

    The full text will be freely available from 2032-07-29 10:41
  • 35.
    Ahnoff, Martin
    et al.
    Univ Gothenburg, Dept Chem Mol Biol, SE-41296 Gothenburg, Sweden.;Denator AB, Gothenburg, Sweden..
    Cazares, Lisa H.
    US Army Med Res Inst Infect Dis, Mol & Translat Sci, Frederick, MD 21702 USA.;US Army Med Res & Mat Command, DoD Biotechnol High Performance Comp Software App, Telemed & Adv Technol Res Ctr, Ft Detrick, MD 21702 USA..
    Sköld, Karl
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Cancer Pharmacology and Computational Medicine. Denator AB, Gothenburg, Sweden.;Uppsala Univ, Dept Med Sci Canc Pharmacol & Computat Med, Uppsala, Sweden..
    Thermal inactivation of enzymes and pathogens in biosamples for MS analysis2015In: Bioanalysis, ISSN 1757-6180, E-ISSN 1757-6199, Vol. 7, no 15, p. 1885-1899Article, review/survey (Refereed)
    Abstract [en]

    Protein denaturation is the common basis for enzyme inactivation and inactivation of pathogens, necessary for preservation and safe handling of biosamples for downstream analysis. While heat-stabilization technology has been used in proteomic and peptidomic research since its introduction in 2009, the advantages of using the technique for simultaneous pathogen inactivation have only recently been addressed. The time required for enzyme inactivation by heat (approximate to 1 min) is short compared with chemical treatments, and inactivation is irreversible in contrast to freezing. Heat stabilization thus facilitates mass spectrometric studies of biomolecules with a fast conversion rate, and expands the chemical space of potential biomarkers to include more short-lived entities, such as phosphorylated proteins, in tissue samples as well as whole-blood (dried blood sample) samples.

  • 36. Aili, Daniel
    et al.
    Enander, Karin
    Baltzer, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry.
    Liedberg, Bo
    Synthetic de novo designed polypeptides for control of nanoparticle assembly and biosensing2007In: Biochemical Society Transactions, ISSN 0300-5127, E-ISSN 1470-8752, Vol. 35, no 3, p. 532-534Article in journal (Refereed)
    Abstract [en]

    This contribution describes how de novo designed synthetic helix–loop–helix polypeptides are utilized tocontrol the assembly of gold nanoparticles and as scaffolds for biosensing. The synthetic polypeptides aredesigned to fold into a four-helix bundle upon dimerization. When immobilized on gold nanoparticles,dimerization and folding occur between peptides on neighbouring particles as an effect of particleaggregation and the folded polypeptides are rigid enough to keep the particles separated at a distancecorresponding to the size of the four-helix bundle. Moreover, peptide dimerization offers a convenientroute to assemble nanoparticles into hybrid multilayers on planar substrates. The drastic change in theresonance conditions of the localized nanoparticle surface plasmon upon particle aggregation is shown tobe useful for optical detection of biomolecular interactions.

  • 37.
    Akaberi, Dario
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Identification of protease inhibitors against Flaviviruses and Coronaviruses2023Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Vector-borne flaviviruses and coronaviruses of zoonotic origins are important human pathogens and represent a serious threat to public health worldwide. Flaviviruses can be found on all continents, apart from Antarctica, where they are transmitted by arthropod vectors causing millions of infections every year. While most of the infections are mild or asymptomatic, flaviviruses like dengue and yellow fever viruses can cause potentially lethal hemorrhagic fever and shock syndrome. Neurotropic flaviviruses like West Nile, Japanese encephalitis, and Tick-borne encephalitis (TBEV) can cause meningoencephalitis with long-term symptoms.  Coronaviruses, and in particular betacoronaviruses of zoonotic origin like SARS (2003) and MERS (2012), have been periodically emerging since the early 2000s causing outbreaks of severe respiratory syndrome. The latest example is SARS-CoV-2 that after causing a cluster of infection in the Chinese city of Wuhan, spread all over the world causing at present over 6.9 million deaths. Although vaccines are essential in preventing infections or severe disease and hospitalization in the case of SARS-CoV-2, antivirals represent an extremely valuable tool for treatment and prevention of current and future flavivirus and coronavirus infections. In the work presented in this thesis we have used a combination of in silico and in vitro techniques to identify and test the activity of potential inhibitors of viral proteases. 

    In our first study (paper 1) we unexpectedly identified an HIV protease inhibitor with in vitro activity against ZIKV NS2B-NS3 protease. The inhibitor was identified by virtual screening of a library of known protease inhibitors, evaluated by molecular dynamics simulation and finally tested against recombinant ZIKV protease using a FRET-based enzymatic assay. The same combination of molecular docking and molecular dynamics simulations were also used to correctly predict the activity of a known pan-Flavivirus protease inhibitor against TBEV protease (paper 2). As a result, we were the first to report peptide-based compounds with in vitro activity against TBEV. 

    After the outbreak of the COVID-19 we switched our attention to SARS-CoV-2. We first tested the inhibitory effect of the broad-spectrum antiviral nitric oxide (NO) and found that the NO-releasing compound SNAP had a dose dependent inhibitory effect on SARS-CoV-2 replication in cell-based assays (paper 3). We speculated that SNAP could inhibit SARS-COV-2 protease by trans-nitration of the catalytic Cys145 of SARS-CoV-2 main protease and found that SNAP had a dose dependent inhibitory effect on recombinant SARS-CoV-2 Mpro protease activity in an in vitro enzymatic assay. In our last study (paper 4) we identified a new class of potent SARS-CoV-2 protease inhibitors through the affinity screening of DNA-encoded-chemical libraries containing 4.2 billion compounds. The identified compounds inhibited recombinant SARS-CoV-2 protease with IC50 as low as 25 nM and had a dose dependent antiviral effect in the low micromolar range in infected Calu-3 and Caco-2 cell lines. 

    List of papers
    1. Targeting the NS2B-NS3 protease of tick-borne encephalitis virus with pan-flaviviral protease inhibitors
    Open this publication in new window or tab >>Targeting the NS2B-NS3 protease of tick-borne encephalitis virus with pan-flaviviral protease inhibitors
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    2021 (English)In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 190, article id 105074Article in journal (Refereed) Published
    Abstract [en]

    Tick-borne encephalitis (TBE) is a severe neurological disorder caused by tick-borne encephalitis virus (TBEV), a member of the Flavivirus genus. Currently, two vaccines are available in Europe against TBEV. However, TBE cases have been rising in Sweden for the past twenty years, and thousands of cases are reported in Europe, emphasizing the need for antiviral treatments against this virus. The NS2B-NS3 protease is essential for flaviviral life cycle and has been studied as a target for the design of inhibitors against several well-known flaviviruses, but not TBEV. In the present study, Compound 86, a known tripeptidic inhibitor of dengue (DENV), West Nile (WNV) and Zika (ZIKV) proteases, was predicted to be active against TBEV protease using a combination of in silico techniques. Further, Compound 86 was found to inhibit recombinant TBEV protease with an IC50 = 0.92 mu M in the in vitro enzymatic assay. Additionally, two more peptidic analogues were synthetized and they displayed inhibitory activities against both TBEV and ZIKV proteases. In particular, Compound 104 inhibited ZIKV protease with an IC50 = 0.25 mu M. These compounds represent the first reported inhibitors of TBEV protease to date and provides valuable information for the further development of TBEV as well as pan-flavivirus protease inhibitors.

    Place, publisher, year, edition, pages
    ElsevierELSEVIER, 2021
    Keywords
    Tick-borne encephalitis virus, Zika virus, NS2B-NS3 serine protease, Docking, MD simulations, Peptide hybrids
    National Category
    Infectious Medicine
    Identifiers
    urn:nbn:se:uu:diva-447918 (URN)10.1016/j.antiviral.2021.105074 (DOI)000657783600004 ()33872674 (PubMedID)
    Funder
    Swedish National Infrastructure for Computing (SNIC), SNIC 2017/1-213Swedish National Infrastructure for Computing (SNIC), SNIC 2018/3-252Swedish National Infrastructure for Computing (SNIC), SNIC 2019/3-312
    Available from: 2021-09-02 Created: 2021-09-02 Last updated: 2024-01-15Bibliographically approved
    2. Identification of a C2-symmetric diol based human immunodeficiency virus protease inhibitor targeting Zika virus NS2B-NS3 protease
    Open this publication in new window or tab >>Identification of a C2-symmetric diol based human immunodeficiency virus protease inhibitor targeting Zika virus NS2B-NS3 protease
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    2020 (English)In: Journal of Biomolecular Structure and Dynamics, ISSN 0739-1102, E-ISSN 1538-0254, Vol. 38, no 18, p. 5526-5536Article in journal (Refereed) Published
    Abstract [en]

    Zika virus (ZIKV) is an emerging mosquito-borne flavivirus and infection by ZIKV Asian lineage is known to cause fetal brain anomalies and Guillain-Barrés syndrome. The WHO declared ZIKV a global public health emergency in 2016. However, currently neither vaccines nor antiviral prophylaxis/treatments are available. In this study, we report the identification of a C2-symmetric diol-based Human immunodeficiency virus type-1 (HIV) protease inhibitor active against ZIKV NS2B-NS3 protease. The compound, referred to as 9b, was identified by in silico screening of a library of 6265 protease inhibitors. Molecular dynamics (MD) simulation studies revealed that compound 9b formed a stable complex with ZIKV protease. Interaction analysis of compound 9b's binding pose from the cluster analysis of MD simulations trajectories predicted that 9b mostly interacted with ZIKV NS3. Although designed as an aspartyl protease inhibitor, compound 9b was found to inhibit ZIKV serine protease in vitro with IC50 = 143.25 ± 5.45 µM, in line with the in silico results. Additionally, linear interaction energy method (LIE) was used to estimate binding affinities of compounds 9b and 86 (a known panflavivirus peptide hybrid with IC50 = 1.64 ± 0.015 µM against ZIKV protease). The LIE method correctly predicted the binding affinity of compound 86 to be lower than that of 9b, proving to be superior to the molecular docking methods in scoring and ranking compounds. Since most of the reported ZIKV protease inhibitors are positively charged peptide-hybrids, with our without electrophilic warheads, compound 9b represents a less polar and more drug-like non-peptide hit compound useful for further optimization.Communicated by Ramaswamy Sarma.

    Keywords
    In silico screening, NS2B-NS3 protease, Zika virus (ZIKV), protease inhibitors, structure-based drug discovery
    National Category
    Medicinal Chemistry
    Identifiers
    urn:nbn:se:uu:diva-430553 (URN)10.1080/07391102.2019.1704882 (DOI)000504558200001 ()31880199 (PubMedID)
    Funder
    Kjell and Marta Beijer Foundation
    Available from: 2021-01-11 Created: 2021-01-11 Last updated: 2023-08-10Bibliographically approved
    3. Mitigation of the replication of SARS-CoV-2 by nitric oxide in vitro
    Open this publication in new window or tab >>Mitigation of the replication of SARS-CoV-2 by nitric oxide in vitro
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    2020 (English)In: Redox Biology, E-ISSN 2213-2317, Vol. 37, article id 101734Article in journal (Refereed) Published
    Abstract [en]

    The ongoing SARS-CoV-2 pandemic is a global public health emergency posing a high burden on nations' health care systems and economies. Despite the great effort put in the development of vaccines and specific treatments, no prophylaxis or effective therapeutics are currently available. Nitric oxide (NO) is a broad-spectrum antimicrobial and a potent vasodilator that has proved to be effective in reducing SARS-CoV replication and hypoxia in patients with severe acute respiratory syndrome. Given the potential of NO as treatment for SARS-CoV-2 infection, we have evaluated the in vitro antiviral effect of NO on SARS-CoV-2 replication. The NO-donor S-nitroso-N-acetylpenicillamine (SNAP) had a dose dependent inhibitory effect on SARS-CoV-2 replication, while the non S-nitrosated NAP was not active, as expected. Although the viral replication was not completely abolished (at 200 μM and 400 μM), SNAP delayed or completely prevented the development of viral cytopathic effect in treated cells, and the observed protective effect correlated with the level of inhibition of the viral replication. The capacity of the NO released from SNAP to covalently bind and inhibit SARS-CoV-2 3CL recombinant protease in vitro was also tested. The observed reduction in SARS-CoV-2 protease activity was consistent with S-nitrosation of the enzyme active site cysteine.

    Place, publisher, year, edition, pages
    Elsevier, 2020
    Keywords
    3CL protease, COVID-19, FRET, Nitric oxide, SARS-CoV-2
    National Category
    Infectious Medicine
    Identifiers
    urn:nbn:se:uu:diva-430554 (URN)10.1016/j.redox.2020.101734 (DOI)000605007700001 ()33007504 (PubMedID)
    Funder
    Swedish Research Council, 2017-05807Swedish Research Council, 2018-02569Knut and Alice Wallenberg FoundationScience for Life Laboratory, SciLifeLab
    Available from: 2021-01-11 Created: 2021-01-11 Last updated: 2024-01-04Bibliographically approved
    4. Identification of unique and potent inhibitors of SARS-CoV-2 main protease from DNA-encoded chemical libraries
    Open this publication in new window or tab >>Identification of unique and potent inhibitors of SARS-CoV-2 main protease from DNA-encoded chemical libraries
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    (English)Manuscript (preprint) (Other academic)
    Keywords
    SARS-CoV-2, antivirals, protease inhibitors
    National Category
    Infectious Medicine Medicinal Chemistry
    Research subject
    Biology with specialization in Microbiology
    Identifiers
    urn:nbn:se:uu:diva-508906 (URN)
    Available from: 2023-08-10 Created: 2023-08-10 Last updated: 2023-08-14Bibliographically approved
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  • 38. Akpiroro Peters, Marie Berit
    et al.
    Kassa, Eszter
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Yau, Wai-Lok
    Lindqvist, Richard
    Nilsson, Emma
    Siljedahl, Michaela
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Lundmark, Richard
    Överby, Anna K.
    Tick-borne flaviviruses recruits the pro viral factor NUP153 to the replication siteManuscript (preprint) (Other academic)
  • 39.
    Aksoy, N. H.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Aksaray Univ, Dept Biochem, Aksaray, Turkey..
    Mannervik, B.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Inhibitory effects of ethacrynic acid on glutathione S-transferase A1-1 from Callithrix jacchus2015In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, p. 348-348Article in journal (Other academic)
  • 40.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Box 7011, SE-75007 Uppsala, Sweden..
    Fu, Zhirong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Wernersson, Sara
    Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Box 7011, SE-75007 Uppsala, Sweden..
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    The Evolutionary History of the Chymase Locus -a Locus Encoding Several of the Major Hematopoietic Serine Proteases2021In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 22, no 20, article id 10975Article, review/survey (Refereed)
    Abstract [en]

    Several hematopoietic cells of the immune system store large amounts of proteases in cytoplasmic granules. The absolute majority of these proteases belong to the large family of chymotrypsin-related serine proteases. The chymase locus is one of four loci encoding these granule-associated serine proteases in mammals. The chymase locus encodes only four genes in primates, (1) the gene for a mast-cell-specific chymotryptic enzyme, the chymase; (2) a T-cell-expressed asp-ase, granzyme B; (3) a neutrophil-expressed chymotryptic enzyme, cathepsin G; and (4) a T-cell-expressed chymotryptic enzyme named granzyme H. Interestingly, this locus has experienced a number of quite dramatic expansions during mammalian evolution. This is illustrated by the very large number of functional protease genes found in the chymase locus of mice (15 genes) and rats (18 genes). A separate expansion has also occurred in ruminants, where we find a new class of protease genes, the duodenases, which are expressed in the intestinal region. In contrast, the opossum has only two functional genes in this locus, the mast cell (MC) chymase and granzyme B. This low number of genes may be the result of an inversion, which may have hindered unequal crossing over, a mechanism which may have been a major factor in the expansion within the rodent lineage. The chymase locus can be traced back to early tetrapods as genes that cluster with the mammalian genes in phylogenetic trees can be found in frogs, alligators and turtles, but appear to have been lost in birds. We here present the collected data concerning the evolution of this rapidly evolving locus, and how these changes in gene numbers and specificities may have affected the immune functions in the various tetrapod species.

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  • 41.
    Akula, Srinivas
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Welinder, Charlotte
    Lund Univ, Dept Clin Sci Lund, Div Mass Spectrometry, SE-22100 Lund, Sweden..
    Fu, Zhirong
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Olsson, Anna-Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hellman, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Microbiology and Immunology.
    Identification of the Major Protein Components of Human and Cow Saliva2023In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 24, no 23, article id 16838Article in journal (Refereed)
    Abstract [en]

    Cows produce saliva in very large quantities to lubricate and facilitate food processing. Estimates indicate an amount of 50-150 L per day. Human saliva has previously been found to contain numerous antibacterial components, such as lysozyme, histatins, members of the S-100 family and lactoferrin, to limit pathogen colonization. Cows depend on a complex microbial community in their digestive system for food digestion. Our aim here was to analyze how this would influence the content of their saliva. We therefore sampled saliva from five humans and both nose secretions and saliva from six cows and separated the saliva on SDS-PAGE gradient gels and analyzed the major protein bands with LC-MS/MS. The cow saliva was found to be dominated by a few major proteins only, carbonic anhydrase 6, a pH-stabilizing enzyme and the short palate, lung and nasal epithelium carcinoma-associated protein 2A (SPLUNC2A), also named bovine salivary protein 30 kDa (BSP30) or BPIFA2B. This latter protein has been proposed to play a role in local antibacterial response by binding bacterial lipopolysaccharides (LPSs) and inhibiting bacterial growth but may instead, according to more recent data, primarily have surfactant activity. Numerous peptide fragments of mucin-5B were also detected in different regions of the gel in the MS analysis. Interestingly, no major band on gel was detected representing any of the antibacterial proteins, indicating that cows may produce them at very low levels that do not harm the microbial flora of their digestive system. The nose secretions of the cows primarily contained the odorant protein, a protein thought to be involved in enhancing the sense of smell of the olfactory receptors and the possibility of quickly sensing potential poisonous food components. High levels of secretory IgA were also found in one sample of cow mouth drippings, indicating a strong upregulation during an infection. The human saliva was more complex, containing secretory IgA, amylase, carbonic anhydrase 6, lysozyme, histatins and a number of other less abundant proteins, indicating a major difference to the saliva of cows that show very low levels of antibacterial components, most likely to not harm the microbial flora of the rumen.

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  • 42.
    Alabud, Arwa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Proteasomens roll för ligand inducerad fragmentation av PDGF-β receptorn2021Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
    Abstract [sv]

    Bakgrund: Platelet derived growth factor receptorn (PDGF receptorn) är en receptor i kroppen som tillhör typ III av Recetor tyrosine kinas (RTK) familjen. PDGF-β receptorn är en typ av dessa receptorer som enligt en studie klyvs i två delar efter ligandbindning med PDGF-BB ligand: till en extracellulär del och en intracellulär del. Hypotesen är att den extracellulära delen går till lysosomen medan den intracellulära delen går till proteasomen efter klyvningen.

    Syfte: Att undersöka rollen för proteasomen i ligandinducerad fragmentation av PDGF-β receptorn och se om det finns ko-lokalisation mellan receptorns extracellulära del och proteasomen. Dessutom ska det undersökas hur eventuell ko-lokalisering av PDGF receptorns extracellulära fragment med proteasomet påverkas när proteasomerna i cellerna inhiberas.

    Metod: Bj-hTERT celler stimulerades med PDGF-BB ligand under fyra olika stimuleringstidspunkter (0, 30, 60 och 90 min) och med hjälp av immunofluorescens undersöktes det om det fanns ko-lokalisering mellan proteasomen och PDGF-β receptorns extracellulära del. I ett annat experiment inhiberades även proteasomens aktivitet med inhibitoren MG132 och ko-lokalisationen mättes och jämfördes med kontrollceller behandlat med DMSO för de fyra stimuleringstidspunkterna.

    Resultat: För det första ko-lokalisationsexperimentet visades ingen större ko-lokalisering mellan proteasomen och receptorns extracellulära del för alla fyra tidspunkter. För det andra ko-lokalisationsexperimentet visades ingen skillnad i ko-lokalisationen mellan proteasomen och receptorns extracellulära fragment för cellerna vars proteasomaktivitet inhiberats och kontrollcellerna. Det visade sig däremot att proteasomen spelade roll för internaliseringen av receptorns extracellulära del in i cellen.

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  • 43.
    Al-Ani, Abdullah
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. University of Northampton.
    Investigating the subcellular localisation and function(s) of dystrophin protein Dp71 isoforms in glioma2022Independent thesis Advanced level (degree of Master (Two Years)), 30 credits / 45 HE creditsStudent thesis
    Abstract [en]

    The Dp71 protein is the most expressed product of the DMD gene in the nervous system. Mutation in the region codes dystrophin protein (Dp71) linked to cognitive disturbances in Duchenne muscular dystrophy (DMD) patients. There is growing evidence that the gene is contributing to the development of Central Nervous System related cancers. The aim of this study is to characterise the role of the main 4 Dp71 isoforms by investigating its subcellular localisation and putative cellular functions in glioblastoma cells, the most aggressive and common type of glioma. By transfecting the four GFP-Dp71 constructs into a well characterised human glioblastoma cell line – U251-MG. Immunoblotting was used to assess Dp71 expression in human glioblastoma cell line. Moreover, we examined the subcellular localisation and the effect of Dp71 over expression on the nuclear Lamin B1, cell migration immunofluorescence, and scratch assay. A 71 kDa endogenous Dp71 was expressed in all glioblastoma cells and only GFP-Dp71a (99 kDa) isoform was overexpressed in the transfected cells. Lower Lamin B1 fluorescence intensity and abnormal nuclear shape was observed in cells overexpressing GFP-Dp71a. Furthermore, cytoplasmic and nuclear localisation of Dp71 isoform was found in both cytoplasm and nucleus, but higher in the nucleus. Overexpression of Dp71a transfected cells reduced the cell migration and covering the scratched tissue gaps with 5% and 6%, whilst that of the control cells were 29% and 50% at time 24 and 48 hours, respectively compared to the that at time 0. Dp71ab transfected cells showed similar cell migration to the control. We concluded that Dp71 overexpression had a clear effect on the expression of Lamin B1 and cell migration. Additionally, localisation of Dp71a was higher in the nucleus than in the cytoplasm.

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  • 44.
    Albers, Suki
    et al.
    Univ Hamburg, Inst Biochem & Mol Biol, Hamburg, Germany..
    Beckert, Bertrand
    Univ Hamburg, Inst Biochem & Mol Biol, Hamburg, Germany..
    Matthies, Marco C.
    Univ Hamburg, Ctr Bioinformat, Hamburg, Germany..
    Mandava, Chandra Sekhar
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Schuster, Raphael
    Univ Hamburg, Inst Organ Chem, Hamburg, Germany..
    Seuring, Carolin
    Ctr Struct & Syst Biol, Hamburg, Germany..
    Riedner, Maria
    Univ Hamburg, Inst Organ Chem, Hamburg, Germany..
    Sanyal, Suparna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
    Torda, Andrew E.
    Univ Hamburg, Ctr Bioinformat, Hamburg, Germany..
    Wilson, Daniel N.
    Univ Hamburg, Inst Biochem & Mol Biol, Hamburg, Germany..
    Ignatova, Zoya
    Univ Hamburg, Inst Biochem & Mol Biol, Hamburg, Germany..
    Repurposing tRNAs for nonsense suppression2021In: Nature Communications, E-ISSN 2041-1723, Vol. 12, article id 3850Article in journal (Refereed)
    Abstract [en]

    Three stop codons (UAA, UAG and UGA) terminate protein synthesis and are almost exclusively recognized by release factors. Here, we design de novo transfer RNAs (tRNAs) that efficiently decode UGA stop codons in Escherichia coli. The tRNA designs harness various functionally conserved aspects of sense-codon decoding tRNAs. Optimization within the T Psi C-stem to stabilize binding to the elongation factor, displays the most potent effect in enhancing suppression activity. We determine the structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon in the A site at 2.9 angstrom resolution. In the context of the suppressor tRNA, the conformation of the UGA codon resembles that of a sense-codon rather than when canonical translation termination release factors are bound, suggesting conformational flexibility of the stop codons dependent on the nature of the A-site ligand. The systematic analysis, combined with structural insights, provides a rationale for targeted repurposing of tRNAs to correct devastating nonsense mutations that introduce a premature stop codon. Here, the authors report de novo design, optimization and characterization of tRNAs that decode UGA stop codons in E. coli. The structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon suggests that distinct A-site ligands (tRNAs versus release factors) induce distinct conformation of the stop codon within the mRNA in the decoding center.

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  • 45.
    Alderborn, Anders
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Sundström, Jens
    Soeria-Atmadja, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences.
    Sandberg, Martin
    Andersson, H. Christer
    Hammerling, Ulf
    Genetically modified plants for non-food or non-feed purposes: straightforward screening for their appearance in food and feed2010In: Food and Chemical Toxicology, ISSN 0278-6915, E-ISSN 1873-6351, Vol. 48, no 2, p. 453-464Article in journal (Refereed)
    Abstract [en]

    Genetically modified (GM) plants aimed at producing food/feed are part of regular agriculture in many areas of the World. Commodity plants have also found application as bioreactors, designated non-food/non-feed GM (NFGM) plants, thereby making raw material for further refinement to industrial, diagnostic or pharmaceutical preparations. Many among them may pose health challenge to consumers or livestock animals, if occurring in food/feed. NFGM plants are typically released into the environment, but are grown under special oversight and any among several containment practices, none of which provide full protection against accidental dispersal. Adventitious admixture with food or feed can occur either through distributional mismanagement or as a consequence of gene flow to plant relatives. To facilitate NFGM surveillance we propose a new mandatory tagging of essentially all such plants, prior to cultivation or marketing in the European Union. The suggested tag--Plant-Made Industrial or Pharmaceutical Products Tag (PMIP-T)--is envisaged to occur as a transgenic silent DNA identifier in host plants and designed to enable technically simple identification and characterisation of any NFGM. Implementation of PMIP-T would permit inexpensive, reliable and high-throughput screening for NFGM specifically. The paper outlines key NFGM prospects and challenges as well as the PMIP-T concept.

  • 46.
    Alehagen, Urban
    et al.
    Division of Cardiovascular Medicine, Department of Medical and Health Sciences, Linköping University, SE-581 85 Linköping, Sweden..
    Opstad, Trine B
    Center for Clinical Heart Research, Department of Cardiology, Oslo University Hospital Ullevål, P.O. Box 4950 Nydalen, N-0424 Oslo, Norway..
    Alexander, Jan
    Norwegian Institute of Public Health, P.O. Box 222 Skøyen, N-0213 Oslo, Norway..
    Larsson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Chemistry.
    Aaseth, Jan
    Department of Research, Innlandet Hospital Trust, P.O. Box 104, N-2381 Brumunddal, Norway..
    Impact of Selenium on Biomarkers and Clinical Aspects Related to Ageing: A Review2021In: Biomolecules, E-ISSN 2218-273X, Vol. 11, no 10, article id 1478Article in journal (Refereed)
    Abstract [en]

    Selenium (Se) is an essential dietary trace element that plays an important role in the prevention of inflammation, cardiovascular diseases, infections, and cancer. Selenoproteins contain selenocysteine in the active center and include, i.a., the enzymes thioredoxin reductases (TXNRD1-3), glutathione peroxidases (GPX1-4 and GPX6) and methionine sulfoxide reductase, involved in immune functions, metabolic homeostasis, and antioxidant defense. Ageing is an inevitable process, which, i.a., involves an imbalance between antioxidative defense and reactive oxygen species (ROS), changes in protein and mitochondrial renewal, telomere attrition, cellular senescence, epigenetic alterations, and stem cell exhaustion. These conditions are associated with mild to moderate inflammation, which always accompanies the process of ageing and age-related diseases. In older individuals, Se, by being a component in protective enzymes, operates by decreasing ROS-mediated inflammation, removing misfolded proteins, decreasing DNA damage, and promoting telomere length. Se-dependent GPX1-4 and TXNRD1-3 directly suppress oxidative stress. Selenoprotein H in the cell nucleus protects DNA, and selenoproteins residing in the endoplasmic reticulum (ER) assist in the removal of misfolded proteins and protection against ER stress. In this review, we highlight the role of adequate Se status for human ageing and prevention of age-related diseases, and further its proposed role in preservation of telomere length in middle-aged and elderly individuals.

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  • 47.
    Aleix-Mata, Gael
    et al.
    Univ Jaen, Dept Biol Expt, Area Genet, Campus Las Lagunillas S-N, Jaen 23071, Spain.;Univ Jaen, Dept Biol Anim Biol Vegetal & Ecol, Campus Las Lagunillas S-N, Jaen 23071, Spain..
    Gutierrez, Juana
    Univ Jaen, Dept Biol Expt, Area Genet, Campus Las Lagunillas S-N, Jaen 23071, Spain..
    Ruiz-Ruano, Francisco J.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Systematic Biology.
    Lorite, Pedro
    Univ Jaen, Dept Biol Expt, Area Genet, Campus Las Lagunillas S-N, Jaen 23071, Spain..
    Marchal, Juan A.
    Univ Jaen, Dept Biol Expt, Area Genet, Campus Las Lagunillas S-N, Jaen 23071, Spain..
    Sanchez, Antonio
    Univ Jaen, Dept Biol Expt, Area Genet, Campus Las Lagunillas S-N, Jaen 23071, Spain..
    The complete mitochondrial genome of Talpa aquitania (Talpidae; Insectivora), a mole species endemic to northern Spain and southern France2020In: Molecular Biology Reports, ISSN 0301-4851, E-ISSN 1573-4978, Vol. 47, no 3, p. 2397-2403Article in journal (Refereed)
    Abstract [en]

    The complete mitogenome sequence of Talpa aquitania, a recently described Talpa species, was assembled using whole-genome sequencing data. It varies in length from 16,776 to 16,846 bp, contains 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, one origin of L-strand replication, and a control region. In the control region, which varied from 1320 to 1390 bp, we identified the extended termination-associated sequence (ETAS-1 and ETAS-2) and the conserved sequence blocks (CSB-1, 2, 3, B, C, D, E, F). In addition, this region includes a 10 bp tandem repeat DNA sequence, with a variable number of repeats that suggest the existence of heteroplasmy. Phylogeny reconstructions based on Maximum Likelihood, Neighbor-joining and Bayesian inference analyses yielded phylogenies with similar topologies demonstrating that T. aquitania and T. occidentalis are sister species.

  • 48.
    Alejandro Zamora-Briseno, Jesus
    et al.
    Inst Politecn Nacl, Unidad Merida, Ctr Invest & Estudios Avanzados, Lab Inmunol & Biol Mol,Dept Recursos Mar, Merida, Yucatan, Mexico..
    Ruiz-May, Eliel
    Cluster Cientif & Tecnol BioMimic, Inst Ecol, Red Estudios Mol Avanzados, Xalapa, Veracruz, Mexico..
    Miguel Elizalde-Contreras, Jose
    Cluster Cientif & Tecnol BioMimic, Inst Ecol, Red Estudios Mol Avanzados, Xalapa, Veracruz, Mexico..
    Margarita Hernandez-Velazquez, Ioreni
    Inst Politecn Nacl, Unidad Merida, Ctr Invest & Estudios Avanzados, Lab Inmunol & Biol Mol,Dept Recursos Mar, Merida, Yucatan, Mexico..
    Hernandez-Perez, Ariadne
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
    Guadalupe Fuentes-Garcia, Ana
    Inst Politecn Nacl, Unidad Merida, Ctr Invest & Estudios Avanzados, Lab Inmunol & Biol Mol,Dept Recursos Mar, Merida, Yucatan, Mexico..
    Herrera-Salvatierra, Nancy
    Inst Politecn Nacl, Unidad Merida, Ctr Invest & Estudios Avanzados, Lab Inmunol & Biol Mol,Dept Recursos Mar, Merida, Yucatan, Mexico..
    Briones-Fourzan, Patricia
    Univ Nacl Autonoma Mexico, Isnt Ciencias Mar & Limnol, Unidad Acad Sistemas Arrecifales, Puerto Morelos, Mexico..
    Pascual-Jimenez, Cristina
    Univ Nacl Autonoma Mexico, Unidad Multidisciplinaria Docencia & Invest, Fac Ciencias, Sisal, Mexico..
    Lozano-Alvarez, Enrique
    Univ Nacl Autonoma Mexico, Isnt Ciencias Mar & Limnol, Unidad Acad Sistemas Arrecifales, Puerto Morelos, Mexico..
    Rodriguez-Canul, Rossanna
    Inst Politecn Nacl, Unidad Merida, Ctr Invest & Estudios Avanzados, Lab Inmunol & Biol Mol,Dept Recursos Mar, Merida, Yucatan, Mexico..
    iTRAQ-Based Proteomic Profile Analysis of the Hepatopancreas of Caribbean Spiny Lobsters Infected WithPanulirus argusVirus 1: Metabolic and Physiological Implications2020In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 11, article id 1084Article in journal (Refereed)
    Abstract [en]

    The Caribbean spiny lobsterPanulirus argus(Latreille, 1084) sustains economically valuable fisheries throughout the wider Caribbean region. This species is currently affected by the pathogenic virusPanulirus argusVirus 1 (PaV1) that causes a systemic and chronic-degenerative infection in juvenile spiny lobstersP. argus. To date, there is no available information regarding the host alterations induced by this pathogen at the molecular level. In the present study, comparative proteomic analyses of the changes in the hepatopancreas between infected and non-infected juvenile lobsters were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ) coupled to synchronous precursor selection (SPS)-based MS3. We identified a total of 636 proteins, being 68 down-regulated and 71 up-regulated proteins. Among the down-regulated proteins, we identified several enzymes involved in the metabolism of hormones and lipids, digestive proteases and glycosidases, while proteins associated with the histone core, protein synthesis, immune response and RNA regulation were up-regulated. Several misregulated enzymes involved in the regulation of neuromodulators were also identified. RT-qPCR assays were used to validate the expression of transcripts encoding for selected differential proteins that were in concordance to proteomic data, as well as the tendency observed in the enzymatic activities of trypsin, chymotrypsin, and glycosidase. In a similar way, we observed glycogen reduction in muscle, and an increase in plasma acylglycerides and glucose, which may be explained by proteomic data. This study provides the first insight into the molecular changes in the hepatopancreas of Caribbean spiny lobsters associated to PaV1 infection. Data provided herein would help to clarify the origin of the molecular misregulations observed at macroscopic level in this host-pathogen interaction.

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  • 49.
    Alexis, Laura
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Alftrén, Ida
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Elvmarker, Josefin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Mach, Klara-Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    Nyman, Rebecca
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
    DETEKTERA MERA!: - En studie av LigandTracer® och andra vanliga detektionstekniker för interaktion inom antikroppsutveckling2013Independent thesis Basic level (degree of Bachelor), 10 credits / 15 HE creditsStudent thesis
    Abstract [sv]

    Ridgeview Instruments AB är ett företag, med säte i Uppsala, som bland annat utvecklar mjukvara för detektionsanalys och detektionsinstrumenten LigandTracer®. Instrumenten detekterar cellinteraktioner i realtid och tillhandahåller information om hur interaktion mellan undersökt ligand och receptor sker. LigandTracer® används bland annat vid cancerstudier och affinitetsstudier av antikroppsinbindning till antigener på celler.

     

    LigandTracer® och teknologin bakom den har studerats för att hitta de främsta styrkorna samt utvecklingsområden i jämförelse med andra detektionstekniker. Dessa tekniker är surface plasmon resonance, BioLayer Interferometry, quartz crystal microbalance samt surface acoustic wave. Främsta styrkan hos detektion med LigandTracer® är att stor mängd data som beskriver interaktionerna erhålls. Information av denna typ är viktig för att få en helhetsbild av cellers ytprotein. Därför kan instrumenten exempelvis användas som kvalitetssäkrande steg i antikroppsprocessen. LigandTracer® kan även användas för studier av hela celler och vävnader. Utöver detta finns styrkor som minimerar det laborativa arbetet, så som tvättar i prepareringssteg. Detta gör att både tid och reagens sparas.

     

    En utvecklingsmöjlighet för LigandTracer® är egenskapen att kunna detektera fler antikroppar och cellinjer samtidigt. Då mjukvaran TraceDrawer™ är ett viktigt tillbehör för utvärderingen av detektionen, skulle utveckling med avseende på celldynamiken och receptorernas rörelsemönster kunna förbättra instrumenten ytterligare.

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    Detektera mera!
  • 50.
    Alfredsson Timmins, Jenny
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Functional organisation of the cell nucleus in the fission yeast, Schizosaccharomyces pombe2009Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In eukaryotes the genome adopts a non-random spatial organisation, which is important for gene regulation. However, very little is known about the driving forces behind nuclear organisation. In the simple model eukaryote fission yeast, Schizosaccharomyces pombe, it has been known for a long time that transcriptionally repressed heterochromatin localise to the nuclear membrane (NM); the centromeres attaches to spindle pole body (SPB), while the telomeres are positioned at the NM on the opposite side of the nucleus compared to the SPB. Studies presented in this thesis aimed at advancing our knowledge of nuclear organisation in Schizosaccharomyces pombe.

    We show that the heterochromatic mating-type region localises to the NM in the vicinity of the SPB. This positioning was completely dependent on Clr4, a histone methyl transferase crucial for the formation of heterochromatin. Additional factors important for localisation were also identified: the chromo domain protein Swi6, and the two boundary elements IR-L and IR-R surrounding this locus. We further identify two other chromo domain proteins; Chp1 and Chp2, as crucial factors for correct subnuclear localisation of this region. From these results we suggest that the boundary elements together with chromodomain proteins in balanced dosage and composition cooperate in organising the mating-type chromatin.

    Gene regulation can affect the subnuclear localisation of genes. Using nitrogen starvation in S. pombe as a model for gene induction we determined the subnuclear localisation of two gene clusters repressed by nitrogen: Chr1 and Tel1. When repressed these loci localise to the NM, and this positioning is dependent on the histone deacetylase Clr3. During induction the gene clusters moved towards the nuclear interior in a transcription dependent manner.

    The knowledge gained from work presented in this thesis, regarding nuclear organisation in the S. pombe model system, can hopefully aid to a better understanding of human nuclear organisation.

    List of papers
    1. The Clr4 methyltransferase determines the subnuclear localization of the mating-type region in fission yeast
    Open this publication in new window or tab >>The Clr4 methyltransferase determines the subnuclear localization of the mating-type region in fission yeast
    2007 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 120, no 11, p. 1935-1943Article in journal (Refereed) Published
    Abstract [en]

    The genome has a non-random spatial distribution in the cell nucleus. In Schizosaccharomyces pombe, it has been shown that the centromeres, telomeres and the mating-type region localize to the nuclear membrane (NM), the former by attaching to the spindle pole body (SPB). In addition, reporter genes inserted into these areas are transcriptionally repressed due to the formation of specialized chromatin structures. Performing live cell analysis we found that in a wild-type strain the mating-type region was positioned in the proximity of the SPB, the location where the pericentromeric heterochromatin is also found. In a strain lacking the histone methyltransferase, Clr4, crucial for the formation of heterochromatin, the mating-type region had a random localization in the nucleus. Moreover, in a strain where the two boundary elements IR-L and IR-R had been deleted the mating-type region was displaced from its position at the proximity of the SPB, but remained in the vicinity of the NM. Moreover, in all investigated strains with silencing deficiencies the distance between the mating-type region and the SPB increased. This result indicates a correlation between transcriptional derepression and displacement of the region. Two different models of how the mating-type chromatin is organized in the nucleus are discussed.

    Keywords
    fission yeast, heterochromatin, silencing, subnuclear localization
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-13023 (URN)10.1242/jcs.03457 (DOI)000246665300013 ()17504808 (PubMedID)
    Available from: 2009-08-05 Created: 2009-08-05 Last updated: 2022-01-28Bibliographically approved
    2. Chromo domain proteins in balanced dosage together with boundary elements cooperate in organising the mating-type chromatin in fission yeast
    Open this publication in new window or tab >>Chromo domain proteins in balanced dosage together with boundary elements cooperate in organising the mating-type chromatin in fission yeast
    (English)Manuscript (preprint) (Other (popular science, discussion, etc.))
    Abstract [en]

    The chromatin in the cell nucleus has a spatial organisation. For example, in the fission yeast, Schizosaccharomyces pombe, transcriptionally repressed heterochromatin is found at the nuclear membrane (NM). The centromeres and the mating-type region localise in the proximity of the spindle pole body (SPB), while the telomeres are found on the opposite side of the nucleus in the proximity of the nucleolus. In a previous study we used the mating-type region as a model to study the driving force behind nuclear organisation. We proposed two mutually exclusive models to explain what determines the localisation of the mating-type region. The first model suggests that solely the amount of heterochromatin in the region affects the localisation, while the other model stipulates that the boundary elements together with heterochromatin formation anchor the mating-type region in the NM in the vicinity of the SPB. Here, we present data that disproves the first model. We found that in a strain expressing tripled amounts of the chromodomain protein Swi6, a structural component of heterochromatin, the mating-type region was delocalised from the proximity of the SPB. A strain deleted of the histone deacetylase clr3+ also had a delocalised mating-type locus. Interestingly, a strain with a point-mutation in clr3-735 producing an enzymatically inactive protein in normal amounts showed an intermediate phenotype. Most importantly, we identify the chromodomain proteins, Chp1 and Chp2, as crucial factors for correct subnuclear localisation of the mating-type region. We suggest that boundary elements together with chromodomain proteins in balanced dosage and composition cooperate in organising the mating-type chromatin.

    Identifiers
    urn:nbn:se:uu:diva-107280 (URN)
    Available from: 2009-08-04 Created: 2009-08-04 Last updated: 2010-01-14Bibliographically approved
    3. Reorganization of chromatin is an early response to nitrogen starvation in Schizosaccharomyces pombe
    Open this publication in new window or tab >>Reorganization of chromatin is an early response to nitrogen starvation in Schizosaccharomyces pombe
    Show others...
    2009 (English)In: Chromosoma, ISSN 0009-5915, E-ISSN 1432-0886, Vol. 118, no 1, p. 99-112Article in journal (Refereed) Published
    Abstract [en]

    There are several documented events of changes in subnuclear localization during gene activation. However, there are conflicting data on whether the nuclear periphery is a compartment for gene repression or activation, and whether genes are moved to the pores at the nuclear membrane (NM) or not during gene activation. Nitrogen starvation of fission yeast serves as a good model system for studying gene induction since it causes fast regulation of hundreds of genes. In this study the subnuclear localization of two gene clusters repressed by nitrogen was investigated. During normal growth conditions the gene clusters localized to the nuclear periphery at the opposite side of the nucleus as compared to the spindle pole body (SPB). This constrained localization was dependent on the histone deacetylase Clr3, known to transcriptionally repress genes in these clusters. Already 20 minutes after nitrogen depletion drastic changes in subnuclear localization of the two loci were observed, away from the NM towards the nuclear interior. At least for one of the clusters the movement was clearly transcription dependent. Data presented here illustrates how interconnected events of gene activation and nuclear reorganization are, as well as provides a suggestion of how nuclear organization might be maintained.

    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:uu:diva-107289 (URN)10.1007/s00412-008-0180-6 (DOI)000262504300009 ()
    Available from: 2009-08-05 Created: 2009-08-05 Last updated: 2022-01-28Bibliographically approved
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