| 1. |
- Kovalevsky, Andrey, et al.
(författare)
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"To Be or Not to Be" Protonated : Atomic Details of Human Carbonic Anhydrase-Clinical Drug Complexes by Neutron Crystallography and Simulation
- 2018
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Ingår i: Structure. - : Elsevier BV. - 0969-2126. ; 26:3, s. 3-390
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Tidskriftsartikel (refereegranskat)abstract
- Human carbonic anhydrases (hCAs) play various roles in cells, and have been drug targets for decades. Sequence similarities of hCA isoforms necessitate designing specific inhibitors, which requires detailed structural information for hCA-inhibitor complexes. We present room temperature neutron structures of hCA II in complex with three clinical drugs that provide in-depth analysis of drug binding, including protonation states of the inhibitors, hydration water structure, and direct visualization of hydrogen-bonding networks in the enzyme's active site. All sulfonamide inhibitors studied bind to the Zn metal center in the deprotonated, anionic, form. Other chemical groups of the drugs can remain neutral or be protonated when bound to hCA II. MD simulations have shown that flexible functional groups of the inhibitors may alter their conformations at room temperature and occupy different sub-sites. This study offers insights into the design of specific drugs to target cancer-related hCA isoform IX. Kovalevsky et al. used macromolecular neutron crystallography and molecular dynamics simulations to obtain a detailed picture of clinical inhibitors binding to human carbonic anhydrase II. The study visualized hydrogen atom positions, revealing protonation/deprotonation events and intricate hydrogen-bonding networks, providing insights for drug design.
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| 2. |
- Blakeley, Matthew, et al.
(författare)
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Lectin-Functionalized Polyethylene Glycol for Relief of Mucosal Dryness
- 2022
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Ingår i: Advanced Healthcare Materials. - : Wiley. - 2192-2640 .- 2192-2659. ; 11:2
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Tidskriftsartikel (refereegranskat)abstract
- The importance of lubrication between oral surfaces provided by the salivary film is most acutely apparent when it is disrupted, a prevalent consequence of salivary gland hypofunction experienced with aging, a symptom of certain diseases, or a side effect of some medical interventions. Sufferers report difficulty with speech and oral food processing and collectively is detrimental to quality of life. Polyethylene glycol (PEG) is widely employed as a successful biocompatible boundary lubricant in engineering and biomedical applications. It is hypothesized that the immobilization of PEG to biological materials such as oral epithelial cells and tissue can mimic the salivary film and provide durable relief from the symptoms of mucosal dryness. To do so, PEG is functionalized with a sugar binding lectin (wheat germ agglutinin) to enhance epithelial adhesion through lectin-sugar interactions. Retention and lubricity are characterized on an ex vivo oral tissue tribology rig. WGA-PEG coats and retains on mucin films, oral epithelial cells, and porcine tongue tissue, and offers sustained reduction in coefficient of friction (COF). WGA-PEG could be developed into a useful topical treatment for reducing oral friction and the perception of dry mouth.
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| 3. |
- Drago, Victoria N., et al.
(författare)
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Microgravity crystallization of perdeuterated tryptophan synthase for neutron diffraction
- 2022
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Ingår i: npj Microgravity. - : Springer Science and Business Media LLC. - 2373-8065. ; 8:1
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Tidskriftsartikel (refereegranskat)abstract
- Biologically active vitamin B6-derivative pyridoxal 5′-phosphate (PLP) is an essential cofactor in amino acid metabolic pathways. PLP-dependent enzymes catalyze a multitude of chemical reactions but, how reaction diversity of PLP-dependent enzymes is achieved is still not well understood. Such comprehension requires atomic-level structural studies of PLP-dependent enzymes. Neutron diffraction affords the ability to directly observe hydrogen positions and therefore assign protonation states to the PLP cofactor and key active site residues. The low fluxes of neutron beamlines require large crystals (≥0.5 mm3). Tryptophan synthase (TS), a Fold Type II PLP-dependent enzyme, crystallizes in unit gravity with inclusions and high mosaicity, resulting in poor diffraction. Microgravity offers the opportunity to grow large, well-ordered crystals by reducing gravity-driven convection currents that impede crystal growth. We developed the Toledo Crystallization Box (TCB), a membrane-barrier capillary-dialysis device, to grow neutron diffraction-quality crystals of perdeuterated TS in microgravity. Here, we present the design of the TCB and its implementation on Center for Advancement of Science in Space (CASIS) supported International Space Station (ISS) Missions Protein Crystal Growth (PCG)-8 and PCG-15. The TCB demonstrated the ability to improve X-ray diffraction and mosaicity on PCG-8. In comparison to ground control crystals of the same size, microgravity-grown crystals from PCG-15 produced higher quality neutron diffraction data. Neutron diffraction data to a resolution of 2.1 Å has been collected using microgravity-grown perdeuterated TS crystals from PCG-15.
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| 4. |
- Drago, Victoria N., et al.
(författare)
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Neutron diffraction from a microgravity-grown crystal reveals the active site hydrogens of the internal aldimine form of tryptophan synthase
- 2024
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Ingår i: Cell Reports Physical Science. - 2666-3864. ; 5:2
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Tidskriftsartikel (refereegranskat)abstract
- Pyridoxal 5′-phosphate (PLP), the biologically active form of vitamin B6, is an essential cofactor in many biosynthetic pathways. The emergence of PLP-dependent enzymes as drug targets and biocatalysts, such as tryptophan synthase (TS), has underlined the demand to understand PLP-dependent catalysis and reaction specificity. The ability of neutron diffraction to resolve the positions of hydrogen atoms makes it an ideal technique to understand how the electrostatic environment and selective protonation of PLP regulates PLP-dependent activities. Facilitated by microgravity crystallization of TS with the Toledo Crystallization Box, we report the 2.1 Å joint X-ray/neutron (XN) structure of TS with PLP in the internal aldimine form. Positions of hydrogens were directly determined in both the α- and β-active sites, including PLP cofactor. The joint XN structure thus provides insight into the selective protonation of the internal aldimine and the electrostatic environment of TS necessary to understand the overall catalytic mechanism.
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| 5. |
- Gajdos, Lukas, et al.
(författare)
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Neutron crystallography reveals mechanisms used by Pseudomonas aeruginosa for host-cell binding
- 2022
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The opportunistic pathogen Pseudomonas aeruginosa, a major cause of nosocomial infections, uses carbohydrate-binding proteins (lectins) as part of its binding to host cells. The fucose-binding lectin, LecB, displays a unique carbohydrate-binding site that incorporates two closely located calcium ions bridging between the ligand and protein, providing specificity and unusually high affinity. Here, we investigate the mechanisms involved in binding based on neutron crystallography studies of a fully deuterated LecB/fucose/calcium complex. The neutron structure, which includes the positions of all the hydrogen atoms, reveals that the high affinity of binding may be related to the occurrence of a low-barrier hydrogen bond induced by the proximity of the two calcium ions, the presence of coordination rings between the sugar, calcium and LecB, and the dynamic behaviour of bridging water molecules at room temperature. These key structural details may assist in the design of anti-adhesive compounds to combat multi-resistance bacterial infections.
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| 6. |
- Kelpšas, Vinardas, et al.
(författare)
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Neutron structures of Leishmania mexicana triosephosphate isomerase in complex with reaction-intermediate mimics shed light on the proton-shuttling steps
- 2021
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Ingår i: IUCrJ. - 2052-2525. ; 8:Pt 4, s. 633-643
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Tidskriftsartikel (refereegranskat)abstract
- Triosephosphate isomerase (TIM) is a key enzyme in glycolysis that catalyses the interconversion of glyceraldehyde 3-phosphate and dihydroxy-acetone phosphate. This simple reaction involves the shuttling of protons mediated by protolysable side chains. The catalytic power of TIM is thought to stem from its ability to facilitate the deprotonation of a carbon next to a carbonyl group to generate an enediolate intermediate. The enediolate intermediate is believed to be mimicked by the inhibitor 2-phosphoglycolate (PGA) and the subsequent enediol intermediate by phosphoglycolohydroxamate (PGH). Here, neutron structures of Leishmania mexicana TIM have been determined with both inhibitors, and joint neutron/X-ray refinement followed by quantum refinement has been performed. The structures show that in the PGA complex the postulated general base Glu167 is protonated, while in the PGH complex it remains deprotonated. The deuteron is clearly localized on Glu167 in the PGA-TIM structure, suggesting an asymmetric hydrogen bond instead of a low-barrier hydrogen bond. The full picture of the active-site protonation states allowed an investigation of the reaction mechanism using density-functional theory calculations.
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| 7. |
- Kelpšas, Vinardas, et al.
(författare)
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Perdeuteration, large crystal growth and neutron data collection of Leishmania mexicana triose-phosphate isomerase E65Q variant
- 2019
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Ingår i: Acta crystallographica. Section F, Structural biology communications. - 2053-230X. ; 75:4, s. 260-269
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Tidskriftsartikel (refereegranskat)abstract
- Triose-phosphate isomerase (TIM) catalyses the interconversion of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. Two catalytic mechanisms have been proposed based on two reaction-intermediate analogues, 2-phosphoglycolate (2PG) and phosphoglycolohydroxamate (PGH), that have been used as mimics of the cis-enediol(ate) intermediate in several studies of TIM. The protonation states that are critical for the mechanistic interpretation of these structures are generally not visible in the X-ray structures. To resolve these questions, it is necessary to determine the hydrogen positions using neutron crystallography. Neutron crystallography requires large crystals and benefits from replacing all hydrogens with deuterium. Leishmania mexicana triose-phosphate isomerase was therefore perdeuterated and large crystals with 2PG and PGH were produced. Neutron diffraction data collected from two crystals with different volumes highlighted the importance of crystal volume, as smaller crystals required longer exposures and resulted in overall worse statistics.
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| 8. |
- Koruza, Katarina, et al.
(författare)
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Using neutron crystallography to elucidate the basis of selective inhibition of carbonic anhydrase by saccharin and a derivative
- 2019
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Ingår i: Journal of Structural Biology. - : Elsevier BV. - 1047-8477. ; 205:2, s. 147-154
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Tidskriftsartikel (refereegranskat)abstract
- Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IXmimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IXmimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.
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| 9. |
- Manzoni, Francesco, et al.
(författare)
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Elucidation of Hydrogen Bonding Patterns in Ligand-Free, Lactose- and Glycerol-Bound Galectin-3C by Neutron Crystallography to Guide Drug Design
- 2018
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 1520-4804 .- 0022-2623. ; 61:10, s. 4412-4420
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Tidskriftsartikel (refereegranskat)abstract
- The medically important drug target galectin-3 binds galactose-containing moieties on glycoproteins through an intricate pattern of hydrogen bonds to a largely polar surface-exposed binding site. All successful inhibitors of galectin-3 to date have been based on mono- or disaccharide cores closely resembling natural ligands. A detailed understanding of the H-bonding networks in these natural ligands will provide an improved foundation for the design of novel inhibitors. Neutron crystallography is an ideal technique to reveal the geometry of hydrogen bonds because the positions of hydrogen atoms are directly detected rather than being inferred from the positions of heavier atoms as in X-ray crystallography. We present three neutron crystal structures of the C-terminal carbohydrate recognition domain of galectin-3: the ligand-free form and the complexes with the natural substrate lactose and with glycerol, which mimics important interactions made by lactose. The neutron crystal structures reveal unambiguously the exquisite fine-tuning of the hydrogen bonding pattern in the binding site to the natural disaccharide ligand. The ligand-free structure shows that most of these hydrogen bonds are preserved even when the polar groups of the ligand are replaced by water molecules. The protonation states of all histidine residues in the protein are also revealed and correlate well with NMR observations. The structures give a solid starting point for molecular dynamics simulations and computational estimates of ligand binding affinity that will inform future drug design.
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| 10. |
- Manzoni, Francesco, et al.
(författare)
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Perdeuteration, crystallization, data collection and comparison of five neutron diffraction data sets of complexes of human galectin-3C
- 2016
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Ingår i: Acta Crystallographica Section D: Structural Biology. - 2059-7983. ; 72:11, s. 1194-1202
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Tidskriftsartikel (refereegranskat)abstract
- Galectin-3 is an important protein in molecular signalling events involving carbohydrate recognition, and an understanding of the hydrogen-bonding patterns in the carbohydrate-binding site of its C-terminal domain (galectin-3C) is important for the development of new potent inhibitors. The authors are studying these patterns using neutron crystallography. Here, the production of perdeuterated human galectin-3C and successive improvement in crystal size by the development of a crystal-growth protocol involving feeding of the crystallization drops are described. The larger crystals resulted in improved data quality and reduced data-collection times. Furthermore, protocols for complete removal of the lactose that is necessary for the production of large crystals of apo galectin-3C suitable for neutron diffraction are described. Five data sets have been collected at three different neutron sources from galectin-3C crystals of various volumes. It was possible to merge two of these to generate an almost complete neutron data set for the galectin-3C-lactose complex. These data sets provide insights into the crystal volumes and data-collection times necessary for the same system at sources with different technologies and data-collection strategies, and these insights are applicable to other systems.Perdeuteration, purification and the growth of large crystals of the carbohydrate-recognition domain of galectin-3C are described. Five neutron diffraction data sets have been collected at four neutron sources; these are compared and two are merged.
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| 11. |
- Marczynski, Matthias, et al.
(författare)
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Structural Alterations of Mucins Are Associated with Losses in Functionality
- 2021
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Ingår i: Biomacromolecules. - : AMER CHEMICAL SOC. - 1525-7797 .- 1526-4602. ; 22:4, s. 1600-1613
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Tidskriftsartikel (refereegranskat)abstract
- Commercial mucin glycoproteins are routinely used as a model to investigate the broad range of important functions mucins fulfill in our bodies, including lubrication, protection against hostile germs, and the accommodation of a healthy microbiome. Moreover, purified mucins are increasingly selected as building blocks for multifunctional materials, i.e., as components of hydrogels or coatings. By performing a detailed side-by-side comparison of commercially available and lab-purified variants of porcine gastric mucins, we decipher key molecular motifs that are crucial for mucin functionality. As two main structural features, we identify the hydrophobic termini and the hydrophilic glycosylation pattern of the mucin glycoprotein; moreover, we describe how alterations in those structural motifs affect the different properties of mucins-on both microscopic and macroscopic levels. This study provides a detailed understanding of how distinct functionalities of gastric mucins are established, and it highlights the need for high-quality mucins-for both basic research and the development of mucin-based medical products.
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| 12. |
- Oksanen, Esko, et al.
(författare)
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The neutron structure of urate oxidase resolves a long-standing mechanistic conundrum and reveals unexpected changes in protonation.
- 2014
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Urate oxidase transforms uric acid to 5-hydroxyisourate without the help of cofactors, but the catalytic mechanism has remained enigmatic, as the protonation state of the substrate could not be reliably deduced. We have determined the neutron structure of urate oxidase, providing unique information on the proton positions. A neutron crystal structure inhibited by a chloride anion at 2.3 Å resolution shows that the substrate is in fact 8-hydroxyxanthine, the enol tautomer of urate. We have also determined the neutron structure of the complex with the inhibitor 8-azaxanthine at 1.9 Å resolution, showing the protonation states of the K10-T57-H256 catalytic triad. Together with X-ray data and quantum chemical calculations, these structures allow us to identify the site of the initial substrate protonation and elucidate why the enzyme is inhibited by a chloride anion.
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| 13. |
- Sørensen, H. V., et al.
(författare)
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Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase
- 2023
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Ingår i: ACS Omega. - 2470-1343. ; 8:32, s. 29101-29112
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Tidskriftsartikel (refereegranskat)abstract
- Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen’s large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.
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| 14. |
- Yan, Hongji, et al.
(författare)
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Immune-Informed Mucin Hydrogels Evade Fibrotic Foreign Body Response In Vivo
- 2019
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Ingår i: Advanced Functional Materials. - : WILEY-V C H VERLAG GMBH. - 1616-301X .- 1616-3028.
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Tidskriftsartikel (refereegranskat)abstract
- The immune-mediated foreign body response to biomaterial implants can trigger the formation of insulating fibrotic capsules that can compromise implant function. To address this challenge, the intrinsic bioactivity of the mucin biopolymer, a heavily glycosylated protein that forms the protective mucus gel covering mucosal epithelia, is leveraged. By using a bioorthogonal inverse electron demand Diels-Alder reaction, mucins are crosslinked into implantable hydrogels. It is shown that mucin hydrogels (Muc-gels) modulate the immune response driving biomaterial-induced fibrosis. Muc-gels do not elicit fibrosis 21 days after implantation in the peritoneal cavity of C57Bl/6 mice, whereas medical-grade alginate hydrogels are covered by fibrous tissues. Further, Muc-gels dampen the recruitment of innate and adaptive immune cells to the gel and trigger a pattern of very mild activation marked by a noticeably low expression of the fibrosis-stimulating transforming growth factor beta 1 cytokine. Macrophages recruited to Muc-gels upregulate the gene expression of the protein inhibitor of activated STAT 1 (PIAS1) and SH2-containing phosphatase 1 (SHP-1) cytokine regulatory proteins, which likely contributes to their low cytokine expression profiles. With this advance in mucin materials, an essential tool is provided to better understand mucin bioactivities and to initiate the development of new mucin-based and mucin-inspired "immune-informed" materials for implantable devices subject to fibrotic encapsulation.
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