| 1. |
- Abelein, A, et al.
(author)
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High-yield Production of Amyloid-β Peptide Enabled by a Customized Spider Silk Domain
- 2020
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In: Scientific reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 10:1, s. 235-
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Journal article (peer-reviewed)abstract
- During storage in the silk gland, the N-terminal domain (NT) of spider silk proteins (spidroins) keeps the aggregation-prone repetitive region in solution at extreme concentrations. We observe that NTs from different spidroins have co-evolved with their respective repeat region, and now use an NT that is distantly related to previously used NTs, for efficient recombinant production of the amyloid-β peptide (Aβ) implicated in Alzheimer’s disease. A designed variant of NT from Nephila clavipes flagelliform spidroin, which in nature allows production and storage of β-hairpin repeat segments, gives exceptionally high yields of different human Aβ variants as a solubility tag. This tool enables efficient production of target peptides also in minimal medium and gives up to 10 times more isotope-labeled monomeric Aβ peptides per liter bacterial culture than previously reported.
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| 2. |
- Ahlström, J. Zebialowicz, et al.
(author)
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Synthetic surfactant with a recombinant surfactant protein C analogue improves lung function and attenuates inflammation in a model of acute respiratory distress syndrome in adult rabbits
- 2019
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In: Respiratory Research. - : BMC. - 1465-9921 .- 1465-993X. ; 20
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Journal article (peer-reviewed)abstract
- AimIn acute respiratory distress syndrome (ARDS) damaged alveolar epithelium, leakage of plasma proteins into the alveolar space and inactivation of pulmonary surfactant lead to respiratory dysfunction. Lung function could potentially be restored with exogenous surfactant therapy, but clinical trials have so far been disappointing. These negative results may be explained by inactivation and/or too low doses of the administered surfactant. Surfactant based on a recombinant surfactant protein C analogue (rSP-C33Leu) is easy to produce and in this study we compared its effects on lung function and inflammation with a commercial surfactant preparation in an adult rabbit model of ARDS.MethodsARDS was induced in adult New Zealand rabbits by mild lung-lavages followed by injurious ventilation (V-T 20m/kg body weight) until P/F ratio<26.7kPa. The animals were treated with two intratracheal boluses of 2.5mL/kg of 2% rSP-C33Leu in DPPC/egg PC/POPG, 50:40:10 or poractant alfa (Curosurf (R)), both surfactants containing 80mg phospholipids/mL, or air as control. The animals were subsequently ventilated (V-T 8-9m/kg body weight) for an additional 3h and lung function parameters were recorded. Histological appearance of the lungs, degree of lung oedema and levels of the cytokines TNF alpha IL-6 and IL-8 in lung homogenates were evaluated.ResultsBoth surfactant preparations improved lung function vs. the control group and also reduced inflammation scores, production of pro-inflammatory cytokines, and formation of lung oedema to similar degrees. Poractant alfa improved compliance at 1h, P/F ratio and PaO2 at 1.5h compared to rSP-C33Leu surfactant.ConclusionThis study indicates that treatment of experimental ARDS with synthetic lung surfactant based on rSP-C33Leu improves lung function and attenuates inflammation.
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| 3. |
- Al Adwani, Salma, et al.
(author)
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Citrullination Alters the Antibacterial and Anti-Inflammatory Functions of the Host Defense Peptide Canine Cathelicidin K9CATH In Vitro
- 2021
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In: Journal of Immunology. - : The American Association of Immunologists. - 0022-1767 .- 1550-6606. ; 207:3, s. 974-984
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Journal article (peer-reviewed)abstract
- K9CATH is the sole cathelicidin in canines (dogs) and exhibits broad antimicrobial activity against both Gram-positive and Gram-negative bacteria. K9CATH also modulates inflammatory responses and binds to LPS. These activities depend on the secondary structure and a net-positive charge of the peptide. Peptidylarginine deiminases (PAD) convert cationic peptidyl arginine to neutral citrulline. Thus, we hypothesized that citrullination is a biologically relevant modification of the peptide that would reduce the antibacterial and LPS-binding activities of K9CATH. Recombinant PAD2 and PAD4 citrullinated K9CATH to various extents and circular dichroism spectroscopy revealed that both native and citrullinated K9CATH exhibited similar α-helical secondary structures. Notably, citrullination of K9CATH reduced its bactericidal activity, abolished its ability to permeabilize the membrane of Gram-negative bacteria and reduced the hemolytic capacity. Electron microscopy showed that citrullinated K9CATH did not cause any morphological changes of Gram-negative bacteria, whereas the native peptide caused clear alterations of membrane integrity, concordant with a rapid bactericidal effect. Finally, citrullination of K9CATH impaired its capacity to inhibit LPS-mediated release of proinflammatory molecules from mouse and canine macrophages. In conclusion, citrullination attenuates the antibacterial and the LPS-binding properties of K9CATH, demonstrating the importance of a net positive charge for antibacterial lysis of bacteria and LPS-binding effects and suggests that citrullination is a means to regulate cathelicidin activities.
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| 4. |
- Andersson, Marlene, et al.
(author)
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Biomimetic spinning of artificial spider silk from a chimeric minispidroin
- 2017
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In: Nature Chemical Biology. - : Nature Publishing Group. - 1552-4450 .- 1552-4469. ; 13:3, s. 262-
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Journal article (peer-reviewed)abstract
- Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.
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| 5. |
- Andersson, Marlene, et al.
(author)
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Biomimetic spinning of artificial spider silk from a chimeric minispidroin
- 2017
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In: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 254
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Journal article (peer-reviewed)abstract
- Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.
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| 6. |
- Andersson, Marlene, et al.
(author)
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Carbonic Anhydrase Generates CO2 and H+ That Drive Spider Silk Formation Via Opposite Effects on the Terminal Domains
- 2014
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In: PLoS biology. - : Public Library of Science (PLoS). - 1544-9173 .- 1545-7885. ; 12:8, s. e1001921-
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Journal article (peer-reviewed)abstract
- Spider silk fibers are produced from soluble proteins (spidroins) under ambient conditions in a complex but poorly understood process. Spidroins are highly repetitive in sequence but capped by nonrepetitive N- and C-terminal domains (NT and CT) that are suggested to regulate fiber conversion in similar manners. By using ion selective microelectrodes we found that the pH gradient in the silk gland is much broader than previously known. Surprisingly, the terminal domains respond in opposite ways when pH is decreased from 7 to 5: Urea denaturation and temperature stability assays show that NT dimers get significantly stabilized and then lock the spidroins into multimers, whereas CT on the other hand is destabilized and unfolds into ThT-positive beta-sheet amyloid fibrils, which can trigger fiber formation. There is a high carbon dioxide pressure (pCO(2)) in distal parts of the gland, and a CO2 analogue interacts with buried regions in CT as determined by nuclear magnetic resonance (NMR) spectroscopy. Activity staining of histological sections and inhibition experiments reveal that the pH gradient is created by carbonic anhydrase. Carbonic anhydrase activity emerges in the same region of the gland as the opposite effects on NT and CT stability occur. These synchronous events suggest a novel CO2 and proton-dependent lock and trigger mechanism of spider silk formation.
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| 7. |
- Andersson, Marlene, et al.
(author)
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Morphology and Composition of the Spider Major Ampullate Gland and Dragline Silk
- 2013
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In: Biomacromolecules. - : American Chemical Society (ACS). - 1525-7797 .- 1526-4602. ; 14, s. 2945-2952
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Journal article (peer-reviewed)abstract
- Spider silk is made of unique proteins-spidroins-secreted and stored as a protein solution (dope) in specialized glands. The major ampullate gland, source of the dragline silk, is composed of a tail, a sac and an elongated duct. For this gland, several different types of epithelial cells and granules have been described, but it is largely unknown how they correlate with spidroin production. It is also not settled what parts of the large spidroins end up in the final silk, and it has been suggested that the N-terminal domain (NT) is lacking. Here we show that NT is present in the dope and throughout drag,line silk fibers, including the skin layer, and that the major ampullate tail and sac consist of three different and sharply demarcated zones (A-C), each with a distinct epithelial cell type. Finally, we show that spidroins are produced in the A and B zone epithelia, while the C zone granules lack spidroins.
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| 8. |
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| 9. |
- Andersson, Marlene, et al.
(author)
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Silk Spinning in Silkworms and Spiders
- 2016
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In: International Journal of Molecular Sciences. - : MDPI AG. - 1661-6596 .- 1422-0067. ; 17
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Research review (peer-reviewed)abstract
- Spiders and silkworms spin silks that outcompete the toughness of all natural and manmade fibers. Herein, we compare and contrast the spinning of silk in silkworms and spiders, with the aim of identifying features that are important for fiber formation. Although spiders and silkworms are very distantly related, some features of spinning silk seem to be universal. Both spiders and silkworms produce large silk proteins that are highly repetitive and extremely soluble at high pH, likely due to the globular terminal domains that flank an intermediate repetitive region. The silk proteins are produced and stored at a very high concentration in glands, and then transported along a narrowing tube in which they change conformation in response primarily to a pH gradient generated by carbonic anhydrase and proton pumps, as well as to ions and shear forces. The silk proteins thereby convert from random coil and alpha helical soluble conformations to beta sheet fibers. We suggest that factors that need to be optimized for successful production of artificial silk proteins capable of forming tough fibers include protein solubility, pH sensitivity, and preservation of natively folded proteins throughout the purification and initial spinning processes.
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| 10. |
- Arndt, Tina, et al.
(author)
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Engineered Spider Silk Proteins for Biomimetic Spinning of Fibers with Toughness Equal to Dragline Silks
- 2022
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In: Advanced Functional Materials. - : Wiley. - 1616-301X .- 1616-3028. ; 32:23
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Journal article (peer-reviewed)abstract
- Spider silk is the toughest fiber found in nature, and bulk production of artificial spider silk that matches its mechanical properties remains elusive. Development of miniature spider silk proteins (mini-spidroins) has made large-scale fiber production economically feasible, but the fibers’ mechanical properties are inferior to native silk. The spider silk fiber's tensile strength is conferred by poly-alanine stretches that are zipped together by tight side chain packing in β-sheet crystals. Spidroins are secreted so they must be void of long stretches of hydrophobic residues, since such segments get inserted into the endoplasmic reticulum membrane. At the same time, hydrophobic residues have high β-strand propensity and can mediate tight inter-β-sheet interactions, features that are attractive for generation of strong artificial silks. Protein production in prokaryotes can circumvent biological laws that spiders, being eukaryotic organisms, must obey, and the authors thus design mini-spidroins that are predicted to more avidly form stronger β-sheets than the wildtype protein. Biomimetic spinning of the engineered mini-spidroins indeed results in fibers with increased tensile strength and two fiber types display toughness equal to native dragline silks. Bioreactor expression and purification result in a protein yield of ≈9 g L−1 which is in line with requirements for economically feasible bulk scale production.
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| 11. |
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| 12. |
- Arndt, Tina, et al.
(author)
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Spidroin N-terminal domain forms amyloid-like fibril based hydrogels and provides a protein immobilization platform
- 2022
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In: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13
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Journal article (peer-reviewed)abstract
- Recombinant spider silk proteins (spidroins) have multiple potential applications in development of novel biomaterials, but their multimodal and aggregation-prone nature have complicated production and straightforward applications. Here, we report that recombinant miniature spidroins, and importantly also the N-terminal domain (NT) on its own, rapidly form self-supporting and transparent hydrogels at 37 °C. The gelation is caused by NT α-helix to β-sheet conversion and formation of amyloid-like fibrils, and fusion proteins composed of NT and green fluorescent protein or purine nucleoside phosphorylase form hydrogels with intact functions of the fusion moieties. Our findings demonstrate that recombinant NT and fusion proteins give high expression yields and bestow attractive properties to hydrogels, e.g., transparency, cross-linker free gelation and straightforward immobilization of active proteins at high density.
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| 13. |
- Askarieh, Glareh, et al.
(author)
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Self-assembly of spider silk proteins is controlled by a pH-sensitive relay
- 2010
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In: Nature. - : Springer Nature. - 0028-0836 .- 1476-4687. ; 465:7295, s. 236-8
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Journal article (peer-reviewed)abstract
- Nature's high-performance polymer, spider silk, consists of specific proteins, spidroins, with repetitive segments flanked by conserved non-repetitive domains. Spidroins are stored as a highly concentrated fluid dope. On silk formation, intermolecular interactions between repeat regions are established that provide strength and elasticity. How spiders manage to avoid premature spidroin aggregation before self-assembly is not yet established. A pH drop to 6.3 along the spider's spinning apparatus, altered salt composition and shear forces are believed to trigger the conversion to solid silk, but no molecular details are known. Miniature spidroins consisting of a few repetitive spidroin segments capped by the carboxy-terminal domain form metre-long silk-like fibres irrespective of pH. We discovered that incorporation of the amino-terminal domain of major ampullate spidroin 1 from the dragline of the nursery web spider Euprosthenops australis (NT) into mini-spidroins enables immediate, charge-dependent self-assembly at pH values around 6.3, but delays aggregation above pH 7. The X-ray structure of NT, determined to 1.7 A resolution, shows a homodimer of dipolar, antiparallel five-helix bundle subunits that lack homologues. The overall dimeric structure and observed charge distribution of NT is expected to be conserved through spider evolution and in all types of spidroins. Our results indicate a relay-like mechanism through which the N-terminal domain regulates spidroin assembly by inhibiting precocious aggregation during storage, and accelerating and directing self-assembly as the pH is lowered along the spider's silk extrusion duct.
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| 14. |
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| 15. |
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| 16. |
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| 17. |
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| 18. |
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| 19. |
- Bäcklund, Fredrik G., et al.
(author)
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An Image-Analysis-Based Method for the Prediction of Recombinant Protein Fiber Tensile Strength
- 2022
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In: Materials. - : MDPI AG. - 1996-1944. ; 15:3
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Journal article (peer-reviewed)abstract
- Silk fibers derived from the cocoon of silk moths and the wide range of silks produced by spiders exhibit an array of features, such as extraordinary tensile strength, elasticity, and adhesive properties. The functional features and mechanical properties can be derived from the structural composition and organization of the silk fibers. Artificial recombinant protein fibers based on engineered spider silk proteins have been successfully made previously and represent a promising way towards the large-scale production of fibers with predesigned features. However, for the production and use of protein fibers, there is a need for reliable objective quality control procedures that could be automated and that do not destroy the fibers in the process. Furthermore, there is still a lack of understanding the specifics of how the structural composition and organization relate to the ultimate function of silk-like fibers. In this study, we develop a new method for the categorization of protein fibers that enabled a highly accurate prediction of fiber tensile strength. Based on the use of a common light microscope equipped with polarizers together with image analysis for the precise determination of fiber morphology and optical properties, this represents an easy-to-use, objective non-destructive quality control process for protein fiber manufacturing and provides further insights into the link between the supramolecular organization and mechanical functionality of protein fibers.
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| 20. |
- Chen, G., et al.
(author)
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Abilities of the BRICHOS domain to prevent neurotoxicity and fibril formation are dependent on a highly conserved Asp residue
- 2022
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In: RSC Chemical Biology. - : Royal Society of Chemistry (RSC). - 2633-0679. ; 3:11, s. 1342-1358
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Journal article (peer-reviewed)abstract
- Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but to what extent the respective mechanisms are overlapping is not fully understood. The BRICHOS domain constitutes a disease-associated chaperone family, with activities against amyloid neurotoxicity, fibril formation, and amorphous protein aggregation. Here, we show that the activities of BRICHOS against amyloid-induced neurotoxicity and fibril formation, respectively, are oppositely dependent on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families. The conserved Asp in its ionized state promotes structural flexibility and has a pKa value between pH 6.0 and 7.0, suggesting that chaperone effects can be differently affected by physiological pH variations.
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| 21. |
- Chen, Gefei, et al.
(author)
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Full-Length Minor Ampullate Spidroin Gene Sequence
- 2012
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In: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:12, s. e52293-
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Journal article (peer-reviewed)abstract
- Spider silk includes seven protein based fibers and glue-like substances produced by glands in the spider's abdomen. Minor ampullate silk is used to make the auxiliary spiral of the orb-web and also for wrapping prey, has a high tensile strength and does not supercontract in water. So far, only partial cDNA sequences have been obtained for minor ampullate spidroins (MiSps). Here we describe the first MiSp full-length gene sequence from the spider species Araneus ventricosus, using a multidimensional PCR approach. Comparative analysis of the sequence reveals regulatory elements, as well as unique spidroin gene and protein architecture including the presence of an unusually large intron. The spliced full-length transcript of MiSp gene is 5440 bp in size and encodes 1766 amino acid residues organized into conserved nonrepetitive N- and C-terminal domains and a central predominantly repetitive region composed of four units that are iterated in a non regular manner. The repeats are more conserved within A. ventricosus MiSp than compared to repeats from homologous proteins, and are interrupted by two nonrepetitive spacer regions, which have 100% identity even at the nucleotide level.
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| 22. |
- Davis, B, et al.
(author)
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Voices of chemical biology
- 2021
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In: Nature chemical biology. - : Springer Science and Business Media LLC. - 1552-4469 .- 1552-4450. ; 17:1, s. 1-4
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Journal article (peer-reviewed)
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| 23. |
- Fredriksson, Camilla, et al.
(author)
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Tissue Response to Subcutaneously Implanted Recombinant Spider Silk : An in Vivo Study
- 2009
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In: Materials. - Basel, Switzerland : MDPI AG. - 1996-1944. ; 2:4, s. 1908-1922
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Journal article (peer-reviewed)abstract
- Spider silk is an interesting biomaterial for medical applications. Recently, a method for production of recombinant spider silk protein (4RepCT) that forms macroscopic fibres in physiological solution was developed. Herein, 4RepCT and MersilkTM (control) fibres were implanted subcutaneously in rats for seven days, without any negative systemic or local reactions. The tissue response, characterised by infiltration of macrophages and multinucleated cells, was similar with both fibres, while only the 4RepCT-fibres supported ingrowth of fibroblasts and newly formed capillaries. This in vivo study indicates that 4RepCT-fibres are well tolerated and could be used for medical applications, e.g., tissue engineering.
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| 24. |
- Gault, Joseph, et al.
(author)
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Mass Spectrometry Reveals the Direct Action of a Chemical Chaperone
- 2018
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In: The Journal of Physical Chemistry Letters. - : American Chemical Society (ACS). - 1948-7185. ; 9:14, s. 4082-4086
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Journal article (peer-reviewed)abstract
- Despite their fundamental biological importance and therapeutic potential, the interactions between chemical chaperones and proteins remain difficult to capture due to their transient and nonspecific nature. Using a simple mass spectrometric assay, we are able to follow the interactions between proteins and the chemical chaperone trimethylamine-N-oxide (TMAO). In this manner, we directly observe that the counteraction of TMAO and the denaturant urea is driven by the exclusion of TMAO from the protein surface, whereas the surfactant lauryl dimethylamine-N-oxide cannot be displaced. Our results clearly demonstrate a direct chaperoning mechanism for TMAO, corroborating extensive computational studies, and pave the way for the use of nondenaturing mass spectrometry and related techniques to study chemical chaperones in molecular detail.
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| 25. |
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