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  • 1.
    Abuabaid, Hanan
    et al.
    Örebro University, School of Science and Technology.
    Karlsson, Mattias
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Probiotic Lactobacillus rhamnosus alters inflammatory responses of bladder epithelial and macrophage-like cells in co-cultureManuscript (preprint) (Other academic)
  • 2.
    Alijagic, A.
    et al.
    Örebro University, School of Science and Technology. Inflammatory Response and Infection Susceptibility Center (iRiSC).
    Scherbak, N.
    Örebro University, School of Science and Technology.
    Kotlyar, O.
    Örebro University, School of Science and Technology.
    Karlsson, P.
    Örebro University, School of Science and Technology. Department of Mechanical Engineering.
    Persson, A.
    Örebro University, School of Medical Sciences. Inflammatory Response and Infection Susceptibility Center (iRiSC).
    Hedbrant, A.
    Örebro University, School of Medical Sciences. Inflammatory Response and Infection Susceptibility Center (iRiSC).
    Norinder, U.
    Örebro University, School of Science and Technology.
    Larsson, M.
    Örebro University, School of Science and Technology.
    Felth, J.
    Uddeholms AB, Hagfors, Sweden.
    Andersson, L.
    Örebro University, School of Medical Sciences. Örebro University Hospital. Inflammatory Response and Infection Susceptibility Center (iRiSC); , Department of Occupational and Environmental Medicine.
    Särndahl, E.
    Örebro University, School of Medical Sciences. Inflammatory Response and Infection Susceptibility Center (iRiSC).
    Engwall, M.
    Örebro University, School of Science and Technology.
    Cell Painting unveils cell response signatures to (nano)particles formed in additive manufacturing2022In: Toxicology Letters, ISSN 0378-4274, E-ISSN 1879-3169, P17-01, Vol. 368, no Suppl. 1, p. S226-S227, article id P17-01Article in journal (Other academic)
  • 3.
    Alijagic, Andi
    et al.
    Örebro University, School of Science and Technology. School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Engwall, Magnus
    Örebro University, School of Science and Technology.
    Särndahl, Eva
    Örebro University, School of Medical Sciences.
    Karlsson, Helen
    Department of Health, Medicine and Caring Sciences, Occupational and Environmental Medicine Center in Linköping, Linköping University, Linköping, Sweden.
    Hedbrant, Alexander
    Örebro University, School of Medical Sciences.
    Andersson, Lena
    Örebro University, School of Medical Sciences. Örebro University Hospital. Department of Occupational and Environmental Medicine.
    Karlsson, Patrik
    Örebro University, School of Science and Technology.
    Dalemo, Magnus
    Absolent AB, Lidköping, Sweden.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Färnlund, Kim
    AMEXCI AB, Karlskoga, Sweden.
    Larsson, Maria
    Örebro University, School of Science and Technology.
    Persson, Alexander
    Örebro University, School of Medical Sciences.
    Particle Safety Assessment in Additive Manufacturing: From Exposure Risks to Advanced Toxicology Testing2022In: Frontiers in Toxicology, E-ISSN 2673-3080, Vol. 4, article id 836447Article, review/survey (Refereed)
    Abstract [en]

    Additive manufacturing (AM) or industrial three-dimensional (3D) printing drives a new spectrum of design and production possibilities; pushing the boundaries both in the application by production of sophisticated products as well as the development of next-generation materials. AM technologies apply a diversity of feedstocks, including plastic, metallic, and ceramic particle powders with distinct size, shape, and surface chemistry. In addition, powders are often reused, which may change the particles' physicochemical properties and by that alter their toxic potential. The AM production technology commonly relies on a laser or electron beam to selectively melt or sinter particle powders. Large energy input on feedstock powders generates several byproducts, including varying amounts of virgin microparticles, nanoparticles, spatter, and volatile chemicals that are emitted in the working environment; throughout the production and processing phases. The micro and nanoscale size may enable particles to interact with and to cross biological barriers, which could, in turn, give rise to unexpected adverse outcomes, including inflammation, oxidative stress, activation of signaling pathways, genotoxicity, and carcinogenicity. Another important aspect of AM-associated risks is emission/leakage of mono- and oligomers due to polymer breakdown and high temperature transformation of chemicals from polymeric particles, both during production, use, and in vivo, including in target cells. These chemicals are potential inducers of direct toxicity, genotoxicity, and endocrine disruption. Nevertheless, understanding whether AM particle powders and their byproducts may exert adverse effects in humans is largely lacking and urges comprehensive safety assessment across the entire AM lifecycle-spanning from virgin and reused to airborne particles. Therefore, this review will detail: 1) brief overview of the AM feedstock powders, impact of reuse on particle physicochemical properties, main exposure pathways and protective measures in AM industry, 2) role of particle biological identity and key toxicological endpoints in the particle safety assessment, and 3) next-generation toxicology approaches in nanosafety for safety assessment in AM. Altogether, the proposed testing approach will enable a deeper understanding of existing and emerging particle and chemical safety challenges and provide a strategy for the development of cutting-edge methodologies for hazard identification and risk assessment in the AM industry.

  • 4.
    Alijagic, Andi
    et al.
    Örebro University, School of Science and Technology. Inflammatory Response and Infection Susceptibility Centre (iRiSC), Faculty of Medicine and Health, Örebro University, Örebro, Sweden; School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Kotlyar, Oleksandr
    Örebro University, School of Science and Technology.
    Larsson, Maria
    Örebro University, School of Science and Technology.
    Salihovic, Samira
    Örebro University, School of Medical Sciences. Inflammatory Response and Infection Susceptibility Centre (iRiSC), Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Hedbrant, Alexander
    Örebro University, School of Medical Sciences. Inflammatory Response and Infection Susceptibility Centre (iRiSC), Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Eriksson, Ulrika
    Örebro University, School of Science and Technology.
    Karlsson, Patrik
    Örebro University, School of Science and Technology.
    Persson, Alexander
    Örebro University, School of Medical Sciences. Inflammatory Response and Infection Susceptibility Centre (iRiSC), Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Färnlund, Kim
    AMEXCI AB, Karlskoga, Sweden.
    Engwall, Magnus
    Örebro University, School of Science and Technology.
    Särndahl, Eva
    Örebro University, School of Medical Sciences. Inflammatory Response and Infection Susceptibility Centre (iRiSC), Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Immunotoxic, genotoxic, and endocrine disrupting impacts of polyamide microplastic particles and chemicals2024In: Environment International, ISSN 0160-4120, E-ISSN 1873-6750, Vol. 183, article id 108412Article in journal (Refereed)
    Abstract [en]

    Due to their exceptional properties and cost effectiveness, polyamides or nylons have emerged as widely used materials, revolutionizing diverse industries, including industrial 3D printing or additive manufacturing (AM). Powder-based AM technologies employ tonnes of polyamide microplastics to produce complex components every year. However, the lack of comprehensive toxicity assessment of particulate polyamides and polyamide-associated chemicals, especially in the light of the global microplastics crisis, calls for urgent action. This study investigated the physicochemical properties of polyamide-12 microplastics used in AM, and assessed a number of toxicity endpoints focusing on inflammation, immunometabolism, genotoxicity, aryl hydrocarbon receptor (AhR) activation, endocrine disruption, and cell morphology. Specifically, microplastics examination by means of field emission scanning electron microscopy revealed that work flow reuse of material created a fraction of smaller particles with an average size of 1-5 µm, a size range readily available for uptake by human cells. Moreover, chemical analysis by means of gas chromatography high-resolution mass spectrometry detected several polyamide-associated chemicals including starting material, plasticizer, thermal stabilizer/antioxidant, and migrating slip additive. Even if polyamide particles and chemicals did not induce an acute inflammatory response, repeated and prolonged exposure of human primary macrophages disclosed a steady increase in the levels of proinflammatory chemokine Interleukin-8 (IL-8/CXCL-8). Moreover, targeted metabolomics disclosed that polyamide particles modulated the kynurenine pathway and some of its key metabolites. The p53-responsive luciferase reporter gene assay showed that particles per se were able to activate p53, being indicative of a genotoxic stress. Polyamide-associated chemicals triggered moderate activation of AhR and elicited anti-androgenic activity. Finally, a high-throughput and non-targeted morphological profiling by Cell Painting assay outlined major sites of bioactivity of polyamide-associated chemicals and indicated putative mechanisms of toxicity in the cells. These findings reveal that the increasing use of polyamide microplastics may pose a potential health risk for the exposed individuals, and it merits more attention.

  • 5.
    Alijagic, Andi
    et al.
    Örebro University, School of Science and Technology. Inflammatory Response and Infection Susceptibility Centre (iRiSC), Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Kotlyar, Oleksandr
    Örebro University, School of Science and Technology.
    Karlsson, Patrik
    Örebro University, School of Science and Technology.
    Wang, Xuying
    KTH Royal Institute of Technology, Department of Chemistry, Division of Surface and Corrosion Science, SE-100 44 Stockholm, Sweden.
    Odnevall, Inger
    KTH Royal Institute of Technology, Department of Chemistry, Division of Surface and Corrosion Science, SE-100 44 Stockholm, Sweden; AIMES-Center for the Advancement of Integrated Medical and Engineering Sciences at Karolinska Institutet and KTH Royal Institute of Technology, SE-100 44 Stockholm, Sweden; Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
    Benada, Oldřich
    Institute of Microbiology of the Czech Academy of Sciences, 140 00 Prague, Czech Republic.
    Amiryousefi, Ali
    Örebro University, School of Medical Sciences.
    Andersson, Lena
    Örebro University, School of Medical Sciences. Örebro University Hospital. Inflammatory Response and Infection Susceptibility Centre (iRiSC), Faculty of Medicine and Health, Örebro University, SE-701 82 Örebro, Sweden; Department of Occupational and Environmental Medicine, Örebro University Hospital, Örebro, Sweden.
    Persson, Alexander
    Inflammatory Response and Infection Susceptibility Centre (iRiSC), Faculty of Medicine and Health, Örebro University, SE-701 82 Örebro, Sweden .
    Felth, Jenny
    Uddeholms AB, SE-683 85 Hagfors, Sweden.
    Andersson, Henrik
    Uddeholms AB, SE-683 85 Hagfors, Sweden.
    Larsson, Maria
    Örebro University, School of Science and Technology.
    Hedbrant, Alexander
    Örebro University, School of Medical Sciences. Inflammatory Response and Infection Susceptibility Centre (iRiSC), Faculty of Medicine and Health, Örebro University, SE-701 82 Örebro, Sweden.
    Salihovic, Samira
    Örebro University, School of Medical Sciences. Man-Technology-Environment Research Center (MTM), Örebro University, SE-701 82 Örebro, Sweden; Inflammatory Response and Infection Susceptibility Centre (iRiSC), Faculty of Medicine and Health, Örebro University, SE-701 82 Örebro, Sweden.
    Hyötyläinen, Tuulia
    Örebro University, School of Science and Technology.
    Repsilber, Dirk
    Örebro University, School of Medical Sciences.
    Särndahl, Eva
    Örebro University, School of Medical Sciences. Inflammatory Response and Infection Susceptibility Centre (iRiSC), Faculty of Medicine and Health, Örebro University, SE-701 82 Örebro, Sweden.
    Engwall, Magnus
    Örebro University, School of Science and Technology.
    A Novel Nanosafety Approach Using Cell Painting, Metabolomics, and Lipidomics Captures the Cellular and Molecular Phenotypes Induced by the Unintentionally Formed Metal-Based (Nano)Particles2023In: Cells, E-ISSN 2073-4409, Vol. 12, no 2, article id 281Article in journal (Refereed)
    Abstract [en]

    Additive manufacturing (AM) or industrial 3D printing uses cutting-edge technologies and materials to produce a variety of complex products. However, the effects of the unintentionally emitted AM (nano)particles (AMPs) on human cells following inhalation, require further investigations. The physicochemical characterization of the AMPs, extracted from the filter of a Laser Powder Bed Fusion (L-PBF) 3D printer of iron-based materials, disclosed their complexity, in terms of size, shape, and chemistry. Cell Painting, a high-content screening (HCS) assay, was used to detect the subtle morphological changes elicited by the AMPs at the single cell resolution. The profiling of the cell morphological phenotypes, disclosed prominent concentration-dependent effects on the cytoskeleton, mitochondria, and the membranous structures of the cell. Furthermore, lipidomics confirmed that the AMPs induced the extensive membrane remodeling in the lung epithelial and macrophage co-culture cell model. To further elucidate the biological mechanisms of action, the targeted metabolomics unveiled several inflammation-related metabolites regulating the cell response to the AMP exposure. Overall, the AMP exposure led to the internalization, oxidative stress, cytoskeleton disruption, mitochondrial activation, membrane remodeling, and metabolic reprogramming of the lung epithelial cells and macrophages. We propose the approach of integrating Cell Painting with metabolomics and lipidomics, as an advanced nanosafety methodology, increasing the ability to capture the cellular and molecular phenotypes and the relevant biological mechanisms to the (nano)particle exposure.

  • 6.
    Berg, Håkan
    et al.
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Liimatta, Harri
    Örebro University, School of Science and Technology.
    Hoffmann, Erik
    Karlsson, Johnny
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Characterization of antibodies for quantitative determination of spiggin protein levels in male and female three-spined stickleback (Gasterosteus aculeatus)2009In: Reproductive Biology and Endocrinology, E-ISSN 1477-7827, Vol. 7, no 1, article id 46Article in journal (Refereed)
    Abstract [en]

    Spiggin is an adhesive glycoprotein produced in the kidney of sticklebacks during the breeding season and is subsequently secreted into the urinary bladder from where it is employed for nest building. Since the production of the protein has been shown to be under androgenic control, spiggin has been suggested to be a useful biomarker for androgenic substances in the environment. In this study, two polyclonal spiggin antibodies based on synthetic peptides and one polyclonal antibody directed against native spiggin have been characterized. The antibodies ability to identify spiggin was investigated by quantitative immunoassay. For both peptide antibodies the quantification range was determined to be between 1 and 80 ng spiggin and determination of renal spiggin levels from immature and mature males displayed a 15-fold increase in total spiggin content of the kidney resulting in a 6-fold increase in male kidney weight due to hypertrophy. The kidney somatic index (KSI) was found to correlate well with the total renal spiggin content and therefore it appears that KSI in sticklebacks could be used as an initial method to identify substances displaying androgenic effects. Furthermore, western blot analysis revealed that the polyclonal antibodies recognize different spiggin isoforms and that spiggin can be detected in the urinary bladder and kidney of both males and female sticklebacks. In order to develop a quantitative detection method for native spiggin it is necessary to produce a standard that can be used in a bioassay. Due to the adhesive and polymerization characteristics of spiggin the protein is difficult to use as a standard in bioassays. So far spiggin has been shown to exist in at least 14 isoforms, all of which contain polymerization domains. To overcome the solubility problem we have produced recombinant spiggin gamma, with only one polymerization domain, that can be expressed in E. coli. Western blot analysis demonstrated that the polyclonal antibodies were able to detect recombinant spiggin gamma protein in bacterial cell lysate, suggesting that it may be developed into a useful source of standard spiggin to be used for quantitative determination of androgen induced spiggin production in sticklebacks.

  • 7.
    Blanc, Mélanie
    et al.
    Örebro University, School of Science and Technology. MARBEC, University of Montpellier, CNRS, Ifremer, IRD, Palavas, France; Université Paris-Saclay, AgroParisTech, INRAE, GABI, Domaine de Vilvert, Jouy-en-Josas, France.
    Antczak, Philipp
    Centre for Molecular Medicine Cologne, University of Cologne, Cologne, Germany.
    Cousin, Xavier
    MARBEC, University of Montpellier, CNRS, Ifremer, IRD, Palavas, France; Université Paris-Saclay, AgroParisTech, INRAE, GABI, Domaine de Vilvert, Jouy-en-Josas, France.
    Grunau, Christoph
    IHPE, Univ. Montpellier, CNRS, Ifremer, Univ. Perpignan Via Domitia, Perpignan, France.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Rüegg, Joëlle
    Department of Organismal Biology, Uppsala University, Uppsala, Sweden.
    Keiter, Steffen
    Örebro University, School of Science and Technology.
    The insecticide permethrin induces transgenerational behavioral changes linked to transcriptomic and epigenetic alterations in zebrafish (Danio rerio)2021In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 779, article id 146404Article in journal (Refereed)
    Abstract [en]

    The pyrethroid insecticide permethrin is widely used for agricultural and domestic purposes. Previous data indicated that it acts as a developmental neurotoxicant and can induce transgenerational effects in non-target organisms. However, associated underlying mechanisms remain unclear. The aim of this study was to investigate permethrin-related transgenerational effects in the zebrafish model, and to identify possible molecular mechanisms underlying inheritance. Zebrafish (F0) were exposed to permethrin during early-life (2 h post-fertilization up to 28 days). The F1 and F2 offspring generations were obtained by pairing exposed F0 males and females, and were bred unexposed. Locomotor and anxiety behavior were investigated, together with transcriptomic and epigenomic (DNA methylation) changes in brains. Permethrin exposed F0 fish were hypoactive at adulthood, while males from the F1 and F2 generations showed a specific decrease in anxiety-like behavior. In F0, transcriptomic data showed enrichment in pathways related to glutamatergic synapse activity, which may partly underlie the behavioral effects. In F1 and F2 males, dysregulation of similar pathways was observed, including a subset of differentially methylated regions that were inherited from the F0 to the F2 generation and indicated stable dysregulation of glutamatergic signaling. Altogether, the present results provide novel evidence on the transgenerational neurotoxic effects of permethrin, as well as mechanistic insight: a transient exposure induces persistent transcriptional and DNA methylation changes that may translate into transgenerational alteration of glutamatergic signaling and, thus, into behavioral alterations.

  • 8.
    Blanc, Mélanie
    et al.
    Örebro University, School of Science and Technology.
    Antczak, Philipp
    Centre for Molecular Medicine Cologne, University of Cologne, Cologne, German.
    Cousin, Xavier
    MARBEC, University of Montpellier, CNRS, Ifremer, IRD, Route de Maguelone, Palavas, France; Université Paris-Saclay, AgroParisTech, INRAE, GABI, Domaine de Vilvert, Jouy-en-Josas, France.
    Grunau, Christoph
    IHPE, Univ. Montpellier, CNRS, Ifremer, Univ. Perpignan Via Domitia, Perpignan France.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology. Örebro Life Science Centre, School of Science and Technology, Örebro University, Örebro, Sweden.
    Rüegg, Joëlle
    Department of Organismal Biology, Uppsala University, Uppsala, Sweden.
    Keiter, Steffen
    Örebro University, School of Science and Technology.
    Transgenerational behavioral, transcriptomic, and epigenetic changes in brain after early-life exposure of F0 zebrafish to permethrinManuscript (preprint) (Other academic)
  • 9.
    Blanc, Mélanie
    et al.
    Örebro University, School of Science and Technology.
    Cormier, Bettie
    Örebro University, School of Science and Technology. University of Bordeaux, EPOC UMR CNRS 5805, Pessac, France.
    Hyötyläinen, Tuulia
    Örebro University, School of Science and Technology.
    Krauss, Martin
    Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology. Örebro Life Science Centre, School of Science and Technology, Örebro University, Örebro, Sweden.
    Cousin, Xavier
    MARBEC, Univ. Montpellier, CNRS, Ifremer, IRD, Palavas-les-Flots, France; Univ. Paris-Saclay, AgroParisTech, INRAE, GABI, Jouy-en-Josas, France.
    Keiter, Steffen
    Örebro University, School of Science and Technology.
    Multi- and transgenerational effects following early-life exposure of zebrafish to permethrin and coumarin 47: impact on growth, fertility, behavior and lipid metabolismManuscript (preprint) (Other academic)
  • 10.
    Blanc, Mélanie
    et al.
    Örebro University, School of Science and Technology.
    Cormier, Bettie
    Örebro University, School of Science and Technology. University of Bordeaux, EPOC UMR CNRS, Pessac, France .
    Hyötyläinen, Tuulia
    Örebro University, School of Science and Technology.
    Krauss, Martin
    Helmholtz Centre for Environmental Research - UFZ, Leipzig, Germany.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology. Orebro Life Science Centre.
    Cousin, Xavier
    MARBEC, Univ. Montpellier, CNRS, Ifremer, IRD, Palavas-les-Flots, France; Univ. Paris-Saclay, AgroParisTech, INRAE, GABI, Jouy-en-Josas, France.
    Keiter, Steffen
    Örebro University, School of Science and Technology.
    Multi- and transgenerational effects following early-life exposure of zebrafish to permethrin and coumarin 47: Impact on growth, fertility, behavior and lipid metabolism2020In: Ecotoxicology and Environmental Safety, ISSN 0147-6513, E-ISSN 1090-2414, Vol. 205, article id 111348Article in journal (Refereed)
    Abstract [en]

    Transgenerational effects induced by environmental stressors are a threat to ecosystems and human health. However, there is still limited observation and understanding of the potential of chemicals to influence life outcomes over several generations. In the present study, we investigated the effects of two environmental contaminants, coumarin 47 and permethrin, on exposed zebrafish (FO) and their progeny (F1-F3). Coumarin 47 is commonly found in personal care products and dyes, whereas permethrin is used as a domestic and agricultural pyrethroid insecticide/insect repellent. Zebrafish (F0) were exposed during early development until 28 days post-fertilization and their progeny (F1-F3) were bred unexposed. On one hand, the effects induced by coumarin 47 suggest no multigenerational toxicity. On the other hand, we found that behavior of zebrafish larvae was significantly affected by exposure to permethrin in F1 to F3 generations with some differences depending on the concentration. This suggests persistent alteration of the neural or neuromuscular function. In addition, lipidomic analyses showed that permethrin treatment was partially correlated with lysophosphatidylcholine levels in zebrafish, an important lipid for neurodevelopment. Overall, these results stress out one of the most widely used pyrethroids can trigger long-term, multi- and possibly transgenerational changes in the nervous system of zebrafish. These neurobehavioral changes echo the effects observed under direct exposure to high concentrations of permethrin and therefore call for more research on mechanisms underlying effect inheritance.

  • 11.
    Blanc, Mélanie
    et al.
    Örebro University, School of Science and Technology.
    Kärrman, Anna
    Örebro University, School of Science and Technology.
    Kukučka, Petr
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Keiter, Steffen
    Örebro University, School of Science and Technology.
    Mixture-specific gene expression in zebrafish (Danio rerio) embryos exposed to perfluorooctane sulfonic acid (PFOS), perfluorohexanoic acid (PFHxA) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126)2017In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 590-591, p. 249-257Article in journal (Refereed)
    Abstract [en]

    Perfluorooctane sulfonic acid (PFOS) and 3,3′,4,4′,5-pentachlorobiphenyl (PCB126) are persistent organic pollutants of high concern because of their environmental persistence, bioaccumulation and toxic properties. Besides, the amphiphilic properties of fluorinated compounds such as PFOS and perfluorohexanoic acid (PFHxA) suggest a role in increasing cell membrane permeability and solubilizing chemicals. The present study aimed at investigating whether PFOS and PFHxA are capable of modifying the activation of PCB126 toxicity-related pathways. For this purpose, zebrafish embryos were exposed in semi-static conditions to 7.5 μg/L of PCB126 alone, in the presence of 25 mg/L of PFOS, 15.7 mg/L of PFHxA or in the presence of both PFOS and PFHxA. Quantitative PCR was performed on embryos aged from 24 h post fertilization (hpf) to 96 hpf to investigate expression changes of genes involved in metabolism of xenobiotics (ahr2, cyp1a), oxidative stress (gpx1a, tp53), lipids metabolism (acaa2, osbpl1a), and epigenetic mechanisms (dnmt1, dnmt3ba). Cyp1a and ahr2 expression were significantly induced by the presence of PCB126. However, after 72 and 78 h of exposure, induction of cyp1a expression was significantly lower when embryos were co-exposed to PCB126 + PFOS + PFHxA when compared to PCB126-exposed embryos. Significant upregulation of gpx1a occurred after exposure to PCB126 + PFHxA and to PCB126 + PFOS + PFHxA at 30 and 48 hpf. Besides, embryos appeared more sensitive to PCB126 + PFOS + PFHxA at 78 hpf: acaa2 and osbpl1a were significantly downregulated; dnmt1 was significantly upregulated. While presented as environmentally safe, PFHxA demonstrated that it could affect gene expression patterns in zebrafish embryos when combined to PFOS and PCB126, suggesting that such mixture may increase PCB126 toxicity. This is of particular relevance since PFHxA is persistent and still being ejected into the environment. Moreover, it provides additional information as to the importance to integrate mixture effects of chemicals in risk assessment and biomonitoring frameworks.

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  • 12.
    Blanc, Mélanie
    et al.
    Örebro University, School of Science and Technology.
    Rüegg, Joëlle
    Institute for Environmental Medicine, Karolinska Institutet, Solna, Sweden.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Keiter, Steffen
    Örebro University, School of Science and Technology.
    Environmental chemicals differentially affect epigenetic-related mechanisms in the zebrafish liver (ZF-L) cell line and in zebrafish embryos2019In: Aquatic Toxicology, ISSN 0166-445X, E-ISSN 1879-1514, Vol. 215, article id 105272Article in journal (Refereed)
    Abstract [en]

    A number of chemicals have been shown to affect epigenetic patterning and functions. Since epigenetic mechanisms regulate transcriptional networks, epigenetic changes induced by chemical exposure can represent early molecular events for long-term adverse physiological effects. Epigenetics has thus appeared as a research field of major interest within (eco)toxicological sciences. The present study aimed at measuring effects on epigenetic-related mechanisms of selected environmental chemicals (bisphenols, perfluorinated chemicals, methoxychlor, permethrin, vinclozolin and coumarin 47) in zebrafish embryos and liver cells (ZFL). Transcription of genes related to DNA methylation and histone modifications was measured and global DNA methylation was assessed in ZFL cells using the LUMA assay. The differences in results gathered from both models suggest that chemicals affect different mechanisms related to epigenetics in embryos and cells. In zebrafish embryos, exposure to bisphenol A, coumarin 47, methoxychlor and permethrin lead to significant transcriptional changes in epigenetic factors suggesting that they can impact early epigenome reprogramming related to embryonic development. In ZFL cells, significant transcriptional changes were observed upon exposure to all chemicals but coumarin 47; however, only perfluorooctane sulfonate induced significant effects on global DNA methylation. Notably, in contrast to the other tested chemicals, perfluorooctane sulfonate affected only the expression of the histone demethylase kdm5ba. In addition, kdm5ba appeared as a sensitive gene in zebrafish embryos as well. Taken together, the present results suggest a role for kdm5ba in regulating epigenetic patterns in response to chemical exposure, even though mechanisms remain unclear. To confirm these findings, further evidence is required regarding changes in site-specific histone marks and DNA methylation together with their long-term effects on physiological outcomes.

  • 13.
    Chavan, Swapnil
    et al.
    School of Science and Technology, Örebro University, Örebro, Sweden.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Engwall, Magnus
    Örebro University, School of Science and Technology.
    Repsilber, Dirk
    Örebro University, School of Medical Sciences.
    Predicting Chemical-Induced Liver Toxicity Using High-Content Imaging Phenotypes and Chemical Descriptors: A Random Forest Approach2020In: Chemical Research in Toxicology, ISSN 0893-228X, E-ISSN 1520-5010, Vol. 33, no 9, p. 2261-2275Article in journal (Refereed)
    Abstract [en]

    Hepatotoxicity is a major reason for the withdrawal or discontinuation of drugs from clinical trials. Thus, better tools are needed to filter potential hepatotoxic drugs early in drug discovery. Our study demonstrates utilization of HCI phenotypes, chemical descriptors, and both combined (hybrid) descriptors to construct random forest classifiers (RFCs) for the prediction of hepatotoxicity. HCI data published by Broad Institute provided HCI phenotypes for about 30 000 samples in multiple replicates. Phenotypes belonging to 346 chemicals, which were tested in up to eight replicates, were chosen as a basis for our analysis. We then constructed individual RFC models for HCI phenotypes, chemical descriptors, and hybrid (chemical and HCI) descriptors. The model that was constructed using selective hybrid descriptors showed high predictive performance with 5-fold cross validation (CV) balanced accuracy (BA) at 0.71, whereas within the given applicability domain (AD), independent test set and external test set prediction BAs were equal to 0.61 and 0.60, respectively. The model constructed using chemical descriptors showed a similar predictive performance with a 5-fold CV BA equal to 0.66, a test set prediction BA within the AD equal to 0.56, and an external test set prediction BA within the AD equal to 0.50. In conclusion, the hybrid and chemical descriptor-based models presented here should be considered as a new tool for filtering hepatotoxic molecules during compound prioritization in drug discovery.

  • 14.
    Delbro, Dick
    et al.
    Örebro University, School of Medical Sciences.
    Hallsberg, Lena
    Department of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Peeker, Ralph
    Department of Urology, Institute of Clinical Sciences, Sahlgrenska Academy. University of Gothenburg, Gothenburg, Sweden.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Fall, Magnus
    Department of Urology, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    Godtman, Rebecka Arnsrud
    Department of Urology, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden.
    The extracellular matrix-degrading protein ADAMTS5 is expressed in the nuclei of urothelial cells in healthy rats2018In: Scandinavian journal of urology, ISSN 2168-1805, E-ISSN 2168-1813, Vol. 52, no 2, p. 139-142Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: The aim of this study was to investigate whether protein expression of the extracellular matrix-degrading protease ADAMTS5 can be demonstrated in the urinary bladder of healthy rats, and, if so, to determine the localization of this enzyme.

    MATERIALS AND METHODS: The experiments were conducted with eight inbred male Sprague-Dawley rats. Immunohistochemistry was used to investigate the expression of ADAMTS5 in the urinary bladder. Negative controls were established by either excluding the primary antibody or applying the antibody after it had been preabsorbed with its immunogenic peptide. Confocal microscopy was used to visualize the distribution of ADAMTS5 in the urinary bladder tissue.

    RESULTS: Immunoreactivity for ADAMTS5 was demonstrated in the urothelium and in the detrusor. This expression was localized not only in the cytoplasm, but also in the nuclei. Confocal microscopy corroborated these findings.

    CONCLUSION: Expression of ADAMTS5 was demonstrated in the cytoplasm as well as in the nuclei of the urothelium and detrusor cells, suggesting that it may play a role at the transcriptional level.

  • 15. dos Santos, Daniel
    et al.
    Scherbak, Nikolai
    Örebro University, Department of Natural Sciences.
    Strid, Åke
    Örebro University, Department of Natural Sciences.
    Eriksson, Leif A.
    Örebro University, Department of Natural Sciences.
    Modelling Pisum sativum short-chain dehydrogenase/reductase enzymesManuscript (Other academic)
  • 16.
    Geng, Dawei
    et al.
    Örebro University, School of Science and Technology.
    Musse, Ayan Au
    School of Science and Technology, Örebro University, Örebro, Sweden.
    Wigh, Viktoria
    School of Science and Technology, Örebro University, Örebro, Sweden.
    Carlsson, Cecilia
    School of Science and Technology, Örebro University, Örebro, Sweden.
    Engwall, Magnus
    Örebro University, School of Science and Technology.
    Oresic, Matej
    Örebro University, School of Medical Sciences. Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Hyötyläinen, Tuulia
    Örebro University, School of Science and Technology.
    Effect of perfluorooctanesulfonic acid (PFOS) on the liver lipid metabolism of the developing chicken embryo2019In: Ecotoxicology and Environmental Safety, ISSN 0147-6513, E-ISSN 1090-2414, Vol. 170, p. 691-698Article in journal (Refereed)
    Abstract [en]

    Perfluorooctanesulfonate (PFOS) is a well-known contaminant in the environment and it has shown to disrupt multiple biological pathways, particularly those related with lipid metabolism. In this study, we have studied the impact of in ovo exposure to PFOS on lipid metabolism in livers in developing chicken embryos using lipidomics for detailed characterization of the liver lipidome. We used an avian model (Gallus gallus domesticus) for in ovo treatment at two levels of PFOS. The lipid profile of the liver of the embryo was investigated by ultra-high performance liquid chromatography combined with quadrupole-time-of-flight mass spectrometry and by gas chromatography mass spectrometry. Over 170 lipids were identified, covering phospholipids, ceramides, di- and triacylglycerols, cholesterol esters and fatty acid composition of the lipids. The PFOS exposure caused dose dependent changes in the lipid levels, which included upregulation of specific phospholipids associated with the phosphatidylethanolamine N-methyltransferase (PEMT) pathway, triacylglycerols with low carbon number and double bond count as well as of lipotoxic ceramides and diacylglycerols. Our data suggest that at lower levels of exposure, mitochondrial fatty acid β-oxidation is suppressed while the peroxisomal fatty acid β -oxidation is increased. At higher doses, however, both β -oxidation pathways are upregulated.

  • 17. Hossain, Mohammad Sorowar
    et al.
    Larsson, Anders
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Orban, Laszlo
    Zebrafish androgen receptor: isolation, molecular, and biochemical characterization2008In: Biology of Reproduction, ISSN 0006-3363, E-ISSN 1529-7268, Vol. 78, no 2, p. 361-369Article in journal (Refereed)
    Abstract [en]

    Androgens play an important role in male sexual differentiation and development. They exert their function by binding to and activating the androgen receptor (Ar), a member of the steroid hormone receptor superfamily. Here, we report on the isolation and characterization of zebrafish Ar. The complete transcript of zebrafish ar is 5.3 kb long encoding a putative polypeptide of 868 amino acids. Our experimental and bioinformatic analysis has found a single ar locus in zebrafish. Phylogenetic analysis using the ligand-binding domain showed that the zebrafish Ar clustered with its cyprinid orthologs to form a separate group, which was closer to the beta clade than to the alpha clade. Tissue-specific expression analysis revealed that the ar mRNA was expressed ubiquitously in all adult tissues tested, with sexually dimorphic expression in the gonad and muscle. While the ar transcript was maternally deposited into the embryo, signs of zygotic expression could be detected as early as 24 h after fertilization, and the expression level increased substantially afterwards. When analyzed during gonad development, the expression level of ar mRNA at 4 wk after fertilization was similar in both developing gonads but later became higher in the transforming testis, suggesting a potential role during male gonad differentiation. We also combined theoretical modeling with in vitro experiments to show that the zebrafish Ar is preferentially activated by 11-ketotestosterone.

  • 18.
    Jacobsen, Annette V.
    et al.
    School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, Australia; The Life Science Center, School of Science and Technology, Örebro University, Örebro, Sweden; The Walter and Eliza Hall Institute, Department of Medical Biology, The University of Melbourne, Parkville, Australia.
    Nordén, Marcus
    MTM Research Center, School of Science and Technology, Örebro University, Örebro, Sweden; Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, Mölndal, Sweden.
    Engwall, Magnus
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Effects of perfluorooctane sulfonate on genes controlling hepatic fatty acid metabolism in livers of chicken embryos2018In: Environmental Science and Pollution Research, ISSN 0944-1344, E-ISSN 1614-7499, Vol. 25, no 23, p. 23074-23081Article in journal (Refereed)
    Abstract [en]

    Per- and polyfluoroalkyl substances (PFAS) are synthetic surfactants with a wide variety of applications; however, due to their stability, they are particularly resistant to degradation and, as such, are classed as persistent organic pollutants. Perfluorooctane sulfonate (PFOS) is one such PFAS that is still detectable in a range of different environmental settings, despite its use now being regulated in numerous countries. Elevated levels of PFOS have been detected in various avian species, and the impact of this on avian health is of interest when determining acceptable levels of PFOS in the environment. Due to its similarities to naturally occurring fatty acids, PFOS has potential to disrupt a range of biological pathways, particularly those associated with lipid metabolism, and this has been shown in various species. In this study, we have investigated how in ovo exposure to environmentally relevant levels of PFOS affects expression of genes involved in lipid metabolism of developing chicken embryos. We have found a broad suppression of transcription of genes involved in fatty acid oxidation and PPAR-mediated transcription with more significant effects apparent at lower doses of PFOS. These results highlight the need for more research investigating the biological impacts of low levels of PFAS to properly inform environmental policy governing their regulation.

  • 19.
    Jacobsen, Annette
    et al.
    School of Science and Technology, Örebro University, Örebro, Sweden; School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, Australia.
    Yemaneab, Bisrat
    School of Science and Technology, Örebro University, Örebro, Sweden.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Reference gene selection for qPCR Is dependent on cell type rather than treatment in colonic and vaginal human epithelial cell lines2014In: PLOS ONE, E-ISSN 1932-6203, Vol. 9, no 12, p. e115592-Article in journal (Refereed)
    Abstract [en]

    The ability of commensal bacteria to influence gene expression in host cells under the influence of pathogenic bacteria has previously been demonstrated, however the extent of this interaction is important for understanding how bacteria can be used as probiotics. Real-time quantitative polymerase chain reaction is the most sensitive tool for evaluating relative changes to gene expression levels. However as a result of its sensitivity an appropriate method of normalisation should be used to account for any variation incurred in preparatory experimental procedures. These variations may result from differences in the amount of starting material, quality of extracted RNA, or in the efficiency of the reverse transcriptase or polymerase enzymes. Selection of an endogenous control gene is the preferred method of normalisation, and ideally a proper validation of the gene's appropriateness for the study in question should be performed. In this study we used quantitative polymerase chain reaction data and applied four different algorithms (geNorm, BestKeeper, NormFinder, and comparative ΔCq) to evaluate eleven different genes as to their suitability as endogenous controls for use in studies involving colonic (HT-29) and vaginal (VK2/E6E7) human mucosal epithelial cells treated with probiotic and pathogenic bacteria. We found phosphoglycerate kinase 1 to be most appropriate for HT-29 cells, and ribosomal protein large P0 to be the best choice for VK2/E6E7 cells. We also showed that use of less stable reference genes can lead to less accurate quantification of expression levels of gene of interest (GOI) and also can result in decreased statistical significance for GOI expression levels when compared to control. Additionally, we found the cell type being analysed had greater influence on reference gene selection than the treatment performed. This study provides recommendations for stable endogenous control genes for use in further studies involving colonic and vaginal cell lines after bacterial challenge.

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  • 20.
    Karlsson, Mattias
    et al.
    Örebro University, School of Science and Technology.
    Lam, Simon
    Department of Microbiology and Immunology, University of Western Ontario and The Lawson Health Research Institute, London ON, Canada.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Released substances from lactobacilli influence immune responses in human epithelial cells2010In: In Vivo, ISSN 0258-851X, E-ISSN 1791-7549, Vol. 24, no 3, p. 367-368Article in journal (Refereed)
  • 21.
    Karlsson, Mattias
    et al.
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Khalaf, Hazem
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Olsson, Per-Erik
    Örebro University, School of Science and Technology.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Substances released from probiotic Lactobacillus rhamnosus GR-1 potentiate NF-κB activity in Escherichia coli-stimulated urinary bladder cells2012In: FEMS Immunology and Medical Microbiology, ISSN 0928-8244, E-ISSN 1574-695X, Vol. 66, no 2, p. 147-156Article in journal (Refereed)
    Abstract [en]

    Lactobacillus rhamnosus GR-1 is a probiotic bacterium used to maintain urogenital health. The putative mechanism for its probiotic effect is by modulating the host immunity. Urinary tract infections (UTI) are often caused by uropathogenic Escherichia coli that frequently evade or suppress immune responses in the bladder and can target pathways, including nuclear factor-kappaB (NF-κB). We evaluated the role of L. rhamnosus GR-1 on NF-κB activation in E. coli-stimulated bladder cells. Viable L. rhamnosus GR-1 was found to potentiate NF-κB activity in E. coli-stimulated T24 bladder cells, whereas heat-killed lactobacilli demonstrated a marginal increase in NF-κB activity. Surface components released by trypsin- or LiCl treatment, or the resultant heat-killed shaved lactobacilli, had no effect on NF-κB activity. Isolation of released products from L. rhamnosus GR-1 demonstrated that the induction of NF-κB activity was owing to released product(s) with a relatively large native size. Several putative immunomodulatory proteins were identified, namely GroEL, elongation factor Tu and NLP/P60. GroEL and elongation factor Tu have previously been shown to elicit immune responses from human cells. Isolating and using immune-augmenting substances produced by lactobacilli is a novel strategy for the prevention or treatment of UTI caused by immune-evading E. coli.

  • 22.
    Karlsson, Mattias
    et al.
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Reid, Gregor
    Department of Microbiology and Immunology, University of Western Ontario, London ON, Canada.
    Jass, Jana
    Örebro University, School of Science and Technology.
    Lactobacillus rhamnosus GR-1 enhances NF-kappaB activation in Escherichia coli-stimulated urinary bladder cells through TLR42012In: BMC Microbiology, E-ISSN 1471-2180, Vol. 12, p. 15-Article in journal (Refereed)
    Abstract [en]

    Background: Epithelial cells of the urinary tract recognize pathogenic bacteria through pattern recognition receptors on their surface, such as toll-like receptors (TLRs), and mount an immune response through the activation of the NF-kappaB pathway. Some uropathogenic bacteria can subvert these cellular responses, creating problems with how the host eliminates pathogens. Lactobacillus is a genus of lactic acid bacteria that are part of the microbiota and consist of many probiotic strains, some specifically for urogenital infections. Immunomodulation has emerged as an important mode of action of probiotic and commensal lactobacilli and given the importance of epithelial cells, we evaluated the effect of the urogenital probiotic Lactobacillus rhamnosus GR-1 on epithelial immune activation.

    Results: Immune activation through the NF-kappaB pathway was initiated by stimulation of T24 urothelial cells with heat-killed Escherichia coli and this was further potentiated when cells were co-cultured with live L. rhamnosus GR-1. Heat-killed lactobacilli were poor activators of NF-kappaB. Concomitant stimulation of bladder cells with E. coli and L. rhamnosus GR 1 increased the levels of the pro-inflammatory cytokine TNF, whereas IL-6 and CXCL8 levels were reduced. Another probiotic, L. rhamnosus GG, was also able to potentiate NF-kappaB in these cells although at a significantly reduced level compared to the GR 1 strain. The transcript numbers and protein levels of the lipopolysaccharide receptor TLR4 were significantly increased after co-stimulation with E. coli and lactobacilli compared to controls. Furthermore, inhibition of TLR4 activation by polymixin B completely blocked the lactobacilli potentiation of NF-kappaB.

    Conclusions: The immunological outcome of E. coli challenge of bladder cells was influenced by probiotic L. rhamnosus GR 1, by enhancing the activation of NF-kappaB and TNF release. Thus the urogenital probiotic L. rhamnosus GR-1 modulated the activation of the NF-kappaB through increased levels of TLR4 on the bladder cells and altered subsequent release of cytokines from urothelial cells. By influencing immunological factors such as TLR4, important in the process of fighting pathogens, lactobacilli could facilitate pathogen recognition and infection clearance.

  • 23.
    Khalaf, Hazem
    et al.
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Nakka, Sravya Sowdamini
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden. PEAS Institut AB, Söderleden 1, Linköping.
    Sandén, Camilla
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Svärd, Anna
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Hultenby, Kjell
    Division of Clinical Research Centre, Department of Laboratory Medicine, Karolinska.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Aili, Daniel
    PEAS Institut AB, Söderleden 1, Linköping.
    Bengtsson, Torbjörn
    Örebro University, School of Medicine, Örebro University, Sweden.
    Antibacterial effects of lactobacillus and bacteriocin NC8 αβ on the periodontal pathogen Porphyromonas gingivalisManuscript (preprint) (Other academic)
  • 24.
    Khalaf, Hazem
    et al.
    Örebro University, School of Medical Sciences.
    Nakka, Sravya Sowdamini
    Örebro University, School of Medical Sciences. PEAS Institut AB, Linköping, Sweden.
    Sandén, Camilla
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Svärd, Anna
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Hultenby, Kjell
    Division of Clinical Research Centre, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Aili, Daniel
    Division of Molecular Physics, Department of Physics, Chemistry and Biology (IFM), Linköping University, Linköping, Sweden.
    Bengtsson, Torbjörn
    Örebro University, School of Medical Sciences.
    Antibacterial effects of Lactobacillus and bacteriocin PLNC8 αβ on the periodontal pathogen Porphyromonas gingivalis2016In: BMC Microbiology, E-ISSN 1471-2180, Vol. 16, no 1, article id 188Article in journal (Refereed)
    Abstract [en]

    Background: The complications in healthcare systems associated with antibiotic-resistant microorganisms have resulted in an intense search for new effective antimicrobials. Attractive substances from which novel antibiotics may be developed are the bacteriocins. These naturally occurring peptides are generally considered to be safe and efficient at eliminating pathogenic bacteria. Among specific keystone pathogens in periodontitis, Porphyromonas gingivalis is considered to be the most important pathogen in the development and progression of chronic inflammatory disease. The aim of the present study was to investigate the antimicrobial effects of different Lactobacillus species and the two-peptide bacteriocin PLNC8 αβ on P. gingivalis.

    Results: Growth inhibition of P. gingivalis was obtained by viable Lactobacillus and culture media from L. plantarum NC8 and 44048, but not L. brevis 30670. The two-peptide bacteriocin from L. plantarum NC8 (PLNC8 αβ) was found to be efficient against P. gingivalis through binding followed by permeabilization of the membranes, using Surface plasmon resonance analysis and DNA staining with Sytox Green. Liposomal systems were acquired to verify membrane permeabilization by PLNC8 αβ. The antimicrobial activity of PLNC8 αβ was found to be rapid (1 min) and visualized by TEM to cause cellular distortion through detachment of the outer membrane and bacterial lysis.

    Conclusion: Soluble or immobilized PLNC8 αβ bacteriocins may be used to prevent P. gingivalis colonization and subsequent pathogenicity, and thus supplement the host immune system against invading pathogens associated with periodontitis.

  • 25.
    Krivospitskaya, Olesya
    et al.
    Örebro University, School of Health and Medical Sciences.
    Elmabsout, Ali Ateia
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Sundman, Eva
    Department of Anesthesiology, Surgical Services and Intensive Care Medicine, Karolinska University Hospital Solna, Stockholm, Sweden; Karolinska Institutet, Stockholm, Sweden.
    Söderström, Leif A.
    Department of Anesthesiology, Surgical Services and Intensive Care Medicine, Karolinska University Hospital Solna, Stockholm, Sweden; Karolinska Institutet, Stockholm, Sweden; Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
    Ovchinnikova, Olga
    Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
    Gidlöf, Andreas C.
    Department of Anesthesiology, Surgical Services and Intensive Care Medicine, Karolinska University Hospital Solna, Stockholm, Sweden; Karolinska Institutet, Stockholm, Sweden.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Norata, Giuseppe Danilo
    Atherosclerosis Research Unit, Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden; Department of Pharmacological Sciences University of Milan, Milan, Italy.
    Samnegård, Ann
    Division of Cardiovascular Medicine, Department of Clinical Sciences, Danderyd Hospital, Stockholm, Sweden; Karolinska Institutet, Stockholm, Sweden.
    Törmä, Hans
    Department of Medical Sciences/Dermatology, Uppsala University, Uppsala, Sweden.
    Abdel-Halim, Samy M.
    Division of Respiratory Medicine and Allergology, Department of Clinical Sciences, Danderyd Hospital, Stockholm, Sweden; Karolinska Institutet, Stockholm, Sweden.
    Jansson, Jan-Håkan
    Department of Public Health and Clinical Medicine, Umeå University, Umeå, Sweden; Department of Medicine, Skellefteå Hospital, Skellefteå, Sweden.
    Eriksson, Per
    Atherosclerosis Research Unit, Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden.
    Sirsjö, Allan
    Örebro University, School of Health and Medical Sciences, Örebro University, Sweden.
    Olofsson, Peder S.
    Department of Anesthesiology, Surgical Services and Intensive Care Medicine, Karolinska University Hospital Solna, Stockholm, Sweden; Karolinska Institutet, Stockholm, Sweden; Center for Molecular Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden; Laboratory of Biomedical Science, The Feinstein Institute for Medical Research, North Shore–Long Island Jewish (LIJ) Health System, New York, United States of America.
    A CYP26B1 polymorphism enhances retinoic acid catabolism and may aggravate atherosclerosis2012In: Molecular Medicine, ISSN 1076-1551, E-ISSN 1528-3658, Vol. 18, no 1, p. 712-718Article in journal (Refereed)
    Abstract [en]

    All-trans retinoic acid, controlled by CYP26 enzymes, potentially has beneficial effects in atherosclerosis treatment. This study investigates CYP26B1 in atherosclerosis and effects of a genetic polymorphism in CYP26B1 on retinoid catabolism. We found that CYP26B1 mRNA was induced by retinoic acid in human atherosclerotic arteries and CYP26B1 and the macrophage marker CD68 co-localized in human atherosclerotic lesions. In mice, Cyp26B1 mRNA was higher in atherosclerotic than normal arteries. Databases were queried for non-synonymous CYP26B1 SNPs and rs2241057 selected for further studies. Constructs of the CYP26B1 variants were created and used for production of purified proteins and transfection of macrophage-like cells. The minor variant catabolized retinoic acid with significantly higher efficiency, indicating that rs2241057 is functional and suggesting reduced retinoid availability in tissues with the minor variant. rs2241057 was investigated in a Stockholm Coronary Atherosclerosis Risk Factor (SCARF) subgroup. The minor allele was associated with slightly larger lesions as determined by angiography. In summary, this study identifies the first CYP26B1 polymorphism that alters CYP26B1 capacity to metabolize retinoic acid. CYP26B1 was expressed in macrophage-rich areas of human atherosclerotic lesions, induced by retinoic acid and increased in murine atherosclerosis. Taken together, the results indicate that CYP26B1 capacity is genetically regulated and suggest that local CYP26B1 activity may influence atherosclerosis.

  • 26.
    Lindh, Ingrid
    et al.
    Örebro University, School of Science and Technology.
    Kalbina, Irina
    Örebro University, School of Science and Technology.
    Hedberg, Sara Thulin
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Sävenstrand, Helena
    Örebro University, School of Science and Technology.
    Bråve, Andreas
    Hinkula, Jorma
    Strid, Åke
    Örebro University, School of Science and Technology.
    Andersson, Sören
    Örebro University, School of Science and Technology.
    Feeding of mice with Arabidopsis thaliana expressing the HIV-1 subtype C p24 antigen gives rise to systemic immune responses2008In: Acta Pathologica, Microbiologica et Immunologica Scandinavica (APMIS), ISSN 0903-4641, E-ISSN 1600-0463, Vol. 116, no 11, p. 985-994Article in journal (Refereed)
    Abstract [en]

    Development of transgenic edible plants, to be used as production, storage and delivery systems for recombinant vaccine antigens, is a promising strategy to obtain cost effective vaccines against infectious diseases, not the least for use in developing countries. Therefore, we used Agrobacterium tumefaciens-mediated gene transfer to introduce the p24 gag gene encoding the nucleocapsid protein from HIV-1 subtype C into the Arabidopsis thaliana plant genome. Eighteen plant lines were confirmed positive for the p24 gene by PCR, four of these lines showed an apparent homozygous phenotype when grown on selective medium and these lines also showed transcription of the p24 gene into its corresponding mRNA. The mRNA in all four cases generated the p24 protein in plants, as verified by western blot analysis. The plants were shown to contain between 0.2 µg and 0.5 µg p24 protein per g of fresh tissue. Analysis of the localisation of the p24 protein showed that stem tissue contained the largest amount of protein, more than twice as much as leaf tissue, whereas no p24 protein was detected in roots. By using Southern blotting, we found that 4, 2-3, 2 and 1 T-DNA insertion events took place in the four lines 1, 2, 7, and 10, respectively. The genetic insertions of line 1 were stable from the T1 to the T4 generation and gave rise to the p24 protein in all cases, as verified by western blotting. In mice fed with fresh transgenic A. thaliana (line 10), anti-gag IgG was obtained in serum after a booster injection with recombinant p37Gag. No immune response was observed after equal booster injection of untreated mice or mice fed with A. thaliana WT plants.

  • 27.
    Nilén, Greta
    et al.
    Örebro University, School of Science and Technology.
    Ounoughi, Abir
    Université Clermont Auvergne, Clermont-Ferrand, France.
    Scholz, Stefan
    UFZ Helmholtz Centre for Environmental Research, Leipzig, Germany.
    Keiter, Steffen H.
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Measurements of phenotypical effects caused by priority pollutants using a refined high-content screening approachManuscript (preprint) (Other academic)
  • 28.
    Nilén, Greta
    et al.
    Örebro University, School of Science and Technology.
    Sunder, Supriya
    Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
    Hyötyläinen, Tuulia
    Örebro University, School of Science and Technology.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Keiter, Steffen
    Örebro University, School of Science and Technology.
    A binary, ternary, and quaternary mixture of PFOS, B[a]P, PCB126, and Arsenic alters behavior, gene expression, and lipid content in zebrafish larvae (Danio rerio)Manuscript (preprint) (Other academic)
  • 29.
    Ninyio, Nathaniel
    et al.
    Örebro University, School of Medical Sciences.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Andersson, Sören
    Örebro University, School of Medical Sciences. Folkhälsomyndigheten, Solna, Sweden.
    Development and analysis of prospective anti-HIV probiotic vaccines2022In: 19th Smögen Summer Symposium on Virology: Abstracts, Virus- och Pandemifonden – Swedish Society for Virology , 2022, p. 40-40Conference paper (Other academic)
    Abstract [en]

    Major improvements have been made in the treatment and prevention of HIV/AIDS. However, a prophylactic vaccine is still unavailable, and several vaccine-candidate trials yielded less than favourable results. Given that the HIV pandemic has not slowed down significantly, there is an urgent need for the development of an effective vaccine. The HIV-1 Gag protein, a key player in HIV particle assembly, is a suitable antigen for use in HIV vaccine development since antibodies targeting HIV-1 Gag will interfere with the replication of the virus. In our vaccine development strategy, it was important for us to develop a candidate for mucosal administration. This is because the mucosal route is the major site for HIV transmission and early viral replication, which is associated with extensive and rapid depletion of CD4+ T-cells in the Gut-Associated Lymphoid Tissue (GALT). Here, we transformed probiotic strains of Lactobacillus plantarum and Lactobacillus fermentum with the recombinant plasmid vectors pSIP409 and pSIP411 harbouring the HIV-1 GagM gene. Following electroporation, HIV-1 GagM expression was induced in the probiotics using peptide pheromone. Via PCR and sequencing, the presence of GagM was confirmed in the L. plantarum+ pSIP409-GagM and L. fermentum+ pSIP411-GagM clones. Protein expression was induced with peptide pheromone. Then, protein expression was confirmed by western blotting with goat anti-HIV p24 primary antibody and anti-goat secondary antibody. ELISA was also performed to confirm the antigenicity of the HIV-1 Gag antigen and to also quantify the antigen in the two Lactobacilli clones. Our results show that 1.5×109 CFU of L. plantarum+ pSIP409-GagM expressed 125μg of HIV-1 Gag and 1.9×109 CFU of L. fermentum+ pSIP411-GagM clones expressed 125μg of HIV-1 Gag respectively. In vitro digestion with pepsin, pancreatin and bile salts suggested that partial digestion of the probiotic vaccine candidates may occur when administered orally. Taken together, our probiotic HIV-1 vaccine candidates showed good prospects for further immunological analysis via animal trial.

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  • 30.
    Ninyio, Nathaniel
    et al.
    Örebro University, School of Medical Sciences.
    Schmitt, Katherina
    School of Science and Technology, Life Science Center, Örebro University, Örebro, Sweden; Institute of Virology, Saarland University Medical Center, 66421, Homburg, Germany.
    Sergon, Gladys
    Örebro University, School of Science and Technology.
    Nilsson, Charlotta
    Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; Department of Microbiology, Public Health Agency of Sweden, Solna, Sweden.
    Andersson, Sören
    Örebro University, School of Medical Sciences. Department of Public Health Analysis and Data Management, Unit for Vaccination Programmes, Public Health Agency of Sweden, Solna, Sweden.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Stable expression of HIV-1 MPER extended epitope on the surface of the recombinant probiotic bacteria Escherichia Coli Nissle 1917 using CRISPR/Cas92024In: Microbial Cell Factories, E-ISSN 1475-2859, Vol. 23, no 1, article id 39Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Mucosal vaccines have the potential to induce protective immune responses at the sites of infection. Applying CRISPR/Cas9 editing, we aimed to develop a probiotic-based vaccine candidate expressing the HIV-1 envelope membrane-proximal external region (MPER) on the surface of E. coli Nissle 1917.

    RESULTS: The HIV-1 MPER epitope was successfully introduced in the porin OmpF of the E. coli Nissle 1917 (EcN-MPER) and the modification was stable over 30 passages of the recombinant bacteria on the DNA and protein level. Furthermore, the introduced epitope was recognized by a human anti-HIV-1 gp41 (2F5) antibody using both live and heat-killed EcN-MPER, and this antigenicity was also retained over 30 passages. Whole-cell dot blot suggested a stronger binding of anti-HIV-1 gp41 (2F5) to heat-killed EcN-MPER than their live counterpart. An outer membrane vesicle (OMV) - rich extract from EcN-MPER culture supernatant was equally antigenic to anti-HIV-1 gp41 antibody which suggests that the MPER antigen could be harboured in EcN-MPER OMVs. Using quantitative ELISA, we determined the amount of MPER produced by the modified EcN to be 14.3 µg/108 cfu.

    CONCLUSIONS: The CRISPR/Cas9 technology was an effective method for establishment of recombinant EcN-MPER bacteria that was stable over many passages. The developed EcN-MPER clone was devoid of extraneous plasmids and antibiotic resistance genes which eliminates the risk of plasmid transfer to animal hosts, should this clone be used as a vaccine. Also, the EcN-MPER clone was recognised by anti-HIV-1 gp41 (2F5) both as live and heat-killed bacteria making it suitable for pre-clinical evaluation. Expression of OmpF on bacterial surfaces and released OMVs identifies it as a compelling candidate for recombinant epitope modification, enabling surface epitope presentation on both bacteria and OMVs. By applying the methods described in this study, we present a potential platform for cost-effective and rational vaccine antigen expression and administration, offering promising prospects for further research in the field of vaccine development.

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  • 31.
    Romanenko, PA
    et al.
    LLC SPE "The Fifth Element", Kherson, Ukraine.
    Vinogradova, ON
    N.G. Kholodny Institute of Botany, NAS of Ukraine, Kiev, Ukraine.
    Romanenko, EA
    N.G. Kholodny Institute of Botany, NAS of Ukraine, Kiev, Ukraine.
    Mikhailyuk, TI
    N.G. Kholodny Institute of Botany, NAS of Ukraine, Kiev, Ukraine.
    Babenko, LM
    N.G. Kholodny Institute of Botany, NAS of Ukraine, Kiev, Ukraine.
    Ivannikov, R
    N.N. Grishko National Botanical Garden, Kiev, Ukraine.
    Scherbak, Nikolai
    Örebro University, School of Science and Technology.
    Morphological and Molecular Characterization of the Representative of Brasilonema (Scytonemataceae, Cyanoprokaryota) from the Tropical Greenhouse in Kiev (Ukraine)2020In: International Journal of Algae, ISSN 1521-9429, Vol. 22, no 2, p. 103-122Article in journal (Refereed)
    Abstract [en]

    This paper presents morphological and molecular characterizations of the representative of the pantropic genus Brasilonema Fiore et al. first discovered in a European greenhouse. The genus described in 2007, using an integrated approach, is morphologically close to Scytonema Agardh; differing in the filaments forming ascending bundles, rarely occurring false branching, and the color of cells. To date, 12 species of Brasilonema have been described; all of them occur subaerophytically in humid tropical and subtropical regions. For several years in the greenhouse of the N.N. Grishko National Botanical Garden of the NAS of Ukraine, we observed abundant blackish mats with a fleecy surface, formed by falsely branching filaments of heterocytous cyanobacteria lilac, pale purple or gray in color. Cyanobacteria densely covered the vegetative organs of various tropical epiphytic plants from the Orchidaceae and Bromeliaceae families; sometimes inhabiting concrete and wooden substrates in the greenhouse. The morphological features of this newly discovered cyanobacteria were studied both in natural material greenhouse samples and culture. For cultivation, liquid and agarized N-free BG11 medium was used. The specimens from the mats and the cultures had some differences in thalli habitus, coloration and arrangement of filaments, frequency of false branching, trichome appearance, dimensional limits, etc. A comparative analysis of the original data with descriptions of the known Brasilonema species showed that the Kiev population coincides morphologically and ecologically with several species, of which it's closest relative is B. octagenarum Aguiar et al. Phylogenetic analysis of the nucleotide sequence of the gene encoding 16S rRNA confirmed affiliation of the original strains to Brasilonema. Analysis of the nucleotide sequence of the 16S-23S ITS region, as well as the secondary structure of its most informative helices, showed the closest proximity of the Kiev material to B. octagenarum, which in turn is probably a complex of species whose taxonomic separation is possible in the future. Presumably, cyanobacteria have entered into the Kiev greenhouse with tropical plants, brought by Ukrainian botanists from an expedition to Brazil in 1986.

  • 32.
    Scherbak, Nikolai
    Örebro University, Department of Natural Sciences.
    Characterization of stress-inducible short-chain dehydrogenases/reductases (SDR) in plants: study of a novel small protein family from Pisum sativum (pea)2005Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    In pea (Pisum sativum), the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members (SAD-A, -B, and -C). The SAD genes are transiently expressed in plants after short exposures to ultraviolet-B radiation, which in turn leads to formation of SAD protein in leaf and stem tissue upon prolonged irradiation. SAD gene expression is also seen as a result of wounding stress.

    The recombinant SAD-C protein (which was the most highly over-expressed isoform in Escherichia coli of the isoforms) was shown to be a tetramer that probably consists of a dimer of dimers and which possesses quinone-reducing capability. The enzyme shows approximately the same NADH- and NADPH-dependent activity with 2,5- and 2,6-dimethylbenzoquinone and menadione as substrates.

    Western blotting and immunohistochemistry (IHC) shows that the SAD protein is present to a smaller or larger extent in all the different pea tissues examined. Environmental stress such as UV-B radiation clearly increases the content of SAD in leaf and stem tissue but not in roots. This indicates that increased expression of the SAD genes, as a result of UV-B exposure, is limited to the exposed tissue. This is substantiated by the heterologous GUS expression from the pea SAD-C promoter in Arabidopsis during wounding. Only the wound site and the vicinal tissue show transcription from this promoter. In non-stressed tissue (as well as in UV-B-stressed leaves and stem), SAD predominantly occurred in epidermal or sub-epidermal cells as judged by IHC. The protoderm of the pea seed cotyledonary axis contains the most heavily stained cells. This indicates a possible role for the SAD protein in development as well as in protection against environmental stress. Also, discrete staining was obtained in particular cell types of the ovary.

    To be able to understand the biochemical and physiological role of the SAD enzyme, an in silico modeling study of the SAD protein structure was performed. The simulations of our SAD protein, as well as of related proteins with known crystal structure (3alfa,20beta-hydroxysteroid dehydrogenase and secoisolariciresinol dehydrogenase), allowed us to obtain an energy-minimized structure for the monomeric SAD as well as important data on the cofactor interaction in the active site.

    Crystallization of recombinant SAD-C has been performed. The needle-like crystals, which diffract to 3.5Å, contain probably eight monomers in the asymmetric unit, presumably containing a pair of tetramers.

    SAD enhances the reduction rates of quinones with NADH. However, NADH can also accomplish reduction of certain quinones non-enzymatically. Both theoretical calculations and experimental techniques were used to elucidate the structural and electronic pre-requisites for this non-enzymatic quinone reduction.

    List of papers
    1. Plant SAD proteins: characterization of the tetrameric Pisum sativum protein
    Open this publication in new window or tab >>Plant SAD proteins: characterization of the tetrameric Pisum sativum protein
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology Biological Sciences
    Research subject
    Biochemistry; Biology
    Identifiers
    urn:nbn:se:oru:diva-2939 (URN)
    Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2017-10-18Bibliographically approved
    2. Non-enzymatic oxidation of NADH by quinones
    Open this publication in new window or tab >>Non-enzymatic oxidation of NADH by quinones
    2005 (English)In: Chemical Physics Letters, ISSN 0009-2614, E-ISSN 1873-4448, Vol. 414, no 1-3, p. 243-247Article in journal (Refereed) Published
    Abstract [en]

    Non-enzymatic oxidation of NADH by a large number of different quinones has been explored both theoretically and experimentally. It is concluded that the smaller benzo- and naphtho-quinones are capable of oxidising NADH in aqueous solution, whereas the larger anthraquinone is not. The mechanisms of stepwise electron and proton transfers are explored, and ruled out in favour of direct hydride transfer. For menadione (2-methyl-1,4-naphthoquinone), no reaction is observed experimentally; theoretically we find that there is a very close balance between the energetic cost of hydride removal from NADH and the energy gain of formation of the menadione semiquinone radical anion.

    National Category
    Biological Sciences Biochemistry and Molecular Biology
    Research subject
    Biology; Biochemistry
    Identifiers
    urn:nbn:se:oru:diva-2940 (URN)10.1016/j.cplett.2005.08.067 (DOI)000232460400046 ()2-s2.0-28844504236 (Scopus ID)
    Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2023-12-08Bibliographically approved
    3. Tissue distribution of short-chain alcohol dehydrogenase (SAD) proteins in pea (Pisum sativum) in the absence and presence of UV-B stress, and heterologous stress-induced expression from the SAD promoter
    Open this publication in new window or tab >>Tissue distribution of short-chain alcohol dehydrogenase (SAD) proteins in pea (Pisum sativum) in the absence and presence of UV-B stress, and heterologous stress-induced expression from the SAD promoter
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Biochemistry and Molecular Biology Biological Sciences
    Research subject
    Biochemistry; Biology
    Identifiers
    urn:nbn:se:oru:diva-2941 (URN)
    Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2017-10-18Bibliographically approved
    4. Modelling Pisum sativum short-chain dehydrogenase/reductase enzymes
    Open this publication in new window or tab >>Modelling Pisum sativum short-chain dehydrogenase/reductase enzymes
    (English)Manuscript (Other academic)
    National Category
    Biological Sciences
    Research subject
    Biology
    Identifiers
    urn:nbn:se:oru:diva-2942 (URN)
    Available from: 2005-11-11 Created: 2005-11-11 Last updated: 2017-10-18Bibliographically approved
  • 33.
    Scherbak, Nikolai
    et al.
    Örebro University, School of Science and Technology.
    Ala-Häiväla, Anneli
    Brosché, Mikael
    Helsingfors Universitet, Helsingfors, Finland.
    Böwer, Nathalie
    Strid, Hilja
    Örebro University, School of Health and Medical Sciences.
    Gittins, John R.
    University of Southampton, Southampton, UK.
    Grahn, Elin M.
    Örebro University, School of Science and Technology.
    Eriksson, Leif A.
    National University of Ireland, Galway, Ireland.
    Strid, Åke
    Örebro University, School of Science and Technology.
    The pea SAD short-chain dehydrogenase/reductase: quinone reduction, tissue distribution, and heterologous expression2011In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 155, no 4, p. 1839-1850Article in journal (Refereed)
    Abstract [en]

    The pea (Pisum sativum) tetrameric short-chain alcohol dehydrogenase-like protein (SAD) family consists of at least three highly similar members (SAD-A, -B, and -C). According to mRNA data, environmental stimuli induce SAD expression. The aim of this study was to characterize the SAD proteins by examining their catalytic function, distribution in pea, and induction in different tissues. In enzyme activity assays using a range of potential substrates, the SAD-C enzyme was shown to reduce one- or two-ring-membered quinones lacking long hydrophobic hydrocarbon tails. Immunological assays using a specific antiserum against the protein demonstrated that different tissues and cell types contain small amounts of SAD protein that was predominantly located within epidermal or subepidermal cells and around vascular tissue. Particularly high local concentrations were observed in the protoderm of the seed cotyledonary axis. Two bow-shaped rows of cells in the ovary and the placental surface facing the ovule also exhibited considerable SAD staining. Ultraviolet-B irradiation led to increased staining in epidermal and subepidermal cells of leaves and stems. The different localization patterns of SAD suggest functions both in development and in responses to environmental stimuli. Finally, the pea SAD-C promoter was shown to confer heterologous wound-induced expression in Arabidopsis (Arabidopsis thaliana), which confirmed that the inducibility of its expression is regulated at the transcriptional level.

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  • 34.
    Scherbak, Nikolai
    et al.
    Örebro University, Department of Natural Sciences.
    Ala-Häivälä, Anneli
    Gittins, John R.
    Brosché, Mikael
    Strid, Hilja
    Strid, Åke
    Örebro University, Department of Natural Sciences.
    Tissue distribution of short-chain alcohol dehydrogenase (SAD) proteins in pea (Pisum sativum) in the absence and presence of UV-B stress, and heterologous stress-induced expression from the SAD promoterManuscript (preprint) (Other academic)
  • 35.
    Scherbak, Nikolai
    et al.
    Örebro University, Department of Natural Sciences.
    Brosché, Mikael
    Ala-Häivälä, Anneli
    Olsson, Annika
    Enroth, Cristofer
    Örebro University, Department of Natural Sciences.
    Eriksson, Leif A.
    Örebro University, Department of Natural Sciences.
    Nilsson, Fredrik
    Strid, Åke
    Örebro University, Department of Natural Sciences.
    Plant SAD proteins: characterization of the tetrameric Pisum sativum proteinManuscript (preprint) (Other academic)
  • 36.
    Scherbak, Nikolai
    et al.
    Örebro University, School of Science and Technology.
    Brosché, Mikael
    Ala-Häivälä, Anneli
    Örebro University, School of Science and Technology.
    Strid, Hilja
    Örebro University, School of Health and Medical Sciences.
    Öhrfelt, Annika
    Nilsson, Fredrik
    Strid, Åke
    Örebro University, School of Science and Technology.
    Expression of Pisum sativum SAD polypeptides in production hosts and in planta: Tetrameric organization of the protein2009In: Protein Expression and Purification, ISSN 1046-5928, E-ISSN 1096-0279, Vol. 63, no 1, p. 18-25Article in journal (Refereed)
    Abstract [en]

    In Pisum sativum, the short-chain alcohol dehydrogenase-like protein (SAD) gene family consists of at least three members (SAD-A, -B, and -C). Expression of two of these genes (SAD-A and -C) in Escherichia coli or Pichia pastoris resulted in full-length soluble proteins. Purified SAD-A was used as antigen for antibody production in rabbits. With these antibodies the recombinant SAD-C protein (which was most highly expressed of the two isoforms) was shown to be a tetramer consisting of a dimer of dimers. The SAD genes are transiently expressed in plants by short exposures to ultraviolet-B radiation (UV-B), as judged by northern blotting. In turn, mRNA accumulation leads to formation of SAD protein in leaf and stem tissue upon prolonged UV-B irradiation.

  • 37.
    Scherbak, Nikolai
    et al.
    Örebro University, School of Science and Technology.
    Kruse, Robert
    Örebro University, School of Medical Sciences. Department of Clinical Research Laboratory, Faculty of Medicine and Health, Örebro University, Örebro, Sweden; Inflammatory Response and Infection Susceptibility Center (iRiSC), Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Nyström, Thomas
    Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset, Stockholm, Sweden.
    Jendle, Johan
    Örebro University, School of Medical Sciences.
    Glimepiride Compared to Liraglutide Increases Plasma Levels of miR-206, miR-182-5p, and miR-766-3p in Type 2 Diabetes Mellitus: A Randomized Controlled Trial (Diabetes Metab J 2023;47:668-81)2023In: Diabetes & metabolism journal, ISSN 2233-6079, E-ISSN 2233-6087, Vol. 47, no 6, p. 882-883Article in journal (Refereed)
  • 38.
    Scherbak, Nikolai N.
    et al.
    Örebro University, School of Science and Technology.
    Kruse, Robert
    Örebro University, School of Medical Sciences. Department of Clinical Research Laboratory, Inflammatory Response and Infection Susceptibility Center (iRiSC), Faculty of Medicine and Health, Örebro University, Örebro, Sweden; Karolinska Institutet, Department of Clinical Science and Education, Södersjukhuset, Stockholm, Sweden.
    Nyström, Thomas
    School of Medical Sciences, Faculty of Medicine and Health, Örebro University, Örebro, Sweden.
    Jendle, Johan
    Örebro University, School of Medical Sciences.
    Glimepiride Compared to Liraglutide Increases Plasma Levels of miR-206, miR-182-5p, and miR-766-3p in Type 2 Diabetes Mellitus: A Randomized Controlled Trial2023In: Diabetes & metabolism journal, ISSN 2233-6079, E-ISSN 2233-6087, Vol. 47, no 5, p. 668-681Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Diabetes is a chronic disease with several long-term complications. Several glucose-lowering drugs are used to treat type 2 diabetes mellitus (T2DM), e.g., glimepiride and liraglutide, in which both having different modes of action. Circulating microRNAs (miRNAs) are suggested as potential biomarkers that are associated with the disease development and the effects of the treatment. In the current study we evaluated the effect of glimepiride, liraglutide on the expression of the circulating miRNAs.

    METHODS: The present study is a post hoc trial from a previously randomized control trial comparing liraglutide versus glimepiride both in combination with metformin in subjects with T2DM, and subclinical heart failure. miRNAs were determined in the subjects' serum samples with next generation sequencing. Expression patterns of the circulating miRNAs were analyzed using bioinformatic univariate and multivariate analyses (clinical trial registration: NCT01425580).

    RESULTS: Univariate analyses show that treatment with glimepiride altered expression of three miRNAs in patient serum, miR-206, miR-182-5p, and miR-766-3p. Both miR-182-5p and miR-766-3p were also picked up among the top contributing miRNAs with penalized regularised logistic regressions (Lasso). The highest-ranked miRNAs with respect to Lasso coefficients were miR-3960, miR-31-5p, miR-3613-3p, and miR-378a-3p. Liraglutide treatment did not significantly influence levels of circulating miRNAs.

    CONCLUSION: Present study indicates that glucose-lowering drugs differently affect the expression of circulating miRNAs in serum in individuals with T2DM. More studies are required to investigate possible mechanisms by which glimepiride is affecting the expression of circulating miRNAs.

  • 39.
    Scherbak, Nikolai
    et al.
    Örebro University, School of Science and Technology.
    Ninyio, Nathaniel
    Örebro University, School of Medical Sciences. Örebro University.
    Nilsson, Charlotta
    Karolinska Institutet, Stockholm, Sweden.
    Rybicki, Ed
    University of Cape Town, Cape Town, South Africa.
    Andersson, Sören
    Örebro University, School of Medical Sciences. Public Health Agency of Sweden, Solna, Sweden.
    Production of anti-viral vaccines using probiotic bacteria2022In: 19th Smögen Symposium on Virology: Abstracts: Viral Immunology and Vaccines II and III, Virus- och Pandemifonden – Swedish Society for Virology , 2022, p. 16-16Conference paper (Refereed)
    Abstract [en]

    The mucosal surfaces throughout the body are constantly exposed to microorganisms. The mucosal surfaces accommodate a large part of the body’s immune system. For effective vaccination, it is believed that direct immunization on mucosal surfaces will be more effective than the more conventional systemic immunization. Certain probiotic bacteria provide significant adjuvant effects that may be utilized for the desired immune response. Virus-like particles (VLPs) are complexes of viral proteins that without being infectious are efficient in mimicking the natural viral structures. While presenting the patterns of viral antigens, the VLPs have been shown to efficiently interact with dendritic cells and be effective in triggering the B and T-cell immunity. HIV-1 Gag is one of the most highly conserved structural antigens and contains several immunodominant T- and B-cell epitopes. Mosaic Gag protein matches 74% of 9-amino-acid potential epitopes in global Gag sequences thus maximizing the coverage of potential T-cell epitopes for a viral population.The current study aimed to develop probiotic strains of Lactobacillus plantarum NC8 and E. coli Nissle 1917 that produced recombinant mosaic HIV-1 Gag protein as VLPs.For the transient expression of the mosaic HIV-1 Gag protein (GagM), the gene-carrying expression vectors were transformed into the L. plantarum and in probiotic E.coli Nissle 1917. Protein expression of the GagM was shown to lead to the formation of the VLPs of the recombinant HIV-1 Gag proteins. This was confirmed through immunoblotting and transmission electron microscopy (TEM).Our study shows that probiotic lactobacteria can be developed to express HIV-1 Gag VLPs, which may be used for VLP production and potentially for vaccine delivery. Further evaluation of the concept is merited.

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  • 40.
    Scherbak, Nikolai
    et al.
    Örebro University, Department of Natural Sciences.
    Strid, Åke
    Örebro University, Department of Natural Sciences.
    Eriksson, Leif A.
    Örebro University, Department of Natural Sciences.
    Non-enzymatic oxidation of NADH by quinones2005In: Chemical Physics Letters, ISSN 0009-2614, E-ISSN 1873-4448, Vol. 414, no 1-3, p. 243-247Article in journal (Refereed)
    Abstract [en]

    Non-enzymatic oxidation of NADH by a large number of different quinones has been explored both theoretically and experimentally. It is concluded that the smaller benzo- and naphtho-quinones are capable of oxidising NADH in aqueous solution, whereas the larger anthraquinone is not. The mechanisms of stepwise electron and proton transfers are explored, and ruled out in favour of direct hydride transfer. For menadione (2-methyl-1,4-naphthoquinone), no reaction is observed experimentally; theoretically we find that there is a very close balance between the energetic cost of hydride removal from NADH and the energy gain of formation of the menadione semiquinone radical anion.

1 - 40 of 40
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