| 1. |
- Datta, A., et al.
(författare)
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Role of Aromatic Amino Acids in Lipopolysaccharide and Membrane Interactions of Antimicrobial Peptides for use in Plant Disease Control
- 2016
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 291:25, s. 13301-13317
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Tidskriftsartikel (refereegranskat)abstract
- KYE28(KYEITTIHNLFRKLTHRLFRRNFGYTLR), the representative sequence of helix D of heparin co-factor II, was demonstrated to be potent against agronomically important Gram-negative plant pathogens X. vesicatoria and X. oryzae,capable of inhibiting disease symptoms in detached tomato leaves. NMR studies in presence of lipopolysaccharide provided structural insights into the mechanisms underlying this, notably in relation to outer membrane permeabilisation. The three-dimensional solution structure of KYE28 in LPS is characterised by a N-ter helical segment, an intermediate loop and an extended C-ter. The two termini are in close proximity to each other via aromatic packing interactions, while the positively charged residues formed an exterior polar shell. To further demonstrate the importance of the aromatic residues for this, a mutant peptide KYE28A, with Ala substitutions at F11, F19, F23 and Y25 showed attenuated antimicrobial activity at high salt concentrations, as well as lower membrane disruption and LPS binding abilities compared to KYE28. In contrast to KYE28, KYE28A adopted an opened out helical structure in LPS with extended N- and C-ter and a small break in between the helical segments. Aromatic packing interactions were completely lost, although hydrophobic interaction between the side chains of hydrophobic residues were still partly retained, imparting an amphipathic character and explaining its residual antimicrobial activity and LPS binding as observed from ellipsometry and ITC. We thus present important structural aspects of KYE28, constituting an aromatic zipper, of potential importance, for the development of novel plant protection agents and therapeutic agents.
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| 2. |
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| 3. |
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| 4. |
- Holdbrook, Daniel A., et al.
(författare)
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Multiscale modeling of innate immune receptors : Endotoxin recognition and regulation by host defense peptides
- 2019
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Ingår i: Pharmacological Research. - : Elsevier BV. - 1043-6618. ; 147
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Forskningsöversikt (refereegranskat)abstract
- The innate immune system provides a first line of defense against foreign microorganisms, and is typified by the Toll-like receptor (TLR) family. TLR4 is of particular interest, since over-stimulation of its pathway by excess lipopolysaccharide (LPS) molecules from the outer membranes of Gram-negative bacteria can result in sepsis, which causes millions of deaths each year. In this review, we outline our use of molecular simulation approaches to gain a better understanding of the determinants of LPS recognition, towards the search for novel immunotherapeutics. We first describe how atomic-resolution simulations have enabled us to elucidate the regulatory conformational changes in TLR4 associated with different LPS analogues, and hence a means to rationalize experimental structure-activity data. Furthermore, multiscale modelling strategies have provided a detailed description of the thermodynamics and intermediate structures associated with the entire TLR4 relay – which consists of a number of transient receptor/coreceptor complexes – allowing us trace the pathway of LPS transfer from bacterial membranes to the terminal receptor complex at the plasma membrane surface. Finally, we describe our efforts to leverage these computational models, in order to elucidate previously undisclosed anti-inflammatory mechanisms of endogenous host-defense peptides found in wounds. Collectively, this work represents a promising avenue for the development of novel anti-septic treatments, inspired by nature's innate defense strategies.
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| 5. |
- Huber, Roland G., et al.
(författare)
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A Thermodynamic Funnel Drives Bacterial Lipopolysaccharide Transfer in the TLR4 Pathway
- 2018
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Ingår i: Structure. - : Elsevier BV. - 0969-2126. ; 26:8, s. 4-1161
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Tidskriftsartikel (refereegranskat)abstract
- The Gram-negative bacterial outer membrane contains lipopolysaccharide, which potently stimulates the mammalian innate immune response. This involves a relay of specialized complexes culminating in transfer of lipopolysaccharide from CD14 to Toll-like receptor 4 (TLR4) and its co-receptor MD-2 on the cell surface, leading to activation of downstream inflammatory responses. In this study we develop computational models to trace the TLR4 cascade in near-atomic detail. We demonstrate through rigorous thermodynamic calculations that lipopolysaccharide molecules traversing the receptor cascade fall into a thermodynamic funnel. An affinity gradient for lipopolysaccharide is revealed upon extraction from aggregates or realistic bacterial outer membrane models and transfer through CD14 to the terminal TLR4/MD-2 receptor-co-receptor complex. We subsequently assemble viable CD14/TLR4/MD-2 oligomers at the plasma membrane surface, and observe lipopolysaccharide exchange between CD14 and TLR4/MD-2. Collectively, this work helps to unravel the key structural determinants governing endotoxin recognition in the TLR4 innate immune pathway. Huber et al. develop near-atomic computational models to simulate LPS transfer through the TLR4 pathway. These reveal that LPS recognition is favored by a thermodynamic funnel of increasing affinity along a receptor cascade, terminating in productive transfer of LPS at spontaneously assembled CD14/TLR4/MD-2 membrane complexes.
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| 6. |
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| 7. |
- Malekkhaiat Häffner, Sara, et al.
(författare)
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Interaction of Laponite with Membrane Components - Consequences for Bacterial Aggregation and Infection Confinement
- 2019
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Ingår i: ACS Applied Materials and Interfaces. - : American Chemical Society (ACS). - 1944-8244 .- 1944-8252. ; 11:17, s. 15389-15400
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Tidskriftsartikel (refereegranskat)abstract
- The antimicrobial effects of Laponite nanoparticles with or without loading of the antimicrobial peptide LL-37 was investigated along with their membrane interactions. The study combines data from ellipsometry, circular dichroism, fluorescence spectroscopy, particle size/ζ potential measurements, and confocal microscopy. As a result of the net negative charge of Laponite, loading of net positively charged LL-37 increases with increasing pH. The peptide was found to bind primarily to the outer surface of the Laponite nanoparticles in a predominantly helical conformation, leading to charge reversal. Despite their net positive charge, peptide-loaded Laponite nanoparticles did not kill Gram-negative Escherichia coli bacteria or disrupt anionic model liposomes. They did however cause bacteria flocculation, originating from the interaction of Laponite and bacterial lipopolysaccharide (LPS). Free LL-37, in contrast, is potently antimicrobial through membrane disruption but does not induce bacterial aggregation in the concentration range investigated. Through LL-37 loading of Laponite nanoparticles, the combined effects of bacterial flocculation and membrane lysis are observed. However, bacteria aggregation seems to be limited to Gram-negative bacteria as Laponite did not cause flocculation of Gram-positive Bacillus subtilis bacteria nor did it bind to lipoteichoic acid from bacterial envelopes. Taken together, the present investigation reports several novel phenomena by demonstrating that nanoparticle charge does not invariably control membrane destabilization and by identifying the ability of anionic Laponite nanoparticles to effectively flocculate Gram-negative bacteria through LPS binding. As demonstrated in cell experiments, such aggregation results in diminished LPS-induced cell activation, thus outlining a promising approach for confinement of infection and inflammation caused by such pathogens.
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| 8. |
- Saravanan, Rathi, et al.
(författare)
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Structural basis for endotoxin neutralisation and anti-inflammatory activity of thrombin-derived C-terminal peptides
- 2018
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Ingår i: Nature Communications. - : NATURE PUBLISHING GROUP. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Thrombin-derived C-terminal peptides (TCPs) of about 2 kDa are present in wounds, where they exert anti-endotoxic functions. Employing a combination of nuclear magnetic resonance spectroscopy (NMR), biophysical, mass spectrometry and cellular studies combined with in silico multiscale modelling, we here determine the bound conformation of HVF18 (HVFRLKKWIQKVIDQFGE), a TCP generated by neutrophil elastase, in complex with bacterial lipopolysaccharide (LPS) and define a previously undisclosed interaction between TCPs and human CD14. Further, we show that TCPs bind to the LPS-binding hydrophobic pocket of CD14 and identify the peptide region crucial for TCP interaction with LPS and CD14. Taken together, our results demonstrate the role of structural transitions in LPS complex formation and CD14 interaction, providing a molecular explanation for the previously observed therapeutic effects of TCPs in experimental models of bacterial sepsis and endotoxin shock.
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| 9. |
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| 10. |
- Abdillahi, Suado M., et al.
(författare)
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Collagen VI Contains Multiple Host Defense Peptides with Potent In Vivo Activity
- 2018
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Ingår i: Journal of Immunology. - : AMER ASSOC IMMUNOLOGISTS. - 0022-1767 .- 1550-6606. ; 201:3, s. 1007-1020
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Tidskriftsartikel (refereegranskat)abstract
- Collagen VI is a ubiquitous extracellular matrix component that forms extensive microfibrillar networks in most connective tissues. In this study, we describe for the first time, to our knowledge, that the collagen VI von Willebrand factor type A like domains exhibit a broad-spectrum antimicrobial activity against Gram-positive and Gram-negative bacteria in human skin infections in vivo. In silico sequence and structural analysis of VWA domains revealed that they contain cationic and amphipathic peptide sequence motifs, which might explain the antimicrobial nature of collagen VI. In vitro and in vivo studies show that these peptides exhibited significant antibacterial activity against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa through membrane disruption. Our findings shed new light on the role of collagen VI derived peptides in innate host defense and provide templates for development of peptide-based antibacterial therapies.
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| 11. |
- Banas, Magdalena, et al.
(författare)
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Chemerin Is an Antimicrobial Agent in Human Epidermis
- 2013
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:3
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Tidskriftsartikel (refereegranskat)abstract
- Chemerin, a chemoattractant ligand for chemokine-like receptor 1 (CMKLR1) is predicted to share similar tertiary structure with antibacterial cathelicidins. Recombinant chemerin has antimicrobial activity. Here we show that endogenous chemerin is abundant in human epidermis, and that inhibition of bacteria growth by exudates from organ cultures of primary human skin keratinocytes is largely chemerin-dependent. Using a panel of overlapping chemerin-derived synthetic peptides, we demonstrate that the antibacterial activity of chemerin is primarily mediated by Val 66 -Pro 85, which causes direct bacterial lysis. Therefore, chemerin is an antimicrobial agent in human skin.
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| 12. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 13. |
- Gela, Anele, et al.
(författare)
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Eotaxin-3 (CCL26) exerts innate host defense activities that are modulated by mast cell proteases
- 2015
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Ingår i: Allergy. - : Wiley. - 1398-9995 .- 0105-4538. ; 70:2, s. 161-170
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundDuring bacterial infections of the airways, a Th1-profiled inflammation promotes the production of several host defense proteins and peptides with antibacterial activities including -defensins, ELR-negative CXC chemokines, and the cathelicidin LL-37. These are downregulated by Th2 cytokines of the allergic response. Instead, the eosinophil-recruiting chemokines eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 are expressed. This study set out to investigate whether these chemokines could serve as innate host defense molecules during allergic inflammation. MethodsAntibacterial activities of the eotaxins were investigated using viable count assays, electron microscopy, and methods assessing bacterial permeabilization. Fragments generated by mast cell proteases were characterized, and their potential antibacterial, receptor-activating, and lipopolysaccharide-neutralizing activities were investigated. ResultsCCL11, CCL24, and CCL26 all showed potent bactericidal activity, mediated through membrane disruption, against the airway pathogens Streptococcus pneumoniae, Staphylococcus aureus, Nontypeable Haemophilus influenzae, and Pseudomonas aeruginosa. CCL26 retained bactericidal activity in the presence of salt at physiologic concentrations, and the region holding the highest bactericidal activity was the cationic and amphipathic COOH-terminus. Proteolysis of CCL26 by chymase and tryptase, respectively, released distinct fragments of the COOH- and NH2-terminal regions. The COOH-terminal fragment retained antibacterial activity while the NH2-terminal had potent LPS-neutralizing properties in the order of CCL26 full-length protein. An identical fragment to NH2-terminal fragment generated by tryptase was obtained after incubation with supernatants from activated mast cells. None of the fragments activated the CCR3-receptor. ConclusionsTaken together, the findings show that the eotaxins can contribute to host defense against common airway pathogens and that their activities are modulated by mast cell proteases.
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| 14. |
- Hansen, Finja C., et al.
(författare)
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Differential Internalization of Thrombin-Derived Host Defense Peptides into Monocytes and Macrophages
- 2022
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Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 14:5, s. 418-432
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Tidskriftsartikel (refereegranskat)abstract
- Proteolytic cleavage of thrombin generates C-terminal host defense peptides exerting multiple immunomodulatory effects in response to bacterial stimuli. Previously, we reported that thrombin-derived C-terminal peptides (TCPs) are internalized in monocytes and macrophages in a time- and temperature-dependent manner. In this study, we investigated which endocytosis pathways are responsible for the internalization of TCPs. Using confocal microscopy and flow cytometry, we show that both clathrin-dependent and clathrin-independent pathways are involved in the internalization of the prototypic TCP GKY25 in RAW264.7 and human monocyte-derived M1 macrophages, whereas the uptake of GKY25 in monocytic THP-1 cells is mainly dynamin-dependent. Internalized GKY25 was transported to endosomes and finally lysosomes, where it remained detectable for up to 10 h. Comparison of GKY25 uptake with that of the natural occurring TCPs HVF18 and FYT21 indicates that the pathway of TCP endocytosis is not only cell type-dependent but also depends on the length and composition of the peptide as well as the presence of LPS and bacteria. Finally, using neutron reflectometry, we show that the observed differences between HVF18 and the other 2 TCPs may be explained partially by differences in membrane insertion. Taken together, we show that TCPs are differentially internalized into monocytes and macrophages.
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| 15. |
- Hansen, Finja C., et al.
(författare)
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Thrombin-derived host-defense peptides modulate monocyte/macrophage inflammatory responses to gram-negative bacteria
- 2017
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 8:JUL
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Tidskriftsartikel (refereegranskat)abstract
- Host-defense peptides play a fundamental role in the innate immune system by modulating inflammatory responses. Previously, it was shown that the thrombin derived host-defense peptide GKY25 inhibits LPS-induced responses of monocytes and macrophages in vitro, ex vivo, and in vivo. In this study, the effect of GKY25 on the interaction of monocytes/macrophages with Gram-negative bacteria was explored. Electron microscopy analysis showed that fibrin slough from non-healing wounds, colonized with Staphylococcus aureus and Pseudomonas aeruginosa, contains C-terminal thrombin epitopes associated with these bacteria extracellularly and in phagosomes of leukocytes. Live imaging of RAW 264.7 cell cultures showed binding of GKY25 to Escherichia coli BioParticles extracellularly, and colocalization intracellularly. Although peptide binding did not alter the rate of phagocytosis, GKY25 reduced NF-κB/AP-1 activation and subsequent cytokine release in response to both heat-killed and live bacteria. Notably, preincubation of RAW 264.7 cells with peptide did increase BioParticle uptake in a dose-dependent manner. Taken together, the thrombin-derived host-defense peptide GKY25 binds to bacteria extracellularly and colocalizes with bacteria intracellularly, thereby reducing pro-inflammatory responses.
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| 16. |
- Hartman, Erik, et al.
(författare)
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Bioinformatic Analysis of the Wound Peptidome Reveals Potential Biomarkers and Antimicrobial Peptides
- 2021
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Wound infection is a common and serious medical condition with an unmet need for improved diagnostic tools. A peptidomic approach, aided by mass spectrometry and bioinformatics, could provide novel means of identifying new peptide biomarkers for wound healing and infection assessment. Wound fluid is suitable for peptidomic analysis since it is both intimately tied to the wound environment and is readily available. In this study we investigate the peptidomes of wound fluids derived from surgical drainages following mastectomy and from wound dressings following facial skin grafting. By applying sorting algorithms and open source third party software to peptidomic label free tandem mass spectrometry data we provide an unbiased general methodology for analyzing and differentiating between peptidomes. We show that the wound fluid peptidomes of patients are highly individualized. However, differences emerge when grouping the patients depending on wound type. Furthermore, the abundance of peptides originating from documented antimicrobial regions of hemoglobin in infected wounds may contribute to an antimicrobial wound environment, as determined by in silico analysis. We validate our findings by compiling literature on peptide biomarkers and peptides of physiological significance and cross checking the results against our dataset, demonstrating that well-documented peptides of immunological significance are abundant in infected wounds, and originate from certain distinct regions in proteins such as hemoglobin and fibrinogen. Ultimately, we have demonstrated the power using sorting algorithms and open source software to help yield insights and visualize peptidomic data.
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| 17. |
- Holdbrook, Daniel A., et al.
(författare)
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Influence of pH on the activity of thrombin-derived antimicrobial peptides
- 2018
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1860:11, s. 2374-2384
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Tidskriftsartikel (refereegranskat)abstract
- The wound environment is characterized by physiological pH changes. Proteolysis of thrombin by wound-derived proteases, such as neutrophil elastase, generates antimicrobial thrombin-derived C-terminal peptides (TCPs), such as HVF18 (HVFRLKKWIQKVIDQFGE). Presence of such TCPs in human wound fluids in vivo, as well as the occurrence of an evolutionarily conserved His residue in the primary amino acid sequence of TCPs, prompted us to investigate the pH-dependent antibacterial action of HVF18, as well as of the prototypic GKY25 (GKYGFYTHVFRLKKWIQKVIDQFGE). We show that protonation of this His residue at pH 5.5 increases the antibacterial activity of both TCPs against Gram-negative Escherichia coli by membrane disruption. Physiological salt level (150 mM NaCl) augments antibacterial activity of GKY25 but diminishes for the shorter HVF18. Replacing His with Leu or Ser in GKY25 abolishes the His protonation-dependent increase in antibacterial activity at pH 5.5, whereas substitution with Lys maintains activity at neutral (pH 7.4) and acidic pH. Interestingly, both TCPs display decreased binding affinities to human CD14 with decreasing pH, suggesting a likely switch in mode-of-action, from anti-inflammatory at neutral pH to antibacterial at acidic pH. Together, the results demonstrate that apart from structural prerequisites such as peptide length, charge, and hydrophobicity, the evolutionarily conserved His residue of TCPs influences their antibacterial effects and reveals a previously unknown aspect of TCPs biological action.
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| 18. |
- Malekkhaiat Häffner, Sara, et al.
(författare)
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Membrane interactions and antimicrobial effects of layered double hydroxide nanoparticles
- 2017
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Ingår i: Physical Chemistry, Chemical Physics - PCCP. - : ROYAL SOC CHEMISTRY. - 1463-9076 .- 1463-9084. ; 19:35, s. 23832-23842
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Tidskriftsartikel (refereegranskat)abstract
- Membrane interactions are critical for the successful use of inorganic nanoparticles as antimicrobial agents and as carriers of, or co-actives with, antimicrobial peptides (AMPs). In order to contribute to an increased understanding of these, we here investigate effects of particle size (42-208 nm) on layered double hydroxide (LDH) interactions with both bacteria-mimicking and mammalian-mimicking lipid membranes. LDH binding to bacteria-mimicking membranes, extraction of anionic lipids, as well as resulting membrane destabilization, was found to increase with decreasing particle size, also translating into size-dependent synergistic effects with the antimicrobial peptide LL-37. Due to strong interactions with anionic lipopolysaccharide and peptidoglycan layers, direct membrane disruption of both Gram-negative and Gram-positive bacteria is suppressed. However, LDH nanoparticles cause size-dependent charge reversal and resulting flocculation of both liposomes and bacteria, which may provide a mechanism for bacterial confinement or clearance. Taken together, these findings demonstrate a set of previously unknown behaviors, including synergistic membrane destabilization and dual confinement/killing of bacteria through combined LDH/AMP exposure, of potential therapeutic interest.
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| 19. |
- Manderson, Gavin, et al.
(författare)
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Interactions of histidine-rich glycoprotein with immunoglobulins and proteins of the complement system.
- 2009
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Ingår i: Molecular Immunology. - : Elsevier BV. - 1872-9142 .- 0161-5890. ; Aug 10, s. 3388-3398
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Tidskriftsartikel (refereegranskat)abstract
- This study describes how the serum protein histidine-rich glycoprotein (HRG) affects the complement system. We show that HRG binds strongly to several complement proteins: C1q, factor H and C4b-binding protein and that it is found complexed with these proteins in human sera and synovial fluids of rheumatoid arthritis patients. HRG also binds C8 and to a lesser extent mannose-binding lectin, C4 and C3. However, HRG alone neither activates nor inhibits complement. Both HRG and C1q bind to necrotic cells and increase their phagocytosis. We found that C1q competes weakly with HRG for binding to necrotic cells whilst HRG does not compete with C1q. Furthermore, HRG enhances complement activation on necrotic cells measured as deposition of C3b. We show that HRG inhibits the formation of immune complexes of ovalbumin/anti-ovalbumin, whilst the reverse holds for C1q. Immune complexes formed in the presence of HRG show enhanced complement activation, whilst those formed in the presence of C1q show diminished complement activation. Taken together, HRG may assist in the maintenance of normal immune function by mediating the clearance of necrotic material, inhibiting the formation of insoluble immune complexes and enhancing their ability to activate complement, resulting in faster clearance.
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| 20. |
- Mohanty, T., et al.
(författare)
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Saliva induces expression of antimicrobial peptides and promotes intracellular killing of bacteria in keratinocytes by epidermal growth factor receptor transactivation
- 2017
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Ingår i: British Journal of Dermatology. - : Oxford University Press (OUP). - 0007-0963 .- 1365-2133. ; 176:2, s. 403-412
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Tidskriftsartikel (refereegranskat)abstract
- Background: Wounds in the oral cavity, constantly exposed to both saliva and bacteria, heal quickly without infection. Furthermore, during licking of skin wounds, saliva promotes wound healing and plays a role in keeping the wound free of infection. Objectives: To investigate whether saliva induces expression of antimicrobial peptides (AMPs) in human epidermal keratinocytes and whether saliva promotes clearance of intracellular bacteria in these cells. Methods: Expression of AMPs was investigated in the oral mucosa and ex vivo injured skin by immunohistochemistry. Human beta-defensin-3 expression was investigated in epidermal keratinocytes after saliva stimulation, using real-time polymerase chain reaction and immunofluorescence. Results: We found higher expression of AMPs in the oral mucosa than in the epidermis. Saliva accelerated the injury-induced expression of AMPs in human skin ex vivo and was a potent inducer of the expression of AMPs in epidermal keratinocytes. The expression of AMPs was induced by metalloproteinase-dependent epidermal growth factor receptor (EGFR) transactivation mediated by a salivary lipid. Saliva increased the intracellular clearance of Staphylococcus aureus in keratinocytes through EGFR activation. Conclusions: These findings suggest a previously unreported role of saliva in innate immunity and demonstrate for the first time that saliva induces gene expression in epidermal keratinocytes.
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| 21. |
- Nordström, Randi, 1986-, et al.
(författare)
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Degradable dendritic nanogels as carriers for antimicrobial peptides
- 2019
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Ingår i: Journal of Colloid and Interface Science. - : Elsevier BV. - 0021-9797 .- 1095-7103. ; 554, s. 592-602
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Tidskriftsartikel (refereegranskat)abstract
- In the present study, we investigate degradable anionic dendritic nanogels (DNG) as carriers for antimicrobial peptides (AMPs). In such systems, the dendritic part contains carboxylic acid-based anionic binding sites for cationic AMPs, whereas linear poly(ethylene glycol) (PEG) chains form a shell for promotion of biological stealth. In order to clarify factors influencing membrane interactions of such systems, we here address effects of nanogel charge, cross-linking, and degradation on peptide loading/release, as well as consequences of these factors for lipid membrane interactions and antimicrobial effects. The DNGs were found to bind the AMPs LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) and DPK-060 (GKHKNKGKKNGKHNGWKWWW). For the smaller DPK-060 peptide, loading was found to increase with increasing nanogel charge density. For the larger LL-37, on the other hand, peptide loading was largely insensitive to nanogel charge density. In line with this, results on the secondary structure, as well as on the absence of stabilization from proteolytic degradation by the nanogels, show that the larger LL-37 is unable to enter into the interior of the nanogels. While 40–60% nanogel degradation occurred over 10 days, promoted at high ionic strength and lower cross-linking density/higher anionic charge content, peptide release at physiological ionic strength was substantially faster, and membrane destabilization not relying on nanogel degradation. Ellipsometry and liposome leakage experiments showed both free peptide and peptide/DNG complexes to cause membrane destabilization, indicated also by antimicrobial activities being comparable for nanogel-bound and free peptide. Finally, the DNGs were demonstrated to display low toxicity towards erythrocytes even at peptide concentrations of 100 µM.
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| 22. |
- Nordström, Randi, et al.
(författare)
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Membrane interactions of microgels as carriers of antimicrobial peptides
- 2018
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Ingår i: Journal of Colloid and Interface Science. - : Academic Press Inc.. - 0021-9797 .- 1095-7103. ; 513, s. 141-150
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Tidskriftsartikel (refereegranskat)abstract
- Microgels are interesting as potential delivery systems for antimicrobial peptides. In order to elucidate membrane interactions of such systems, we here investigate effects of microgel charge density on antimicrobial peptide loading and release, as well as consequences of this for membrane interactions and antimicrobial effects, using ellipsometry, circular dichroism spectroscopy, nanoparticle tracking analysis, dynamic light scattering and z-potential measurements. Anionic poly(ethyl acrylate-co-methacrylic acid) microgels were found to incorporate considerable amounts of the cationic antimicrobial peptides LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) and DPK-060 (GKHKNKGKKNGKHNGWKWWW) and to protect incorporated peptides from degradation by infection-related proteases at high microgel charge density. As a result of their net negative z-potential also at high peptide loading, neither empty nor peptide-loaded microgels adsorb at supported bacteria-mimicking membranes. Instead, membrane disruption is mediated almost exclusively by peptide release. Mirroring this, antimicrobial effects against several clinically relevant bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Pseudomonas aeruginosa) were found to be promoted by factors facilitating peptide release, such as decreasing peptide length and decreasing microgel charge density. Microgels were further demonstrated to display low toxicity towards erythrocytes. Taken together, the results demonstrate some interesting opportunities for the use of microgels as delivery systems for antimicrobial peptides, but also highlight several key factors which need to be controlled for their successful use.
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| 23. |
- Nyström, Lina, et al.
(författare)
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Peptide-Loaded Microgels as Antimicrobial and Anti-Inflammatory Surface Coatings
- 2018
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Ingår i: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 19:8, s. 3456-3466
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Tidskriftsartikel (refereegranskat)abstract
- Here we report on covalently immobilized poly(ethyl acrylate- co-methacrylic acid) microgels loaded with the host defense peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR), which is derived from human heparin cofactor II, as well as its poly(ethylene glycol)-conjugated (PEGylated) version, KYE28PEG. Peptide loading and release, as well as the consequences of these processes on the microgel and peptide properties, were studied by in situ ellipsometry, confocal microscopy, zeta potential measurements, and circular dichroism spectroscopy. The results show that the microgel-peptide interactions are electrostatically dominated, thus promoted at higher microgel charge density, while PEGylation suppresses peptide binding. PEGylation also enhances the α-helix induction observed for KYE28 upon microgel incorporation. Additionally, peptide release is facilitated at physiological salt concentration, particularly so for KYE28PEG, which illustrates the importance of electrostatic interactions. In vitro studies on Escherichia coli show that the microgel-modified surfaces display potent antifouling properties in both the absence and presence of the incorporated peptide. While contact killing dominates at low ionic strength for the peptide-loaded microgels, released peptides also provide antimicrobial activity in bulk at a high ionic strength. Additionally, KYE28- and KYE28PEG-loaded microgels display anti-inflammatory effects on human monocytes. Taken together, these results not only show that surface-bound microgels offer an interesting approach for local drug delivery of host defense peptides but also illustrate the need to achieve high surface loads of peptides for efficient biological effects.
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| 24. |
- Petrlova, Jitka, et al.
(författare)
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Aggregation of thrombin-derived C-terminal fragments as a previously undisclosed host defense mechanism
- 2017
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 114:21, s. E4213-E4222
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Tidskriftsartikel (refereegranskat)abstract
- Effective control of endotoxins and bacteria is crucial for normal wound healing. During injury, the key enzyme thrombin is formed, leading to generation of fibrin. Here, we show that human neutrophil elastase cleaves thrombin, generating 11-kDa thrombin-derived C-terminal peptides (TCPs), which bind to and form amorphous amyloid-like aggregates with both bacterial lipopolysaccharide (LPS) and gram-negative bacteria. In silico molecular modeling using atomic resolution and coarse-grained simulations corroborates our experimental observations, altogether indicating increased aggregation through LPS-mediated intermolecular contacts between clusters of TCP molecules. Upon bacterial aggregation, recombinantly produced TCPs induce permeabilization of Escherichia coli and phagocytic uptake. TCPs of about 11 kDa are present in acute wound fluids as well as in fibrin sloughs from patients with infected wounds. We noted aggregation and colocalization of LPS with TCPs in such fibrin material, which indicates the presence of TCP-LPS aggregates under physiological conditions. Apart from identifying a function of proteolyzed thrombin and its fragments, our findings provide an interesting link between the coagulation system, innate immunity, LPS scavenging, and protein aggregation/amyloid formation.
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| 25. |
- Petrlova, Jitka, et al.
(författare)
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Thrombin-derived C-terminal fragments aggregate and scavenge bacteria and their proinflammatory products
- 2020
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 295:11, s. 3417-3430
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Tidskriftsartikel (refereegranskat)abstract
- Thrombin-derived C-terminal peptides (TCPs), including a major 11-kDa fragment (TCP96), are produced through cleavage by human neutrophil elastase and aggregate lipopolysaccharide (LPS) and the Gram-negative bacterium Escherichia coli. However, the physiological roles of TCP96 in controlling bacterial infections and reducing LPS-induced inflammation are unclear. Here, using various biophysical methods, in silico molecular modeling, microbiological and cellular assays, and animal models, we examined the structural features and functional roles of recombinant TCP96 (rTCP96) in the aggregation of multiple bacteria and the Toll-like receptor (TLR) agonists they produce. We found that rTCP96 aggregates both Gram-negative and Gram-positive bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa, and their cell-wall components LPS, lipid A, and lipoteichoic acid (LTA). The Gram-negative bacteria E. coli and P. aeruginosa were particularly sensitive to aggregation-induced bacterial permeabilization and killing. As a proof of concept, we show that rTCP96 reduces LPS-induced NF-κB activation in human monocytes, as well as in mouse models of LPS-induced subcutaneous inflammation. Moreover, in a mouse model of subcutaneous inoculation with P. aeruginosa, rTCP96 reduced bacterial levels. Together, these results link TCP-mediated aggregation of endotoxins and bacteria in vitro to attenuation of inflammation and bacterial levels in vivo.
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| 26. |
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| 27. |
- Saravanan, Rathi, et al.
(författare)
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Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo
- 2017
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.
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| 28. |
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| 29. |
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| 30. |
- Strömstedt, Adam A., et al.
(författare)
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Evaluation of strategies for improving proteolytic resistance of antimicrobial peptides by using variants of EFK17, an internal segment of LL-37
- 2009
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Ingår i: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 53:2, s. 593-602
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Tidskriftsartikel (refereegranskat)abstract
- Methods for increasing the proteolytic stability of EFK17 (EFKRIVQRIKDFLRNLV), a new peptide sequence with antimicrobial properties derived from LL-37, were evaluated. EFK17 was modified by four d-enantiomer or tryptophan (W) substitutions at known protease cleavage sites as well as by terminal amidation and acetylation. The peptide variants were studied in terms of proteolytic resistance, antibacterial potency, and cytotoxicity but also in terms their adsorption at model lipid membranes, liposomal leakage generation, and secondary-structure behavior. The W substitutions resulted in a marked reduction in the proteolytic degradation caused by human neutrophil elastase, Staphylococcus aureus aureolysin, and V8 protease but not in the degradation caused by Pseudomonas aeruginosa elastase. For the former two endoproteases, amidation and acetylation of the terminals also reduced proteolytic degradation but only when used in combination with W substitutions. The d-enantiomer substitutions rendered the peptides indigestible by all four proteases; however, those peptides displayed little antimicrobial potency. The W- and end-modified peptides, on the other hand, showed an increased bactericidal potency compared to that of the native peptide sequence, coupled with a moderate cytotoxicity that was largely absent in serum. The bactericidal, cytotoxic, and liposome lytic properties correlated with each other as well as with the amount of peptide adsorbed at the lipid membrane and the extent of helix formation associated with the adsorption. The lytic properties of the W-substituted peptides were less impaired by increased ionic strength, presumably by a combination of W-mediated stabilization of the largely amphiphilic helix conformation and a nonelectrostatic W affinity for the bilayer interface. Overall, W substitutions constitute an interesting means to reduce the proteolytic susceptibility of EFK17 while also improving antimicrobial performance.
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| 31. |
- Strömstedt, Adam A., et al.
(författare)
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Interaction between amphiphilic peptides and phospholipid membranes
- 2010
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Ingår i: Current Opinion in Colloid & Interface Science. - : Elsevier BV. - 1359-0294 .- 1879-0399. ; 15:6, s. 467-478
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Forskningsöversikt (refereegranskat)abstract
- This brief review aims at providing some illustrative examples on the interaction between amphiphilic peptides and phospholipid membranes an area of significant current interest Focusing on antimicrobial peptides factors affecting peptide-membrane interactions are addressed including effects of peptide length charge hydrophobicity secondary structure and topology Effects of membrane composition are also illustrated including effects of membrane charge nature of the polar headgroup and presence of cholesterol and other sterols Throughout novel insights on the importance of peptide adsorption density on membrane stability are emphasized as is the correlation between peptide adsorption peptide induced leakage in model liposome systems peptide-induced lysis of bacteria and bacteria killing.
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| 32. |
- Strömstedt, Adam A., 1977-, et al.
(författare)
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Oligotryptophan-tagged antimicrobial peptides and the role of the cationic sequence
- 2009
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier BV. - 0005-2736 .- 1879-2642. ; 1788:9, s. 1916-1923
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Tidskriftsartikel (refereegranskat)abstract
- The effects of varying the cationic sequence of oligotryptophan-tagged antimicrobial peptides were investigated in terms of peptide adsorption to model lipid membranes, liposome leakage induction, and antibacterial potency. Heptamers of lysine (K7) and arginine (R7) were lytic against Escherichia coli bacteria at low ionic strength. In parallel, both peptides adsorbed on to bilayers formed by E. coli phospholipids, and caused leakage in the corresponding liposomes. K7 was the more potent of the two peptides in causing liposome leakage, although the adsorption of this peptide on E. coli membranes was lower than that of R7. The bactericidal effect, liposome lysis, and membrane adsorption were all substantially reduced at physiological ionic strength. When a tryptophan pentamer tag was linked to the C-terminal end of these peptides, substantial peptide adsorption, membrane lysis, and bacterial killing was observed also at high ionic strength, and also for a peptide of lower cationic charge density (KNKGKKN-W5). Strikingly, the order of membrane lytic potential of the cationic peptides investigated was reversed when tagged. This and other aspects of peptide behavior and adsorption, in conjunction with effects on liposomes and bacteria, suggest that tagged and untagged peptides act by different lytic mechanisms, which to some extent counterbalance each other. Thus, while the untagged peptides act by generating negative curvature strain in the phospholipid membrane, the tagged peptides cause positive curvature strain. The tagged heptamer of arginine, R7W5, was the best candidate for E. coli membrane lysis at physiological salt conditions and proved to be an efficient antibacterial agent.
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| 33. |
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| 34. |
- van der Plas, Mariena J.A., et al.
(författare)
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Method development and characterisation of the low-molecular-weight peptidome of human wound fluids
- 2021
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Ingår i: eLife. - 2050-084X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- The normal wound healing process is characterised by proteolytic events, whereas infection results in dysfunctional activations by endogenous and bacterial proteases. Peptides, downstream reporters of these proteolytic actions, could therefore serve as a promising tool for diagnosis of wounds. Using mass-spectrometry analyses, we here for the first time characterise the peptidome of human wound fluids. Sterile post-surgical wound fluids were found to contain a high degree of peptides in comparison to human plasma. Analyses of the peptidome from uninfected healing wounds and Staphylococcus aureus-infected wounds identify unique peptide patterns of various proteins, including coagulation and complement factors, proteases, and antiproteinases. Together, the work defines a workflow for analysis of peptides derived from wound fluids and demonstrates a proof-of-concept that such fluids can be used for analysis of qualitative differences of peptide patterns from larger patient cohorts, providing potential biomarkers for wound healing and infection.
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