| 1. |
- Thalén, Niklas, 1985-, et al.
(author)
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Systems biology greatly improve activity of secreted therapeutic sulfatase in CHO bioprocess
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Other publication (other academic/artistic)abstract
- Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. However, such recombinant production of sulfatases suffers greatly from low product activity and yield, further limiting accessibility for patient groups. Here, we have addressed this problem by defining key-proteins necessary for active sulfatase secretion by comparison of CHO clones with different levels of production of active sulfatase. Quantitative transcriptomic analysis highlighted 14 key genes associated with sulfatase production, and experimental validation by co-expression improved the sulfatase enzyme activity by up to 150-fold. Furthermore, a correlation between product mRNA levels and sulfatase activity were observed and expression with lower activity promoters showed an increased in sulfatase activity. The workflow devised is general and we propose it to be useful for resolving bottlenecks in cellular machineries for improvement of cell factories for other biologics as well.
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| 2. |
- Thalén, Niklas, et al.
(author)
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Tuning of CHO secretional machinery improve activity of secreted therapeutic sulfatase 150-fold
- 2024
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In: Metabolic engineering. - : Elsevier BV. - 1096-7176 .- 1096-7184. ; 81, s. 157-166
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Journal article (peer-reviewed)abstract
- Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. The recombinant production of such sulfatases suffers greatly from both low product activity and yield, further limiting accessibility for patient groups. To mitigate the low product activity, we have investigated cellular properties through computational evaluation of cultures with varying media conditions and comparison of two CHO clones with different levels of one active sulfatase variant. Transcriptome analysis identified 18 genes in secretory pathways correlating with increased sulfatase production. Experimental validation by upregulation of a set of three key genes improved the specific enzymatic activity at varying degree up to 150-fold in another sulfatase variant, broadcasting general production benefits. We also identified a correlation between product mRNA levels and sulfatase activity that generated an increase in sulfatase activity when expressed with a weaker promoter. Furthermore, we suggest that our proposed workflow for resolving bottlenecks in cellular machineries, to be useful for improvements of cell factories for other biologics as well.
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| 3. |
- Fagerström, Anna, 1980-
(author)
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Long-term molecular epidemiology of extended-spectrum β-lactamase producing Escherichia coli in a low-endemic setting
- 2020
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Doctoral thesis (other academic/artistic)abstract
- Escherichia coli is a commensal inhabitant in the gastro-intestinal tract of humans and animals but it is also the most common bacterial species causing urinary tract infection, which ranges in severity from distal cystitis to urosepsis and septic shock. During the past decades, the prevalence of antibiotic resistant E. coli has increased worldwide. Extended-spectrum β-lactamases (ESBL) causes resistance to β-lactam antibiotics, the most widely used class of antibiotics. The genes encoding ESBL, bla, are usually carried on conjugative plasmids, which can be transferred between different bacterial lineages and different species. These plasmids frequently also carry resistance genes to additional antibiotic classes, and ESBL-producing E. coli are therefore often multidrug-resistant. The aim of this thesis was to describe the long-term molecular epidemiology of ESBL-producing E. coli in Örebro County during the time when they first started to emerge. In addition, potential transmission to the environment was investigated by performing a comparative analysis on ESBL-producing E. coli isolated from patients and from the aquatic environment in Örebro city. In general, the E. coli population was genetically diverse, but the pandemic lineage ST131, first identified in 2004, appears to have been responsible for the dramatic increase of CTX-M-15-producing E.coli observed during the late 2000s. CTX-M-15 was the most prevalent ESBL-type followed by CTX-M-14 and these genes were mainly found on plasmids belonging to the IncF or IncI1 families. Continuous horizontal transmission of IncI1 ST31 and ST37 plasmids between diverse E. coli lineages have also contributed to the dissemination of blaCTX-M-15 in Örebro County. Extended spectrum β-lactamase-producing E. coli were found to be common in the aquatic environment in Örebro city and E. coli lineages genetically similar to those causing infections in humans were present in environmental waters indicating that transmission of ESBL-producing E. colifrom humans to the aquatic environment likely has occurred.
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| 4. |
- Gast, Veronica, 1992, et al.
(author)
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Engineering Saccharomyces cerevisiae for the production and secretion of Affibody molecules
- 2022
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In: Microbial Cell Factories. - : Springer Science and Business Media LLC. - 1475-2859. ; 21:1
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Journal article (peer-reviewed)abstract
- BACKGROUND: Affibody molecules are synthetic peptides with a variety of therapeutic and diagnostic applications. To date, Affibody molecules have mainly been produced by the bacterial production host Escherichia coli. There is an interest in exploring alternative production hosts to identify potential improvements in terms of yield, ease of production and purification advantages. In this study, we evaluated the feasibility of Saccharomyces cerevisiae as a production chassis for this group of proteins. RESULTS: We examined the production of three different Affibody molecules in S. cerevisiae and found that these Affibody molecules were partially degraded. An albumin-binding domain, which may be attached to the Affibody molecules to increase their half-life, was identified to be a substrate for several S. cerevisiae proteases. We tested the removal of three vacuolar proteases, proteinase A, proteinase B and carboxypeptidase Y. Removal of one of these, proteinase A, resulted in intact secretion of one of the targeted Affibody molecules. Removal of either or both of the two additional proteases, carboxypeptidase Y and proteinase B, resulted in intact secretion of the two remaining Affibody molecules. The produced Affibody molecules were verified to bind their target, human HER3, as potently as the corresponding molecules produced in E. coli in an in vitro surface-plasmon resonance binding assay. Finally, we performed a fed-batch fermentation with one of the engineered protease-deficient S. cerevisiae strains and achieved a protein titer of 530 mg Affibody molecule/L. CONCLUSION: This study shows that engineered S. cerevisiae has a great potential as a production host for recombinant Affibody molecules, reaching a high titer, and for proteins where endotoxin removal could be challenging, the use of S. cerevisiae obviates the need for endotoxin removal from protein produced in E. coli.
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| 5. |
- Hendrikse, Natalie, et al.
(author)
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Ancestral lysosomal enzymes with increased activity harbor therapeutic potential for treatment of Hunter syndrome
- 2021
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In: ISCIENCE. - : Elsevier BV. - 2589-0042. ; 24:3
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Journal article (peer-reviewed)abstract
- We show the successful application of ancestral sequence reconstruction to enhance the activity of iduronate-2-sulfatase (IDS), thereby increasing its therapeutic potential for the treatment of Hunter syndrome-a lysosomal storage disease caused by impaired function of IDS. Current treatment, enzyme replacement therapy with recombinant human IDS, does not alleviate all symptoms, and an unmet medical need remains. We reconstructed putative ancestral sequences of mammalian IDS and compared them with extant IDS. Some ancestral variants displayed up to 2-fold higher activity than human IDS in in vitro assays and cleared more substrate in ex vivo experiments in patient fibroblasts. This could potentially allow for lower dosage or enhanced therapeutic effect in enzyme replacement therapy, thereby improving treatment outcomes and cost efficiency, as well as reducing treatment burden. In summary, we showed that ancestral sequence reconstruction can be applied to lysosomal enzymes that function in concert with modern enzymes and receptors in cells.
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| 6. |
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| 7. |
- Mebrahtu, Aman, et al.
(author)
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Co-culture platform for tuning of cancer receptor density allows for evaluation of bispecific immune cell engagers
- 2024
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In: New Biotechnology. - : Elsevier BV. - 1871-6784 .- 1876-4347. ; 79, s. 120-126
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Journal article (peer-reviewed)abstract
- Cancer immunotherapy, where a patient's immune system is harnessed to eradicate cancer cells selectively, is a leading strategy for cancer treatment. However, successes with immune checkpoint inhibitors (ICI) are hampered by reported systemic and organ-specific toxicities and by two-thirds of the patients being non-responders or subsequently acquiring resistance to approved ICIs. Hence substantial efforts are invested in discovering novel targeted immunotherapies aimed at reduced side-effects and improved potency. One way is utilizing the dual targeting feature of bispecific antibodies, which have made them increasingly popular for cancer immunotherapy. Easy and predictive screening methods for activation ranking of candidate drugs in tumor contra non-tumor environments are however lacking. Herein, we present a cell-based assay mimicking the tumor microenvironment by co-culturing B cells with engineered human embryonic kidney 293 T cells (HEK293T), presenting a controllable density of platelet-derived growth factor receptor β (PDGFRβ). A target density panel with three different surface protein levels on HEK293T cells was established by genetic constructs carrying regulatory elements limiting RNA translation of PDGFRβ. We employed a bispecific antibody-affibody construct called an AffiMab capable of binding PDGFRβ on cancer cells and CD40 expressed by B cells as a model. Specific activation of CD40-mediated signaling of immune cells was demonstrated with the two highest receptor-expressing cell lines, Level 2/3 and Level 4, while low-to-none in the low-expressing cell lines. The concept of receptor tuning and the presented co-culture protocol may be of general utility for assessing and developing novel bi-specific antibodies for immuno-oncology applications.
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| 8. |
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| 9. |
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| 10. |
- Mega, Alessandro, et al.
(author)
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A PDGFRB- and CD40-targeting bispecific AffiMab induces stroma-targeted immune cell activation
- 2023
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In: mAbs. - : Informa UK Limited. - 1942-0862 .- 1942-0870. ; 15:1
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Journal article (peer-reviewed)abstract
- CD40 agonism by systemic administration of CD40 monoclonal antibodies has been explored in clinical trials for immunotherapy of cancer, uncovering enormous potential, but also dosing challenges in terms of systemic toxicity. CD40-dependent activation of antigen presenting cells is dependent on crosslinking of the CD40 receptor. Here we exploited this requisite by coupling crosslinking to cancer-receptor density by dual-targeting of CD40 and platelet-derived growth factor receptor beta (PDGFRB), which is highly expressed in the stroma of various types of tumors. A novel PDGFRBxCD40 Fc-silenced bispecific AffiMab was developed to this end to test whether it is possible to activate CD40 in a PDGFRB-targeted manner. A PDGFRB-binding Affibody molecule was fused to each heavy chain of an Fc-silenced CD40 agonistic monoclonal antibody to obtain a bispecific "AffiMab". Binding of the AffiMab to both PDGFRB and CD40 was confirmed by surface plasmon resonance, bio-layer interferometry and flow cytometry, through analysis of cells expressing respective target. In a reporter assay, the AffiMab displayed increased CD40 potency in the presence of PDGFRB-conjugated beads, in a manner dependent on PDGFRB amount/bead. To test the concept in immunologically relevant systems with physiological levels of CD40 expression, the AffiMab was tested in human monocyte-derived dendritic cells (moDCs) and B cells. Expression of activation markers was increased in moDCs specifically in the presence of PDGFRB-conjugated beads upon AffiMab treatment, while the Fc-silenced CD40 mAb did not stimulate CD40 activation. As expected, the AffiMab did not activate moDCs in the presence of unconjugated beads. Finally, in a co-culture experiment, the AffiMab activated moDCs and B cells in the presence of PDGFRB-expressing cells, but not in co-cultures with PDGFRB-negative cells. Collectively, these results suggest the possibility to activate CD40 in a PDGFRB-targeted manner in vitro. This encourages further investigation and the development of such an approach for the treatment of solid cancers.
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| 11. |
- Müller, Vilhelm, 1990, et al.
(author)
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Cultivation-Free Typing of Bacteria Using Optical DNA Mapping
- 2020
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In: Acs Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 6:5, s. 1076-1084
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Journal article (peer-reviewed)abstract
- A variety of pathogenic bacteria can infect humans, and rapid species identification is crucial for the correct treatment. However, the identification process can often be time-consuming and depend on the cultivation of the bacterial pathogen(s). Here, we present a stand-alone, enzyme-free, optical DNA mapping assay capable of species identification by matching the intensity profiles of large DNA molecules to a database of fully assembled bacterial genomes (>10 000). The assay includes a new data analysis strategy as well as a general DNA extraction protocol for both Gram-negative and Gram-positive bacteria. We demonstrate that the assay is capable of identifying bacteria directly from uncultured clinical urine samples, as well as in mixtures, with the potential to be discriminative even at the subspecies level. We foresee that the assay has applications both within research laboratories and in clinical settings, where the time-consuming step of cultivation can be minimized or even completely avoided.
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| 12. |
- Nyblom, My, 1995, et al.
(author)
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Bacterial identification by optical mapping of genomic DNA in nanofluidic channels
- 2019
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In: 23rd International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2019. - 9781733419000 ; , s. 821-822
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Conference paper (peer-reviewed)abstract
- A variety of pathogenic bacteria can infect humans and the increase in bacteria resistant to common antibiotics is a large threat to human health worldwide. This work presents a method, based on optical DNA mapping (ODM) in nanofluidic channels, that can detect the type of bacterial present in a sample by matching the obtained maps of large DNA molecules to a database of fully assembled bacterial genomes. The extraction and labelling protocol has been designed to work for both Gram-positive and Gram-negative bacteria, not requiring any prior knowledge about the sample content.
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| 13. |
- Nyblom, My, 1995, et al.
(author)
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Strain-level bacterial typing directly from patient samples using optical DNA mapping
- 2023
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In: COMMUNICATIONS MEDICINE. - : Springer Science and Business Media LLC. - 2730-664X. ; 3:31
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Journal article (peer-reviewed)abstract
- For bacterial infections, it is important to rapidly and accurately identify and characterize the type of bacteria involved so that optimal antibiotic treatment can be given quickly to the patient. However, current diagnostic methods are sometimes slow and cannot be used for mixtures of bacteria. We have, therefore, developed a method to identify bacteria directly from patient samples. The method was tested on two common species of disease-causing bacteria - Escherichia coli and Klebsiella pneumoniae - and it could correctly identify the bacterial strain or subtype in both urine samples and mixtures. Hence, the method has the potential to provide fast diagnostic information for choosing the most suited antibiotic, thereby reducing the risk of death and suffering. Nyblom, Johnning et al. develop an optical DNA mapping approach for bacterial strain typing of patient samples. They demonstrate rapid identification of clinically relevant E. coli and K. pneumoniae strains, without the need for cultivation. BackgroundIdentification of pathogens is crucial to efficiently treat and prevent bacterial infections. However, existing diagnostic techniques are slow or have a too low resolution for well-informed clinical decisions.MethodsIn this study, we have developed an optical DNA mapping-based method for strain-level bacterial typing and simultaneous plasmid characterisation. For the typing, different taxonomical resolutions were examined and cultivated pure Escherichia coli and Klebsiella pneumoniae samples were used for parameter optimization. Finally, the method was applied to mixed bacterial samples and uncultured urine samples from patients with urinary tract infections. Results We demonstrate that optical DNA mapping of single DNA molecules can identify Escherichia coli and Klebsiella pneumoniae at the strain level directly from patient samples. At a taxonomic resolution corresponding to E. coli sequence type 131 and K. pneumoniae clonal complex 258 forming distinct groups, the average true positive prediction rates are 94% and 89%, respectively. The single-molecule aspect of the method enables us to identify multiple E. coli strains in polymicrobial samples. Furthermore, by targeting plasmid-borne antibiotic resistance genes with Cas9 restriction, we simultaneously identify the strain or subtype and characterize the corresponding plasmids. Conclusion The optical DNA mapping method is accurate and directly applicable to polymicrobial and clinical samples without cultivation. Hence, it has the potential to rapidly provide comprehensive diagnostics information, thereby optimizing early antibiotic treatment and opening up for future precision medicine management.
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| 14. |
- Olsson, Anna, et al.
(author)
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Efficacy of Antibiotic Combinations against Multidrug-Resistant Pseudomonas aeruginosa in Automated Time-Lapse Microscopy and Static Time-Kill Experiments
- 2020
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In: Antimicrobial Agents and Chemotherapy. - : AMER SOC MICROBIOLOGY. - 0066-4804 .- 1098-6596. ; 64:6
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Journal article (peer-reviewed)abstract
- Antibiotic combination therapy is used for severe infections caused by multidrug-resistant (MDR) Gram-negative bacteria, yet data regarding which combinations are most effective are lacking. This study aimed to evaluate the in vitro efficacy of polymyxin B in combination with 13 other antibiotics against four clinical strains of MDR Pseudomonas aeruginosa. We evaluated the interactions of polymyxin B in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, linezolid, meropenem, minocycline, rifampin, temocillin, thiamphenicol, or trimethoprim by automated time-lapse microscopy using predefined cutoff values indicating inhibition of growth (<= 10(6) CFU/ml) at 24 h. Promising combinations were subsequently evaluated in static time-kill experiments. All strains were intermediate or resistant to polymyxin B, antipseudomonal beta-lactams, ciprofloxacin, and amikacin. Genes encoding beta-lactamases (e.g., bla(PAO) and bla(OXA-50)) and mutations associated with permeability and efflux were detected in all strains. In the time-lapse microscopy experiments, positive interactions were found with 39 of 52 antibiotic combination/bacterial strain setups. Enhanced activity was found against all four strains with polymyxin B used in combination with aztreonam, cefepime, fosfomycin, minocycline, thiamphenicol, and trimethoprim. Time-kill experiments showed additive or synergistic activity with 27 of the 39 tested polymyxin B combinations, most frequently with aztreonam, cefepime, and meropenem. Positive interactions were frequently found with the tested combinations, against strains that harbored several resistance mechanisms to the single drugs, and with antibiotics that are normally not active against P. aeruginosa. Further study is needed to explore the clinical utility of these combinations.
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| 15. |
- Olsson, Anna
(author)
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Exploring the interactions of antibiotic combinations against multidrug-resistant Gram-negative bacteria
- 2021
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Doctoral thesis (other academic/artistic)abstract
- Antimicrobial resistance is emerging and available treatment options are lacking. Antibiotics is a cornerstone in modern medicine where medical procedures such as surgery, care of premature babies or treatment of cancer is dependent on efficient drugs. The need for novel treatment alternatives is increasing as bacteria continue to develop new resistance mechanisms.The main goal of this thesis was to screen for antimicrobial combinations efficient against Gram-negative bacteria. The complex membrane structure of Gram-negative bacteria is very protective against antimicrobial activity making many antibiotics ineffective. Polymyxin B was therefore used as a main component in the combinations evaluated due to its membrane disruptive mode of action. Previously neglected or disused antibiotics was used in combination with polymyxin B as a part of a Joint Programming Initiative on Antimicrobial Resistance (JPIAMR) called CO-ACTION. The prevailing theory of polymyxin B combinations is that the membrane disruptive effect will facilitate entry of a second antibiotic and reduce efflux activity. In contrast, the combination will not be as efficient in the presence of bacterial enzymatic activity as the resistance mechanism is not affected by changes in the membrane composition. To increase knowledge on underlying mechanisms behind the success of antimicrobial combinations an extensive genetic analysis was performed. Several promising polymyxin B combinations were found which could offer a treatment option in caring for severely ill patients for which few alternatives exist. Associations between genetic background and efficient bacterial killing was also established. The chance of synergistic effect by the combination was increased if the antibiotic used in combination with polymyxin B could normally not enter the bacterial cell or in presence of resistance mechanisms increasing efflux activity. This thesis highlights the fact that only phenotypical antimicrobial susceptibility testing would not be used in forecasting the success of antimicrobial combinations. Information on antimicrobial susceptibility in combination with knowledge on resistance mechanisms present and how it influences the antibiotics used in combination is equally important. With this work increased knowledge on genetic background of resistance mechanisms and bacterial killing by polymyxin B combinations and was provided. Antimicrobial combinations offer an interesting feature when no other treatment alternatives are available. The lack of diagnostics in forecasting the success of combination therapy in a clinical microbiology lab is of concern. The knowledge obtained in this work contributes to the general knowledge on antimicrobial combinations and provides an example of how to evaluate their effect.
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| 16. |
- Palica, Katarzyna, 1992-, et al.
(author)
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Metallo-β-Lactamase Inhibitor Phosphonamidate Monoesters
- 2022
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In: ACS Omega. - : American Chemical Society (ACS). - 2470-1343. ; 7:5, s. 4550-4562
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Journal article (peer-reviewed)abstract
- Being the second leading cause of death and the leading cause of disability-adjusted life years worldwide, infectious diseases remain─contrary to earlier predictions─a major consideration for the public health of the 21st century. Resistance development of microbes to antimicrobial drugs constitutes a large part of this devastating problem. The most widely spread mechanism of bacterial resistance operates through the degradation of existing β-lactam antibiotics. Inhibition of metallo-β-lactamases is expected to allow the continued use of existing antibiotics, whose applicability is becoming ever more limited. Herein, we describe the synthesis, the metallo-β-lactamase inhibition activity, the cytotoxicity studies, and the NMR spectroscopic determination of the protein binding site of phosphonamidate monoesters. The expression of single- and double-labeled NDM-1 and its backbone NMR assignment are also disclosed, providing helpful information for future development of NDM-1 inhibitors. We show phosphonamidates to have the potential to become a new generation of antibiotic therapeutics to combat metallo-β-lactamase-resistant bacteria.
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| 17. |
- Pratim Bose, Partha, et al.
(author)
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Poly-N-methylated Amyloid β-Peptide (Aβ) C-Terminal Fragments Reduce Aβ Toxicity in Vitro and in Drosophila melanogaster
- 2009
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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 52:24, s. 8002-8009
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Journal article (peer-reviewed)abstract
- Alzheimer’s disease (AD), an age related neurodegenerative disorder, threatens to become a major health-economic problem. Assembly of 40- or 42-residue amyloid β-peptides (Aβ) into neurotoxic oligo-/polymeric β-sheet structures is an important pathogenic feature in AD, thus, inhibition of this process has been explored to prevent or treat AD. The C-terminal part plays an important role in Aβ aggregation, but most Aβ aggregation inhibitors have targeted the central region around residues 16−23. Herein, we synthesized hexapeptides with varying extents of N-methylation based on residues 32−37 of Aβ, to target its C-terminal region. We measured the peptides' abilities to retard β-sheet and fibril formation of Aβ and to reduce Aβ neurotoxicity. A penta-N-methylated peptide was more efficient than peptides with 0, 2, or 3 N-methyl groups. This penta-N-methylated peptide moreover increased life span and locomotor activity in Drosophila melanogaster flies overexpressing human Aβ1−42.
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| 18. |
- Roos, Anna, 1961-, et al.
(author)
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The otter (Lutra lutra) in Sweden - population trends in relation to ΣDDT and total PCB concentrations during 1968-99
- 2001
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In: Environmental Pollution. - 0269-7491 .- 1873-6424. ; 111, s. 457-469
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Journal article (peer-reviewed)abstract
- Polychlorinated biphenyl (PCB) has been suggested as the major cause of the European otter (Lutra lutra) population decline. This study investigates temporal and geographical changes in otter numbers as well as total PCB and ΣDDT concentrations in otters and their food in diferent parts of Sweden with various pollution impacts during three decades. In Sweden, rare species belong to the State and carcasses of such specimens are sent to the authorities. Two-hundred and eight specimens have been used to investigate temporal and spatial variation in the otter populations during 1968 and 1999. One-hundred and twentyfive of them have been chemically analysed. The population trends in northern Sweden coincide with the temporal trends of the contaminants. When concentrations of PCBs decrease in the environment, otter population increases. Neither aldrin/dieldrin nor mercury pollution seem to explain why the Swedish otter populations decreased dramatically during the 1960s-1980s. In southern Sweden total PCB concentrations are still high and the indications of improvement of the population are weak.
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