Academic literature on the topic 'Protein scaffolds'
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Journal articles on the topic "Protein scaffolds"
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Ortiz-Muñoz, Andrés, Héctor F. Medina-Abarca, and Walter Fontana. "Combinatorial protein–protein interactions on a polymerizing scaffold." Proceedings of the National Academy of Sciences 117, no. 6 (January 24, 2020): 2930–37. http://dx.doi.org/10.1073/pnas.1912745117.
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Scaffold proteins organize cellular processes by bringing signaling molecules into interaction, sometimes by forming large signalosomes. Several of these scaffolds are known to polymerize. Their assemblies should therefore not be understood as stoichiometric aggregates, but as combinatorial ensembles. We analyze the combinatorial interaction of ligands loaded on polymeric scaffolds, in both a continuum and discrete setting, and compare it with multivalent scaffolds with fixed number of binding sites. The quantity of interest is the abundance of ligand interaction possibilities—the catalytic potential Q—in a configurational mixture. Upon increasing scaffold abundance, scaffolding systems are known to first increase opportunities for ligand interaction and then to shut them down as ligands become isolated on distinct scaffolds. The polymerizing system stands out in that the dependency of Q on protomer concentration switches from being dominated by a first order to a second order term within a range determined by the polymerization affinity. This behavior boosts Q beyond that of any multivalent scaffold system. In addition, the subsequent drop-off is considerably mitigated in that Q decreases with half the power in protomer concentration than for any multivalent scaffold. We explain this behavior in terms of how the concentration profile of the polymer-length distribution adjusts to changes in protomer concentration and affinity. The discrete case turns out to be similar, but the behavior can be exaggerated at small protomer numbers because of a maximal polymer size, analogous to finite-size effects in bond percolation on a lattice.
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Bari, Elia, Franca Scocozza, Sara Perteghella, Marzio Sorlini, Ferdinando Auricchio, Maria Luisa Torre, and Michele Conti. "3D Bioprinted Scaffolds Containing Mesenchymal Stem/Stromal Lyosecretome: Next Generation Controlled Release Device for Bone Regenerative Medicine." Pharmaceutics 13, no. 4 (April 8, 2021): 515. http://dx.doi.org/10.3390/pharmaceutics13040515.
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Three-dimensional printing of poly(ε-caprolactone) (PCL) is a consolidated scaffold manufacturing technique for bone regenerative medicine. Simultaneously, the mesenchymal stem/stromal cell (MSC) secretome is osteoinductive, promoting scaffold colonization by cells, proliferation, and differentiation. The present paper combines 3D-printed PCL scaffolds with lyosecretome, a freeze-dried formulation of MSC secretome, containing proteins and extracellular vesicles (EVs). We designed a lyosecretome 3D-printed scaffold by two loading strategies: (i) MSC secretome adsorption on 3D-printed scaffold and (ii) coprinting of PCL with an alginate-based hydrogel containing MSC secretome (at two alginate concentrations, i.e., 6% or 10% w/v). A fast release of proteins and EVs (a burst of 75% after 30 min) was observed from scaffolds obtained by absorption loading, while coprinting of PCL and hydrogel, encapsulating lyosecretome, allowed a homogeneous loading of protein and EVs and a controlled slow release. For both loading modes, protein and EV release was governed by diffusion as revealed by the kinetic release study. The secretome’s diffusion is influenced by alginate, its concentration, or its cross-linking modes with protamine due to the higher steric hindrance of the polymer chains. Moreover, it is possible to further slow down protein and EV release by changing the scaffold shape from parallelepiped to cylindrical. In conclusion, it is possible to control the release kinetics of proteins and EVs by changing the composition of the alginate hydrogel, the scaffold’s shape, and hydrogel cross-linking. Such scaffold prototypes for bone regenerative medicine are now available for further testing of safety and efficacy.
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Finch, Anthony, and Jin Kim. "Thermophilic Proteins as Versatile Scaffolds for Protein Engineering." Microorganisms 6, no. 4 (September 25, 2018): 97. http://dx.doi.org/10.3390/microorganisms6040097.
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Literature from the past two decades has outlined the existence of a trade-off between protein stability and function. This trade-off creates a unique challenge for protein engineers who seek to introduce new functionality to proteins. These engineers must carefully balance the mutation-mediated creation and/or optimization of function with the destabilizing effect of those mutations. Subsequent research has shown that protein stability is positively correlated with “evolvability” or the ability to support mutations which bestow new functionality on the protein. Since the ultimate goal of protein engineering is to create and/or optimize a protein’s function, highly stable proteins are preferred as potential scaffolds for protein engineering. This review focuses on the application potential for thermophilic proteins as scaffolds for protein engineering. The relatively high inherent thermostability of these proteins grants them a great deal of mutational robustness, making them promising scaffolds for various protein engineering applications. Comparative studies on the evolvability of thermophilic and mesophilic proteins have strongly supported the argument that thermophilic proteins are more evolvable than mesophilic proteins. These findings indicate that thermophilic proteins may represent the scaffold of choice for protein engineering in the future.
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Simunovic, Mijo, Emma Evergren, Ivan Golushko, Coline Prévost, Henri-François Renard, Ludger Johannes, Harvey T. McMahon, Vladimir Lorman, Gregory A. Voth, and Patricia Bassereau. "How curvature-generating proteins build scaffolds on membrane nanotubes." Proceedings of the National Academy of Sciences 113, no. 40 (September 21, 2016): 11226–31. http://dx.doi.org/10.1073/pnas.1606943113.
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Bin/Amphiphysin/Rvs (BAR) domain proteins control the curvature of lipid membranes in endocytosis, trafficking, cell motility, the formation of complex subcellular structures, and many other cellular phenomena. They form 3D assemblies that act as molecular scaffolds to reshape the membrane and alter its mechanical properties. It is unknown, however, how a protein scaffold forms and how BAR domains interact in these assemblies at protein densities relevant for a cell. In this work, we use various experimental, theoretical, and simulation approaches to explore how BAR proteins organize to form a scaffold on a membrane nanotube. By combining quantitative microscopy with analytical modeling, we demonstrate that a highly curving BAR protein endophilin nucleates its scaffolds at the ends of a membrane tube, contrary to a weaker curving protein centaurin, which binds evenly along the tube’s length. Our work implies that the nature of local protein–membrane interactions can affect the specific localization of proteins on membrane-remodeling sites. Furthermore, we show that amphipathic helices are dispensable in forming protein scaffolds. Finally, we explore a possible molecular structure of a BAR-domain scaffold using coarse-grained molecular dynamics simulations. Together with fluorescence microscopy, the simulations show that proteins need only to cover 30–40% of a tube’s surface to form a rigid assembly. Our work provides mechanical and structural insights into the way BAR proteins may sculpt the membrane as a high-order cooperative assembly in important biological processes.
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Pham, Phuong Ngoc, Maroš Huličiak, Lada Biedermannová, Jiří Černý, Tatsiana Charnavets, Gustavo Fuertes, Štěpán Herynek, et al. "Protein Binder (ProBi) as a New Class of Structurally Robust Non-Antibody Protein Scaffold for Directed Evolution." Viruses 13, no. 2 (January 27, 2021): 190. http://dx.doi.org/10.3390/v13020190.
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Engineered small non-antibody protein scaffolds are a promising alternative to antibodies and are especially attractive for use in protein therapeutics and diagnostics. The advantages include smaller size and a more robust, single-domain structural framework with a defined binding surface amenable to mutation. This calls for a more systematic approach in designing new scaffolds suitable for use in one or more methods of directed evolution. We hereby describe a process based on an analysis of protein structures from the Protein Data Bank and their experimental examination. The candidate protein scaffolds were subjected to a thorough screening including computational evaluation of the mutability, and experimental determination of their expression yield in E. coli, solubility, and thermostability. In the next step, we examined several variants of the candidate scaffolds including their wild types and alanine mutants. We proved the applicability of this systematic procedure by selecting a monomeric single-domain human protein with a fold different from previously known scaffolds. The newly developed scaffold, called ProBi (Protein Binder), contains two independently mutable surface patches. We demonstrated its functionality by training it as a binder against human interleukin-10, a medically important cytokine. The procedure yielded scaffold-related variants with nanomolar affinity.
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Wang, Hong Xin, Zheng Xiang Xue, Mei Hong Wei, Deng Long Chen, and Min Li. "A Novel Scaffold from Recombinant Spider Silk Protein in Tissue Engineering." Advanced Materials Research 152-153 (October 2010): 1734–44. http://dx.doi.org/10.4028/www.scientific.net/amr.152-153.1734.
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As a new biomaterial, recombinant spider silk protein has attracted much attention in tissue engineering. The pNSR-16/ BL21(DE3)pLysS strains fermented and produced the recombinant spider silk protein, which was then cast into scaffolds. NIH-3T3 cells were cultivated with extractions of the scaffolds in vitro. The cytotoxicity of scaffolds was analyzed with a MTT assay. The performances of cells adhesion, growth and expression on the scaffolds were observed with SEM, HE staining and immunohistochemistry. Compared with the control, the extract fluid of materials culturing the NIH-3T3 cells was not apparently different. NIH-3T3 cells could adhere and grow on the scaffolds and secret FGF-2. The pNSR-16 recombinant spider silk protein scaffolds has satisfactory cytocompatibility and the scaffolds are ideal scaffold material for tissue engineering.
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Lin, Peng, Hui Yang, Eiji Nakata, and Takashi Morii. "Mechanistic Aspects for the Modulation of Enzyme Reactions on the DNA Scaffold." Molecules 27, no. 19 (September 24, 2022): 6309. http://dx.doi.org/10.3390/molecules27196309.
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Cells have developed intelligent systems to implement the complex and efficient enzyme cascade reactions via the strategies of organelles, bacterial microcompartments and enzyme complexes. The scaffolds such as the membrane or protein in the cell are believed to assist the co-localization of enzymes and enhance the enzymatic reactions. Inspired by nature, enzymes have been located on a wide variety of carriers, among which DNA scaffolds attract great interest for their programmability and addressability. Integrating these properties with the versatile DNA–protein conjugation methods enables the spatial arrangement of enzymes on the DNA scaffold with precise control over the interenzyme distance and enzyme stoichiometry. In this review, we survey the reactions of a single type of enzyme on the DNA scaffold and discuss the proposed mechanisms for the catalytic enhancement of DNA-scaffolded enzymes. We also review the current progress of enzyme cascade reactions on the DNA scaffold and discuss the factors enhancing the enzyme cascade reaction efficiency. This review highlights the mechanistic aspects for the modulation of enzymatic reactions on the DNA scaffold.
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Thanyaphoo, Suphannee, and Jasadee Kaewsrichan. "A new biocompatible delivery scaffold containing heparin and bone morphogenetic protein 2." Acta Pharmaceutica 66, no. 3 (September 1, 2016): 373–85. http://dx.doi.org/10.1515/acph-2016-0026.
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Abstract Silicon-substituted calcium phosphate (Si-CaP) was developed in our laboratory as a biomaterial for delivery in bone tissue engineering. It was fabricated as a 3D-construct of scaffolds using chitosan-trisodium polyphosphate (TPP) cross-linked networks. In this study, heparin was covalently bonded to the residual -NH2 groups of chitosan on the scaffold applying carbodiimide chemistry. Bonded heparin was not leached away from scaffold surfaces upon vigorous washing or extended storage. Recombinant human bone morphogenetic protein 2 (rhBMP-2) was bound to conjugated scaffolds by ionic interactions between the negatively charged SO42- clusters of heparin and positively charged amino acids of rhBMP-2. The resulting scaffolds were inspected for bone regenerative capacity by subcutaneous implanting in rats. Histological observation and mineralization assay were performed after 4 weeks of implantation. Results from both in vitro and in vivo experiments suggest the potential of the developed scaffolds for bone tissue engineering applications in the future.
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Ford, Audrey C., Hans Machula, Robert S. Kellar, and Brent A. Nelson. "Characterizing the mechanical properties of tropoelastin protein scaffolds." MRS Proceedings 1569 (2013): 45–50. http://dx.doi.org/10.1557/opl.2013.1059.
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ABSTRACTThis paper reports on mechanical characterization of electrospun tissue scaffolds formed from varying blends of collagen and human tropoelastin. The electrospun tropoelastin-based scaffolds have an open, porous structure conducive to cell attachment and have been shown to exhibit strong biocompatibility, but the mechanical character is not well known. Mechanical properties were tested for scaffolds consisting of 100% tropoelastin and 1:1 tropoelastin-collagen blends. The results showed that the materials exhibited a three order of magnitude change in the initial elastic modulus when tested dry vs. hydrated, with moduli of 21 MPa and 0.011 MPa respectively. Noncrosslinked and crosslinked tropoelastin scaffolds exhibited the same initial stiffness from 0 to 50% strain, and the noncrosslinked scaffolds exhibited no stiffness at strains >∼50%. The elastic modulus of a 1:1 tropoelastin-collagen blend was 50% higher than that of a pure tropoelastin scaffold. Finally, the 1:1 tropoelastin-collagen blend was five times stiffer from 0 to 50% strain when strained at five times the ASTM standard rate. By systematically varying protein composition and crosslinking, the results demonstrate how protein scaffolds might be manipulated as customized biomaterials, ensuring mechanical robustness and potentially improving biocompatibility through minimization of compliance mismatch with the surrounding tissue environment. Moreover, the demonstration of strain-rate dependent mechanical behavior has implications for mechanical design of tropoelastin-based tissue scaffolds.
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Chen, Cheng-Yu, Ming-You Shie, Alvin Kai-Xing Lee, Yun-Ting Chou, Chun Chiang, and Chun-Pin Lin. "3D-Printed Ginsenoside Rb1-Loaded Mesoporous Calcium Silicate/Calcium Sulfate Scaffolds for Inflammation Inhibition and Bone Regeneration." Biomedicines 9, no. 8 (July 28, 2021): 907. http://dx.doi.org/10.3390/biomedicines9080907.
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Bone defects are commonly found in the elderly and athletic population due to systemic diseases such as osteoporosis and trauma. Bone scaffolds have since been developed to enhance bone regeneration by acting as a biological extracellular scaffold for cells. The main advantage of a bone scaffold lies in its ability to provide various degrees of structural support and growth factors for cellular activities. Therefore, we designed a 3D porous scaffold that can not only provide sufficient mechanical properties but also carry drugs and promote cell viability. Ginsenoside Rb1 (GR) is an extract from panax ginseng, which has been used for bone regeneration and repair since ancient Chinese history. In this study, we fabricated scaffolds using various concentrations of GR with mesoporous calcium silicate/calcium sulfate (MSCS) and investigated the scaffold’s physical and chemical characteristic properties. PrestoBlue, F-actin staining, and ELISA were used to demonstrate the effect of the GR-contained MSCS scaffold on cell proliferation, morphology, and expression of the specific osteogenic-related protein of human dental pulp stem cells (hDPSCs). According to our data, hDPSCs cultivated in GR-contained MSCS scaffold had preferable abilities of proliferation and higher expression of the osteogenic-related protein and could effectively inhibit inflammation. Finally, in vivo performance was assessed using histological results that revealed the GR-contained MSCS scaffolds were able to further achieve more effective hard tissue regeneration than has been the case in the past. Taken together, this study demonstrated that a GR-containing MSCS 3D scaffold could be used as a potential alternative for future bone tissue engineering studies and has good potential for clinical use.
Dissertations / Theses on the topic "Protein scaffolds"
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Rodriguez, Marin Silvia. "Multifunctional scaffolds for selective protein-protein inhibition." Thesis, University of Leeds, 2016. http://etheses.whiterose.ac.uk/17299/.
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Protein-protein interactions (PPIs) play an important role in numerous biological processes. Consequently, modulating PPIs is fundamental for understanding and manipulating mechanisms that govern many diseases. Among the wide range of topographies that PPIs display, the α-helix is the most common secondary structure in nature and thus represents a good generic template for inhibitor design.1 Some of the most relevant approaches in this field are the proteomimetic approach, which recapitulate the key binding residues of an α-helix on a non-peptidic scaffold; and the constrained peptides, which aim to reproduce the helical structure by stabilising a helical peptide. Both approaches have generated potent inhibitors of a great diversity of α-helix mediated PPIs. However, developing a better understanding of the key features that govern the modulation of protein recognition is necessary to further advance the field and fully exploit each class of foldamer. In that context, we developed functionalised aromatic oligoamide backbones to mimic residues located on multiple faces of an -helix to target the ER/co-activator PPI. The novel scaffolds are based on bis-benzamide and N-(4-aminophenyl)terephthalamidic acid backbones functionalised with isobutyl groups to reproduce the key side chains of the co-activator α-helix. Conformational studies in combination with molecular modeling and docking analysis provide evidence that the new oligomers can adopt conformations that mimic the residues at i, i+3 and i+4 positions of the native co-activator α-helix. In addition, the rules that govern molecular recognition of protein surfaces were further investigated through the optimisation of the oligobenzamide hybrid scaffold using a structure-activity relationship (SAR) study. A library of compound analogues has been synthesised incorporating five variable sites. The modifications focused on size, polarity and stereochemistry to obtain more potent and selective proteomimetic inhibitors of the p53/hDM2 and Mcl-1/NOXA B PPIs. Finally, using existing methodologies a 3-O-alkylated proteomimetic scaffold and hydrocarbon stapling peptide strategy, have been used to design inhibitors of the Asf1/H3 interaction. The application of both approaches allowed the different inhibitor designs to be directly compared when targeting the same PPI.
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Machado, Roque Ana Isabel. "Protein scaffolds for cell culture." Thesis, University of Newcastle Upon Tyne, 2013. http://hdl.handle.net/10443/1843.
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We report here the design, purification and structural characterization of a new protein scaffold for cell culture. Prior studies in our group revealed the structure of the bacterial protein Caf1 to be flexible protein nanofibres, up to 1.5 μm. The existing Cafl expression system was cumbersome and difficult to mutate, we have now produced a system containing the caf operon which allows for the incorporation of specific peptide motifs. The small peptide, RGDS from fibronectin was inserted into 5 different surface loops of Caf1. The Caf1 mutants were expressed and purified and a structural characterization by biophysical methods was conducted. This revealed permissive sites into which new motifs can be inserted. The characterised proteins were sterilised and used to coat 96 well plates for cell culture. In this study we used mammalian cell lines such as 3T3 fibroblasts, PC12 neuronal cells and primary osteoblasts to understand how they behave in the presence of this biomaterial, in particular the formation of focal adhesions, changes in cytoskeleton rearrangement and nuclear and cell morphology. The controlled engineering of sites within the polymer allowed us to study their implication in cell attachment, survival and proliferation. Our preliminary results have shown that cells interact poorly with the unmodified protein e.g. without any motif associated. This reveals that the polymer is inert and does not influence cell growth by itself. In contrast, the incorporation of RGDS, can invert the scenario of cell growth; promoting cell attachment, survival and proliferation. In a second stage of the project we designed a separate compatible plasmid encoding caf1 gene and used it with the previous plasmid to co-express hybrid Caf1 polymers. The long fibres can also be crosslinked with a non-toxic and non-immunogenic chemical compound – NHS-PEG. Thus a protein hydrogel composed of interchangeable folding units which can be used to incorporate different cell interacting peptide motifs. It is robust and, in the unmodified state highly protease resistant. Future studies will elucidate the versatility and potentiality for this peptide hydrogel in stem cell differentiation.
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Badger, David B. "Design and Synthesis of Protein-Protein Interaction Inhibitor Scaffolds." Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/3964.
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Many currently relevant diseases such as cancer arise from altered biological pathways that rely on protein-protein interactions. The proteins involved in these interactions contain certain functional domains that are responsible for the protein's biological activities. These domains consist of secondary structural elements such as α-helices and Β-sheets which are at the heart of the protein's biological activity. Therefore, designing drugs that inhibit protein-protein interactions by binding to these key secondary structural elements should provide an effective treatment for many diseases. Presented in this dissertation are the designs, syntheses, and biological evaluations for both novel α-helix and novel Β-sheet mimics.
The α-helix mimics were designed to inhibit the interactions between the tumor suppressor protein p53 and its inhibitor protein, MDM2. We also targeted the interactions between the Bak/Bcl-xL proteins. Using the knowledge gained from Hamilton's 1,4-terphenylene scaffold, we designed our inhibitors to be non-peptidic small molecule α-helix mimics. These molecules were designed to bind to the NH2-terminal domain of MDM2 protein thus preventing it from binding to the p53 protein thereby allowing p53 to induce apoptosis. The α-helix mimetic scaffold is designed around a central functionalized pyridazine ring while maintaining the appropriate distances between the ith, ith+4, and ith+7 positions of a natural alpha helix.
The Β-sheet mimics were designed as inhibitors for the integrin mediated extracellular matrix cell adhesion found in Multiple Myeloma. We have designed, synthesized, and incorporated novel Β-turns to induce the formation of Β-hairpins as well as to cyclize the peptides in order to increase their binding affinities and reduce proteolytic cleavage. Given that many protein-protein interactions occur through hydrophobic interactions; our primary Β-turn promoter was designed with the ability to alter the Β-hairpin's hydrophobicity depending on the sulfonyl group used in the turn. The synthesis of several different sulfonyl chlorides for use in our Β-turn promoter is included in this section. We have also provided a detailed structural analysis and characterization of these new cyclic peptides via NMR and CD spectrometry. Using standard 2D NMR methods, we have elucidated the 3D conformation of several peptides in solution. We have also studied the structure activity relationships (SAR) for these cyclic peptides and then correlated these results with those obtained from the NMR studies.
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Haji, Ruslan Khairunnisa Nabilah. "Protein hydrogels as tissue engineering scaffolds." Thesis, University of Manchester, 2015. https://www.research.manchester.ac.uk/portal/en/theses/protein-hydrogels-as-tissue-engineering-scaffolds(45ff4e72-49ea-46df-9e7b-b9113576c096).html.
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Hydrogels aim to mimic the natural living environment by entrapping large amount of water or biological fluids in their polymeric network. There has been growing interest in the development of peptide and protein hydrogels, due to their improved biocompatibility, biodegradability and biological properties in comparison to purely synthetic polymer hydrogels. Under the appropriate conditions, biomacromolecular protein hydrogels can self-assemble into ordered meso- to macroscopic supramolecules with better resulting networks that promote tissue development. The work presented here mainly focuses on producing protein hydrogels with controlled physical properties useful for tissue regeneration process and drug delivery applications. Hen egg white lysozyme (HEWL) hydrogels were studied in the presence of water and different reducing agents forming three HEWL systems including HEWL/water, HEWL/DTT and HEWL/TCEP gels. Strong, self-supporting HEWL gels were successfully prepared in the range of pH 2 to 7, using a temperature of 85°C. At pH 2, the protein denaturation in water was relatively slow resulting in a high percentage of turn structure (~50%) that promotes HEWL gelation after 3 days of heating. No lysozyme gelation in water was observed at pH 3, 4 and 7 even after 21 days of heating. A small quantity of DTT (~20 mM) was added to encourage lysozyme unfolding and HEWL/DTT samples formed gels at higher pH including at physiological pH. The pH 2 HEWL/water gel was found to be stronger but more brittle than pH 7 HEWL/DTT gel. It was observed there were some irregularities in the distribution of pH 2 fibrils (~7µm in length) that form large pore sizes within the network. The pH 7 sample contained shorter and stiff fibrils with repetitive polygon-shaped mesh network. The use of TCEP, which is a stronger reductant than DTT, led to the formation of self-supporting HEWL gels between pH 3.5 and 5.5. The highest storage modulus was observed at pH 5, which is related to the high β-sheet content of the sample (~45%). In addition, a promising strategy has been devised to form thermoresponsive HEWL hydrogels by synthesising and incorporating a small fraction of lysozyme-PNIPAAm bioconjugates into the major protein matrix. Results show the thermoresponsive nature of PNIPAAm was conferred to HEWL protein that exhibits higher storage stability in response to changing temperature.
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Wang, Hua. "Control of protein-surface, protein-protein, and cell-matrix interactions for biomaterials as tissue engineering scaffolds /." Thesis, Connect to this title online; UW restricted, 2005. http://hdl.handle.net/1773/9894.
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Lu, Zhengsun. "Electrospun nanofiber scaffolds and crosslinked protein membranes as scaffold materials in tissue engineering." Thesis, Queen Mary, University of London, 2015. http://qmro.qmul.ac.uk/xmlui/handle/123456789/15023.
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Scaffold materials play an essential role in tissue engineering field due to its function of accommodate and guide cell proliferation. In this study, I investigated different types of crosslinked protein membranes that can be produced in microfluidic channels and a number of various types of PLGA electrospun composite nanofiber scaffold to examine their potentials as scaffold materials in tissue engineering. A simplified fabrication technique has been developed to produce a large surface area of crosslinked protein membranes to fulfill the purpose of cell culture experiments. Bovine serum albumin is used along with two acyl chloride crosslinkers, i.e. TCL and IDCL, respectively to accomplish the cross-linking. On the other hand, PLGA is dissolved in HFIP and enhanced with silk fibroin and carbon nanotubes to make composite electrospun materials. The morphology, physicochemical properties and biocompatibility of the membranes are studied. The biocompatibility of the membranes is investigated using cell proliferation of the PC12, ADSCs and neurons cultured on the membranes. Our results show that compared to crosslinked protein membranes, the electrospun materials are easier to prepare, less toxic and more suitable for mass production. Moreover, the electrospun materials are seen to have better biocompatibility in our cell culture study. Furthermore, the composite electrospun materials with high CNTs concentrations demonstrate positive effects on the proliferation of neurons.
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Lee, Maximillian. "Pyridazinediones : versatile scaffolds for site-selective protein modification." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10040797/.
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Disulfide bonds represent an important target for site-selective protein modification, particularly via the strategy of functional re-bridging. Reduction of interchain disulfide bonds, followed by their re-bridging allows proteins to be functionalised in a site-selective manner whilst retaining the stability and integrity offered by the original bridge. This work describes the design and development of two distinct pyridazinedione-based technologies that, through the conduit of functional disulfide re-bridging, enable the synthesis of antibody – drug conjugates with hitherto unmet levels of control and homogeneity. As proteins often contain multiple disulfide bonds that are critical to conformation and stability, reagents that allow functional disulfide re-bridging without disulfide scrambling (non-native disulfide re-bridging) in multiple disulfide containing systems are critical for the success of this method. The first presented technology is a molecule that is capable of both reducing and re-bridging disulfide bonds, enabling a rapid and efficient one-reagent protocol for the functionalisation of disulfide containing proteins, moreover, it does so in such a way that native disulfide configuration is retained via a high local concentration effect. This novel pyridazinedione scaffold has been shown to functionalise a variety of therapeutically relevant proteins, including the widely used mAb HerceptinTM, enabling the synthesis of homogenous antibody – drug conjugates from a native mAb. Shifting focus from homogeneity to control over drug loading, the second presented technology is a single pyridazinedione-based molecule that contains four cysteine reactive centres and only one bioorthogonal reactive handle, which enables the generation of antibody conjugates with a loading of two modules. A loading of two is desirable for many reasons, especially in the context of large, hydrophobic payloads, which are increasingly popular for use in antibody-drug conjugates. A loading of two drugs per antibody has been shown to provide an optimal balance between efficacy and biophysical properties in many cases. A reliable method based on a native antibody scaffold without the use of enzymes or harsh oxidative conditions has hitherto not been achieved. The use of native antibodies has several advantages in terms of cost, practicality, accessibility and time. Thus, a novel, reliable method of furnishing antibody conjugates with a loading of two modules starting from a native antibody scaffold was developed.
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Norville, Julie Erin 1980. "Synthetic scaffolds and protein assemblies for engineering applications." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/28737.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2004.Includes bibliographical references (p. 57-63).
S-layer proteins, which naturally self-assemble on the exterior of cells, provide an interesting basis for the creation of synthetic scaffolds. In this thesis, I created a plasmid which produces a recombinant form of a well characterized S layer protein, sbpA, which has a number of properties ideal for nanotechnology applications. I also explored purification of both the native and recombinant forms of sbpA. Together these preliminary studies are the first, necessary, steps towards quantitative generation of crystallization conditions and the ultimate modifications of the protein form for a wide variety of engineering applications.
by Julie Erin Norville.
S.M.
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Hewitt, Sarah Helen. "Multivalent scaffolds for use as protein surface mimetics." Thesis, University of Leeds, 2017. http://etheses.whiterose.ac.uk/18027/.
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The development of ligands for protein surfaces to inhibit protein-protein interactions (PPI)s is challenging, as protein surfaces often lack the clefts and pockets associated with traditionally druggable targets like enzyme active sites. One way in which protein surfaces can be targeted is by the use of protein surface mimetics, whereby a multivalent scaffold is functionalised with many binding groups on its periphery in order to achieve high affinity protein recognition. One such scaffold is a ruthenium(II) tris (bipyridine)s (Ru(II)(bpy)3). The work in this thesis aimed to further develop these Ru(II)(bpy)3 protein surface mimetics; gaining information as to how they interact with proteins, looking at new ways of achieving high affinity protein surface recognition and the development of new applications for these molecules. In Chapter 2 an indepth study of the binding of two Ru(II)(bpy)3 complexes to a model protein, cytochrome c, is presented, looking at the thermodynamic and electrostatic contributions to binding as well as using protein NMR to elucidate the binding site. In Chapter 3 the development of dynamic combinatorial chemistry (DCC) scaffolds based on Ru(II)(bpy)3 complexes and tetraphenyl porphyrins was explored as a potential avenue for new receptor design, enabling the development of biologically compatible DCC systems, prime for protein ligand discovery. Chapter 4 presents another avenue for using the Ru(II)(bpy)3 complexes; using an array approach to discriminate between different protein.
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Sharma, Rajan. "Protein-mediated patterning of DNA scaffolds for nanoscale electronics." Thesis, University of Leeds, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.521527.
Full textBooks on the topic "Protein scaffolds"
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Udit, Andrew K., ed. Protein Scaffolds. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7893-9.
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Bio-glasses: An introduction. Chichester, West Sussex: Wiley, 2012.
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Udit, Andrew K. Protein Scaffolds: Design, Synthesis, and Applications. Springer New York, 2019.
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Protein Scaffolds: Design, Synthesis, and Applications. Humana, 2018.
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Pettersson, Par L. Alpha-Class Glutathione Transferases As Steroid Isomerases & Scaffolds for Protein Redesign. Uppsala Universitet, 2002.
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Li, Yanyan, Sylvie Rebuffat, and Séverine Zirah. Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds. Springer, 2014.
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Li, Yanyan, Sylvie Rebuffat, and Séverine Zirah. Lasso Peptides: Bacterial Strategies to Make and Maintain Bioactive Entangled Scaffolds. Springer London, Limited, 2014.
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Lennon, Rachel, and Neil Turner. The molecular basis of glomerular basement membrane disorders. Edited by Neil Turner. Oxford University Press, 2018. http://dx.doi.org/10.1093/med/9780199592548.003.0320_update_001.
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The glomerular basement membrane (GBM) is a condensed network of extracellular matrix molecules which provides a scaffold and niche to support the function of the overlying glomerular cells. Within the glomerulus, the GBM separates the fenestrated endothelial cells, which line capillary walls from the epithelial cells or podocytes, which cover the outer aspect of the capillaries. In common with basement membranes throughout the body, the GBM contains core components including collagen IV, laminins, nidogens, and heparan sulphate proteoglycans. However, specific isoforms of these proteins are required to maintain the integrity of the glomerular filtration barrier.Across the spectrum of glomerular disease there is alteration in glomerular extracellular matrix (ECM) and a number of histological patterns are recognized. The GBM can be thickened, expanded, split, and irregular; the mesangial matrix may be expanded and glomerulosclerosis represents a widespread accumulation of ECM proteins associated with loss of glomerular function. Whilst histological patterns may follow a sequence or provide diagnostic clues, there remains limited understanding about the mechanisms of ECM regulation and how this tight control is lost in glomerular disease. Monogenic disorders of the GBM including Alport and Pierson syndromes have highlighted the importance of both collagen IV and laminin isoforms and these observations provide important insights into mechanisms of glomerular disease.
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Jones, Julian, and Alexis Clare. Bio-Glasses. Wiley & Sons, Incorporated, John, 2012.
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Jones, Julian, and Alexis Clare. Bio-Glasses: An Introduction. Wiley & Sons, Incorporated, John, 2012.
Find full textBook chapters on the topic "Protein scaffolds"
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Shibata, Tomonori, Yuki Suzuki, Hiroshi Sugiyama, Masayuki Endo, and Hirohide Saito. "Folding RNA–Protein Complex into Designed Nanostructures." In RNA Scaffolds, 169–79. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2730-2_14.
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Shibata, Tomonori, Yuki Suzuki, Hiroshi Sugiyama, Masayuki Endo, and Hirohide Saito. "Folding RNA–Protein Complex into Designed Nanostructures." In RNA Scaffolds, 221–32. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1499-0_16.
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Gerhard, Laura, and Sven Hennig. "FRET Analysis of RNA–Protein Interactions Using Spinach Aptamers." In RNA Scaffolds, 171–97. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1499-0_13.
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Khouri, Margueritte El, Marjorie Catala, Bili Seijo, Johana Chabal, Carine Tisné, Frédéric Dardel, and Luc Ponchon. "Expression and Purification of RNA–Protein Complexes in Escherichia coli." In RNA Scaffolds, 25–31. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2730-2_3.
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Iioka, Hidekazu, and Ian G. Macara. "Detection of RNA–Protein Interactions Using Tethered RNA Affinity Capture." In RNA Scaffolds, 67–73. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2730-2_6.
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El Khouri, Margot, Marjorie Catala, Bili Seijo, Johana Chabal, Frédéric Dardel, Carine Tisné, and Luc Ponchon. "Coexpression and Copurification of RNA–Protein Complexes in Escherichia coli." In RNA Scaffolds, 67–73. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1499-0_6.
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Webster, Kyle, Luigi Sasso, and Laura J. Domigan. "Adding Function to Protein Scaffolds." In Methods in Molecular Biology, 119–47. New York, NY: Springer US, 2019. http://dx.doi.org/10.1007/978-1-4939-9869-2_8.
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McLane, Joshua S., Nicholas J. Schaub, Ryan J. Gilbert, and Lee A. Ligon. "Electrospun Nanofiber Scaffolds for Investigating Cell–Matrix Adhesion." In Adhesion Protein Protocols, 371–88. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-538-5_23.
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Risso, Valeria A., and Jose M. Sanchez-Ruiz. "Resurrected Ancestral Proteins as Scaffolds for Protein Engineering." In Directed Enzyme Evolution: Advances and Applications, 229–55. Cham: Springer International Publishing, 2017. http://dx.doi.org/10.1007/978-3-319-50413-1_9.
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Avrutina, Olga. "Synthetic Cystine-Knot Miniproteins – Valuable Scaffolds for Polypeptide Engineering." In Protein Targeting Compounds, 121–44. Cham: Springer International Publishing, 2015. http://dx.doi.org/10.1007/978-3-319-22473-2_7.
Full textConference papers on the topic "Protein scaffolds"
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Ozbolat, Ibrahim T., A. K. M. B. Khoda, and Bahattin Koc. "Bioadditive Manufacturing of Hybrid Tissue Scaffolds for Controlled Release Kinetics." In ASME 2012 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/imece2012-86218.
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Development of engineered tissue scaffolds with superior control over cell-protein interactions is still very much infancy. Advancing through heterogeneous multifold scaffolds with controlled release fashion enables synchronization of regenerating tissue with the release kinetics of loaded biomolecules. This might be an engineering challenge and promising approach for improved and efficient tissue regeneration. The most critical limitations: the selection of proper protein(s) incorporation, and precise control over concentration gradient and timing should be overcome. Hence, tissue scaffolds need to be fabricated in a way that proteins or growth factors should be incorporated and released in a specific spatial and temporal orientation to mimic the natural tissue regeneration process. Spatial and temporal control over heterogeneous porous tissue scaffolds can be achieved by controlling two important parameters: (i) internal architecture with enhanced fluid transport, and (ii) distribution of scaffold base material and loaded modifiers. In this research, heterogeneous tissue scaffolds are designed considering both the parameters. Firstly, the three-dimensional porous structures of the scaffold are geometrically partition into functionally uniform porosity regions and controlled spatial micro-architecture has been achieved using a functionally gradient porosity function. The bio-fabrication of the designed internal porous architecture has been performed using a single nozzle bioadditive manufacturing system. The internal architecture scheme is developed to enhance fluid transport with continuous base material deposition. Next, the hybrid tissue scaffolds are modeled with varying material characteristics to mediate the release of base material and enclosed biological modifiers are proposed based on tissue engineering requirements. The hybrid scaffolds are fabricated for spatial control of biomolecules and base material to synchronize the release kinetics with tissue regeneration. A pressure-assisted multi-chamber single nozzle bioadditive manufacturing system is used to fabricate hybrid scaffolds.
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Larsen, Melinda, Riffard Jean-Gilles, David Soscia, Sharon Sequeira, Michael Melfi, Anand Gadre, and James Castracane. "Development of Nanofiber Scaffolds for Engineering an Artificial Salivary Gland." In ASME 2010 First Global Congress on NanoEngineering for Medicine and Biology. ASMEDC, 2010. http://dx.doi.org/10.1115/nemb2010-13372.
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There is currently a significant clinical need for artificial salivary glands as a therapeutic option for patients suffering from xerostomia. To achieve unidirectional fluid secretion, the epithelial acinar cells must establish and maintain polarity by partitioning the plasma membrane into distinct apical and basolateral membrane surfaces to achieve unidirectional fluid secretion. Establishment and maintenance of epithelial acinar cell polarity has been difficult to achieve in vitro, and yet is critical saliva secretion in an engineered salivary gland. Physical properties of the scaffold provided to epithelial cells will likely influence their ability to differentiate and achieve apical-basal polarity. We have engineered nanofiber matrices using the biocompatible polymer, PLGA (poly-L-lactic-co-glycolic acid) having differing topology and organization and documented the structure of these scaffolds using SEM. We evaluated the effects of several factors on epithelial cell attachment, self-organization, and apico-basal polarity on the scaffolds using confocal microscopy to examine expression and organization of apical tight junction proteins, ZO-1 and claudins, and basal markers, such as integrin α6 and the ECM protein fibronectin. The surface of the nanofiber matrix was functionalized with chemically-linked ligands to further optimize apical-basal polarity. These studies will identify an optimal scaffold for future use in an engineered functional salivary gland construct.
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Straley, K., and S. C. Heilshorn. "Designer protein-based scaffolds for neural tissue engineering." In 2009 Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE, 2009. http://dx.doi.org/10.1109/iembs.2009.5334310.
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Whitehead, Tonya J., and Harini G. Sundararaghavan. "Electrospun Hyaluronic Acid Scaffolds Containing Microspheres for Protein Delivery to Support Peripheral Nerve Growth." In ASME 2013 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/sbc2013-14630.
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Peripheral nerve injury can cause lifelong pain, loss of function, and decreased quality of life. The gold standard of repair is a nerve autograft; however this requires additional surgeries and can cause donor site morbidity. As an alternative, nerve growth conduits are being developed to guide he existing nerves to cross these injured gaps. Electrospinning has emerged as a popular method to produce fibrous scaffolds for use in tissue engineering applications. However, limited work has been done electrospinning Hyaluronic Acid (HA) a major component of the extra cellular matrix. Cells respond to several factors in their environment including chemical, mechanical, topographical and adhesion cues.1 Using electrospinning along with microspheres allows us to control mechanical, topographical, and chemical signals within our scaffold. Axons are known to respond to topographical cues, prefer ‘soft’ substrates and grow faster in the presence of Nerve Growth Factor (NGF). We can precisely control the mechanics of our scaffold by conjugating methacrylates to the HA backbone and crosslinking under UV light. We also use the rotation speed of the collection mandrel to create fibers that are aligned along one axis. Adhesivity is achieved by coating the finished scaffold with fibronectin. Microspheres are included to release protein and create a chemical signal. These characteristics combined mimic the natural environment of nervous tissue.
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Walti, Christoph, Rajan Sharma, and Giles Davies. "RecA protein mediated nano-scale patterning of DNA scaffolds." In 2010 IEEE 3rd International Nanoelectronics Conference (INEC 2010). IEEE, 2010. http://dx.doi.org/10.1109/inec.2010.5424745.
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Shim, Jin-Hyung, Jong Young Kim, Kyung Shin Kang, Jung Kyu Park, Sei Kwang Hahn, and Dong-Woo Cho. "Development of HA-PLGA Scaffold Encapsulating Intact BMP-2 Using Solid Freeform Fabrication Technology." In ASME 2011 International Manufacturing Science and Engineering Conference. ASMEDC, 2011. http://dx.doi.org/10.1115/msec2011-50259.
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Tissue engineering is an interdisciplinary field that focuses on restoring and repairing tissues or organs. Cells, scaffolds, and biomolecules are recognized as three main components of tissue engineering. Solid freeform fabrication (SFF) technology is required to fabricate three-dimensional (3D) porous scaffolds to provide a 3D environment for cellular activity. SFF technology is especially advantageous for achieving a fully interconnected, porous scaffold. Bone morphogenic protein-2 (BMP-2), an important biomolecule, is widely used in bone tissue engineering to enhance bone regeneration activity. However, methods for the direct incorporation of intact BMP-2 within 3D scaffolds are rare. In this work, 3D porous scaffolds with poly(lactic-co-glycolic acid) chemically grafted hyaluronic acid (HA-PLGA), in which intact BMP-2 was directly encapsulated, were successfully fabricated using SFF technology. BMP-2 was previously protected by poly(ethylene glycol) (PEG), and the BMP-2/PEG complex was incorporated in HA-PLGA using an organic solvent. The HAPLGA/PEG/BMP-2 mixture was dissolved in chloroform and deposited via a multi-head deposition system (MHDS), one type of SFF technology, to fabricate a scaffold for tissue engineering. An additional air blower system and suction were installed in the MHDS for the solvent-based fabrication method. An in vitro evaluation of BMP-2 release was conducted, and prolonged release of intact BMP-2, for up to 28 days, was confirmed. After confirmation of advanced proliferation of pre osteoblasts, a superior differentiation effect of the HA-PLGA/PEG/BMP-2 scaffold was validated by measuring high expression levels of bone-specific markers, such as alkaline phosphatase (ALP) and osteocalcin (OC). We show that our solvent-based fabrication is a non-toxic method for restoring cellular activity. Moreover, the HAPLGA/PEG/BMP-2 scaffold was effective for bone regeneration.
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Yanoso, Laura, Justin Jacobson, Tulin Dadali, David Reynolds, and Hani Awad. "Evaluation of Polylactic Acid/Beta-Tricalcium Phosphate Scaffolds as Segmental Bone Graft Substitutes." In ASME 2008 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2008. http://dx.doi.org/10.1115/sbc2008-192978.
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The use of processed structural allografts for treatment of massive segmental defects in long bones can be complicated by poor incorporation and remodeling of the devitalized graft, foreign-body reaction and micro-damage accumulation which often leads to catastrophic graft failure [1]. It is therefore useful to develop a bioengineered, biodegradable scaffold that is able to stimulate healing of the defect region. The use of bioengineered scaffolds has been limited due to their poor mechanical strength that does not permit withstanding large in vivo loads and due to their poor osteoinductive properties. We therefore investigated the use of rigid polylactic acid/beta-tricalcium phosphate (PLA/βTCP) composites used in conjunction with osteoinductive factors such as growth hormones (parathyroid hormone (PTH)) and growth factors (bone morphogenic protein-2 (BMP-2) & vascular endothelial growth factor (VEGF)) to stimulate bone formation and vessel ingrowth in the segmental defect region. We examined the physical characteristics of the scaffolds, and evaluated their osteoinductive potential in a clinically-relevant mouse model of a femoral segmental defect with or without PTH treatment. Finally, we used an ectopic bone formation model to assess the efficacy of the scaffold in site-specific delivery of bone anabolic factors.
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Gaudet, Ian D., and David I. Shreiber. "Photocrosslinkable Type-I Collagen for In Situ Material Modification." In ASME 2011 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2011. http://dx.doi.org/10.1115/sbc2011-53125.
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Hydrogels are attractive materials for use as tissue engineering scaffolds [1]. Natural hydrogels, such as collagen, are both cytocompatible and highly biofunctional. However, they have somewhat constrained material properties and have an inherently large variability in composition due to their biological origin, making them more difficult to work with from an engineering viewpoint[2–4]. Here, we aim to use type 1 collagen — the most abundant protein in the body that maintains excellent cytocompatibility and can self assemble into a fibrillar network — as a base component for a photocrosslinkable biomaterial. One main advantage of this system over previous studies attempting to photocrosslink collagen is that the collagen retains its ability to self assemble, which provides a stable environment into which localized modifications can be made to the stiffness, porosity, and biochemical properties of the hydrogel scaffold. By taking advantage of the spatial control provided by the system, we can create complex 3-dimensional hydrogel scaffolds that have non-homogenous microenvironments.
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Papa, Antonio, Vincenzo Guarino, Valentina Cirillo, Olimpia Oliviero, and Luigi Ambrosio. "Optimization of protein cross-linking in bicomponent electrospun scaffolds for therapeutic use." In THE SECOND ICRANET CÉSAR LATTES MEETING: Supernovae, Neutron Stars and Black Holes. AIP Publishing LLC, 2015. http://dx.doi.org/10.1063/1.4937286.
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Nguyen, Christopher, Sara Rudolph, David L. Kaplan, and Srivalleesha Mallidi. "Collagen detection in silk protein-based scaffolds through ultrasound and photoacoustic imaging." In Photons Plus Ultrasound: Imaging and Sensing 2022, edited by Alexander A. Oraevsky and Lihong V. Wang. SPIE, 2022. http://dx.doi.org/10.1117/12.2610402.
Full textReports on the topic "Protein scaffolds"
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Morrison, Mark, Joshuah Miron, Edward A. Bayer, and Raphael Lamed. Molecular Analysis of Cellulosome Organization in Ruminococcus Albus and Fibrobacter Intestinalis for Optimization of Fiber Digestibility in Ruminants. United States Department of Agriculture, March 2004. http://dx.doi.org/10.32747/2004.7586475.bard.
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Improving plant cell wall (fiber) degradation remains one of the highest priority research goals for all ruminant enterprises dependent on forages, hay, silage, or other fibrous byproducts as energy sources, because it governs the provision of energy-yielding nutrients to the host animal. Although the predominant species of microbes responsible for ruminal fiber degradation are culturable, the enzymology and genetics underpinning the process are poorly defined. In that context, there were two broad objectives for this proposal. The first objective was to identify the key cellulosomal components in Ruminococcus albus and to characterize their structural features as well as regulation of their expression, in response to polysaccharides and (or) P AA/PPA. The second objective was to evaluate the similarities in the structure and architecture of cellulosomal components between R. albus and other ruminal and non-ruminal cellulolytic bacteria. The cooperation among the investigators resulted in the identification of two glycoside hydrolases rate-limiting to cellulose degradation by Ruminococcus albus (Cel48A and CeI9B) and our demonstration that these enzymes possess a novel modular architecture specific to this bacterium (Devillard et al. 2004). We have now shown that the novel X-domains in Cel48A and Cel9B represent a new type of carbohydrate binding module, and the enzymes are not part of a ceiluiosome-like complex (CBM37, Xu et al. 2004). Both Cel48A and Cel9B are conditionally expressed in response to P AA/PPA, explaining why cellulose degradation in this bacterium is affected by the availability of these compounds, but additional studies have shown for the first time that neither PAA nor PPA influence xylan degradation by R. albus (Reveneau et al. 2003). Additionally, the R. albus genome sequencing project, led by the PI. Morrison, has supported our identification of many dockerin containing proteins. However, the identification of gene(s) encoding a scaffoldin has been more elusive, and recombinant proteins encoding candidate cohesin modules are now being used in Israel to verify the existence of dockerin-cohesin interactions and cellulosome production by R. albus. The Israeli partners have also conducted virtually all of the studies specific to the second Objective of the proposal. Comparative blotting studies have been conducted using specific antibodies prepare against purified recombinant cohesins and X-domains, derived from cellulosomal scaffoldins of R. flavefaciens 17, a Clostridium thermocellum mutant-preabsorbed antibody preparation, or against CbpC (fimbrial protein) of R. albus 8. The data also suggest that additional cellulolytic bacteria including Fibrobacter succinogenes S85, F. intestinalis DR7 and Butyrivibrio fibrisolvens Dl may also employ cellulosomal modules similar to those of R. flavefaciens 17. Collectively, our work during the grant period has shown that R. albus and other ruminal bacteria employ several novel mechanisms for their adhesion to plant surfaces, and produce both cellulosomal and non-cellulosomal forms of glycoside hydrolases underpinning plant fiber degradation. These improvements in our mechanistic understanding of bacterial adhesion and enzyme regulation now offers the potential to: i) optimize ruminal and hindgut conditions by dietary additives to maximize fiber degradation (e.g. by the addition of select enzymes or PAA/PPA); ii) identify plant-borne influences on adhesion and fiber-degradation, which might be overcome (or improved) by conventional breeding or transgenic plant technologies and; iii) engineer or select microbes with improved adhesion capabilities, cellulosome assembly and fiber degradation. The potential benefits associated with this research proposal are likely to be realized in the medium term (5-10 years).
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Fahima, Tzion, and Jorge Dubcovsky. Map-based cloning of the novel stripe rust resistance gene YrG303 and its use to engineer 1B chromosome with multiple beneficial traits. United States Department of Agriculture, January 2013. http://dx.doi.org/10.32747/2013.7598147.bard.
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Research problem: Bread wheat (Triticumaestivum) provides approximately 20% of the calories and proteins consumed by humankind. As the world population continues to increase, it is necessary to improve wheat yields, increase grain quality, and minimize the losses produced by biotic and abiotic stresses. Stripe rust, caused by Pucciniastriiformisf. sp. tritici(Pst), is one of the most destructive diseases of wheat. The new pathogen races are more virulent and aggressive than previous ones and have produced large economic losses. A rich source for stripe-rust resistance genes (Yr) was found in wild emmer wheat populations from Israel. Original Project goals: Our long term goal is to identify, map, clone, characterize and deploy in breeding, novel wild emmer Yr genes, and combine them with multiple beneficial traits. The current study was aiming to map and clone YrG303 and Yr15, located on chromosome 1BS and combine them with drought resistance and grain quality genes. Positional cloning of YrG303/Yr15: Fine mapping of these genes revealed that YrG303 is actually allelic to Yr15. Fine genetic mapping using large segregating populations resulted in reduction of the genetic interval spanning Yr15 to less than 0.1 cM. Physical mapping of the YrG303/Yr15 locus was based on the complete chromosome 1BS physical map of wheat constructed by our group. Screening of 1BS BAC library with Yr15 markers revealed a long BAC scaffold covering the target region. The screening of T. dicoccoidesaccession-specific BAC library with Yr15 markers resulted in direct landing on the target site. Sequencing of T. dicoccoidesBAC clones that cover the YrG303/Yr15 locus revealed a single candidate gene (CG) with conserved domains that may indicate a role in disease resistance response. Validation of the CG was carried out using EMS mutagenesis (loss-of- function approach). Sequencing of the CG in susceptible yr15/yrG303 plants revealed three independent mutants that harbour non-functional yr15/yrG303 alleles within the CG conserved domains, and therefore validated its function as a Pstresistance gene. Evaluation of marker-assisted-selection (MAS) for Yr15. Introgressions of Yr15 into cultivated wheat are widely used now. Recently, we have shown that DNA markers linked to Yr15 can be used as efficient tools for introgression of Yr15 into cultivated wheat via MAS. The developed markers were consistent and polymorphic in all 34 tested introgressions and are the most recommended markers for the introgression of Yr15. These markers will facilitate simultaneous selection for multiple Yr genes and help to avoid escapees during the selection process. Engineering of improved chromosome 1BS that harbors multiple beneficial traits. We have implemented the knowledge and genetic resources accumulated in this project for the engineering of 1B "super-chromosome" that harbors multiple beneficial traits. We completed the generation of a chromosome including the rye 1RS distal segment associated with improved drought tolerance with the Yr gene, Yr15, and the strong gluten allele 7Bx-over-expressor (7Bxᴼᴱ). We have completed the introgression of this improved chromosome into our recently released variety Patwin-515HP and our rain fed variety Kern, as well as to our top breeding lines UC1767 and UC1745. Elucidating the mechanism of resistance exhibited by Yr36 (WKS1). The WHEAT KINASE START1 (WKS1) resistance gene (Yr36) confers partial resistance to Pst. We have shown that wheat plants transformed with WKS1 transcript are resistant to Pst. WKS1 is targeted to the chloroplast where it phosphorylates the thylakoid-associatedascorbateperoxidase (tAPX) and reduces its ability to detoxify peroxides. Based on these results, we propose that the phosphorylation of tAPX by WKS1 reduces the ability of the cells to detoxify ROS and contributes to cell death. Distribution and diversity of WKS in wild emmer populations. We have shown that WKS1 is present only in the southern distribution range of wild emmer in the Fertile Crescent. Sequence analysis revealed a high level of WKS1 conservation among wild emmer populations, in contrast to the high level of diversity observed in NB-LRR genes. This phenomenon shed some light on the evolution of genes that confer partial resistance to Pst. Three new WKS1 haplotypes displayed a resistance response, suggesting that they can be useful to improve wheat resistance to Pst. In summary, we have improved our understanding of cereals’ resistance mechanisms to rusts and we have used that knowledge to develop improved wheat varieties.