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- Lewicka, M, et al.
(författare)
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Recombinant Spider Silk Protein Matrices Facilitate Differentiation of Neural Stem Cells Into Mature and Functional Neurons
- 2021
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Ingår i: FRONTIERS IN MATERIALS. - : Frontiers Media SA. - 2296-8016. ; 7
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Neural stem cells (NSCs) show great promise in drug discovery and clinical application. Yet few efforts have been made to optimize biocompatible materials for such cells to be expanded and used in clinical conditions. We have previously demonstrated that NSCs are readily cultured on substrates of certain recombinant spider silk protein without addition of animal- or human-derived components. The question remains however whether this material allows differentiation into functional neurons, and whether such differentiation can take place also when the NSCs are cultured not only upon but also within the biodegradable material. Here we demonstrate that “foam”-like structures generated from recombinant spider silk protein (4RepCT) provided excellent matrices for the generation and multicellular analysis of functional excitatory neurons from NSCs without addition of animal- or human-derived components. NSCs isolated from the cerebral cortices of rat embryos were cultured at either 4RepCT matrices shaped as foam-like structures without coating, or on conventional polystyrene plates coated with poly-L-ornithine and fibronectin. Upon treatment with recombinant proteins including the extracellular signaling factor BMP4 or a combination of BMP4 and the signaling factor Wnt3a, the cortical NSCs cultured in 4RepCT foam-like structures differentiated efficiently into neurons that responded to glutamate receptor agonists, such as AMPA, to the same extent as control cultures. Matrices derived from recombinant spider silk proteins thus provide a functional microenvironment for neural stem cells with little or no animal- or human-derived components and can be employed in the development of new strategies in stem cell research and tissue engineering.
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- Pavankumar, A. R., et al.
(författare)
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Bioanalytical advantages of a novel recombinant apyrase enzyme in ATP-based bioluminescence methods
- 2018
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Ingår i: Analytica Chimica Acta. - : Elsevier. - 0003-2670 .- 1873-4324. ; 1025, s. 118-123
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Tidskriftsartikel (refereegranskat)abstract
- Ultrasensitive measurements of intracellular ATP (intATP) based on the firefly luciferase reactions are frequently used to enumerate bacterial or mammalian cells. During clinical applications, extracellular ATP (extATP) should be depleted in biological samples since it interferes with intATP and affects the quantification of bacteria. The extATP can be eliminated by ATP-degrading enzymes but complete hydrolysis of extATP remains a challenge for today's commercial enzymes. The catalytic efficiency of ATP-degrading enzymes depends on enzyme characteristics, sample composition and the ability to deplete diphosphates, triphosphates and their complexes generated during the reaction. This phenomenon restricts the usage of bioluminescence-based ATP methods in clinical diagnostics. In light of this, we have developed a recombinant Shigella flexneri apyrase (RSFA) enzyme and analysed its ATP depletion potential with five commercial biochemical sources including potato apyrase, acid phosphatase, alkaline phosphatase, hexokinase and glycerol kinase. The RSFA revealed superior activity by completely eliminating the extracellular ATP and ATP-complexes, even in biological samples like urine and serum. Therefore, our results can potentially unwrap the chemical and bio-analytical applications of ATP-based bioluminescence tests to develop highly sensitive point-of-care diagnostics.
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