Browsing by Subject "POLYMERIZATION"

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  • Colombo, Jessica; Antkowiak, Adrien; Kogan, Konstantin; Kotila, Tommi; Elliott, Jenna; Guillotin, Audrey; Lappalainen, Pekka; Michelot, Alphée (2021)
    Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding of actin dynamics and energetics remains limited due to the lack of high-quality probes. Most current probes affect dynamics of actin or its interactions with actin-binding proteins (ABPs), and cannot track the bound nucleotide. Here, we identify a family of highly sensitive fluorescent nucleotide analogues structurally compatible with actin. We demonstrate that these fluorescent nucleotides bind to actin, maintain functional interactions with a number of essential ABPs, are hydrolyzed within actin filaments, and provide energy to power actin-based processes. These probes also enable monitoring actin assembly and nucleotide exchange with single-molecule microscopy and fluorescence anisotropy kinetics, therefore providing robust and highly versatile tools to study actin dynamics and functions of ABPs.
  • Collins, Scott; Hasan, Galib; Joshi, Anuj; McIndoe, J. Scott; Linnolahti, Mikko (2021)
    Density functional theory calculations on neutral sheet models for methylaluminoxane (MAO) indicate that these structures, containing 5-coordinate and 4-coordinate Al, are likely precursors to ion-pairs seen during the hydrolysis of trimethylaluminum (Me3Al) in the presence of donors such as octamethyltrisiloxane (OMTS). Ionization by both methide ([Me](-)) and [Me2Al](+) abstraction, involving this donor, were studied by polarizable continuum model calculations in fluorobenzene (PhF) and o-difluorobenzene (DFB) media. These studies suggest that low MW, 5-coordinate sheets ionize by [Me2Al](+) abstraction, while [Me](-) abstraction from Me3Al-OMTS is the likely process for higher MW 4-coordinate sheets. Further, comparison of anion stabilities per mole of aluminoxane repeat unit (MeAlO)(n), suggest that anions such as [(MeAlO)(7)(Me3Al)(4)Me](-)=[7,4](-) are especially stable compared to higher homologues, even though their neutral precursors are unstable.
  • Semenyuk, Pavel; Tiainen, Tony; Hietala, Sami; Tenhu, Heikki; Aseyev, Vladimir; Muronetz, Vladimir (2019)
    Stabilization of the enzymes under stress conditions is of special interest for modern biochemistry, bioengineering, as well as for formulation and target delivery of protein-based drugs. Aiming to achieve an efficient stabilization at elevated temperature with no influence on the enzyme under normal conditions, we studied chaperone-like activity of thermoresponsive polymers based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) toward two different proteins, glyceraldehyde-3-phosphate dehydrogenase and chicken egg lysozyme. The polymers has been shown to do not interact with the folded protein at room temperature but form a complex upon heating to either protein unfolding or polymer phase transition temperature. A PDMAEMA-PEO block copolymer with a dodecyl end-group (d-PDMAEMA-PEO) as well as PDMAEMA-PEO without the dodecyl groups protected the denatured protein against aggregation in contrast to PDMAEMA homopolymer. No effect of the polymers on the enzymatic activity of the client protein was observed at room temperature. The polymers also partially protected the enzyme against inactivation at high temperature. The results provide a platform for creation of artificial chaperones with unfolded protein recognition which is a major feature of natural chaperones. (C) 2018 Elsevier B.V. All rights reserved.
  • Zippel, Christoph; Spuling, Eduard; Hassan, Zahid; Polamo, Mika; Nieger, Martin; Bräse, Stefan (2020)
    Selective activation/functionalization of C-H bonds has emerged as an atom- and step-economical process at the forefront of modern synthetic chemistry. This work reports palladium-catalyzed exclusivelypara-selective C-H activation/aryl-aryl bond formation with a preference overN-arylation under the Buchwald-Hartwig amination reaction of 4-phenylamino[2.2]paracyclophane. This innovative synthetic strategy allows a facile preparation of [2.2]paracyclophane derivatives featuring disparatepara-substitutions at C-4 and C-7 positions in a highly selective manner, gives access to a series of potential candidates for [2.2]paracyclophane-derived new planar chiral ligands. The unprecedented behavior in reactivity and preferential selectivity of C-C coupling over C-N bond formation via C-H activation is unique to the [2.2]paracyclophane scaffold compared to the non-cyclophane analogue under the same reaction conditions. Selective C-H activation/aryl-aryl bond formation and sequential C-N coupling product formation is evidenced unambiguously by X-ray crystallography.
  • Tykesson, Emil; Hassinen, Antti; Zielinska, Katarzyna; Thelin, Martin A.; Frati, Giacomo; Ellervik, Ulf; Westergren-Thorsson, Gunilla; Malmström, Anders; Kellokumpu, Sakari; Maccarana, Marco (2018)
    During the biosynthesis of chondroitin/dermatan sulfate (CS/DS), a variable fraction of glucuronic acid is converted to iduronic acid through the activities of two epimerases, dermatan sulfate epimerases 1 (DS-epi1) and 2 (DS-epi2). Previous in vitro studies indicated that without association with other enzymes, DS-epi1 activity produces structures that have only a few adjacent iduronic acid units. In vivo, concomitant with epimerization, dermatan 4-O-sulfotransferase 1 (D4ST1) sulfates the GalNAc adjacent to iduronic acid. This sulfation facilitates DS-epi1 activity and enables the formation of long blocks of sulfated iduronic acid-containing domains, which can be major components of CS/DS. In this report, we used recombinant enzymes to confirm the concerted action of DS-epi1 and D4ST1. Confocal microscopy revealed that these two enzymes colocalize to the Golgi, and FRET experiments indicated that they physically interact. Furthermore, FRET, immunoprecipitation, and cross-linking experiments also revealed that DS-epi1, DS-epi2, and D4ST1 form homomers and are all part of a hetero-oligomeric complex where D4ST1 directly interacts with DS-epi1, but not with DS-epi2. The cooperation of DS-epi1 with D4ST1 may therefore explain the processive mode of the formation of iduronic acid blocks. In conclusion, the iduronic acid-forming enzymes operate in complexes, similar to other enzymes active in glycosaminoglycan biosynthesis. This knowledge shed light on regulatory mechanisms controlling the biosynthesis of the structurally diverse CS/DS molecule.
  • Burley, Jonathan C.; Motherwell, W. D. Sam; Maaranen, Janne; Ringwald, Markus (2007)
  • Hahn, Lukas; Maier, Matthias; Stahlhut, Philipp; Beudert, Matthias; Flegler, Vanessa; Forster, Stefan; Altmann, Alexander; Toeppke, Fabian; Fischer, Karl; Seiffert, Sebastian; Boettcher, Bettina; Luehmann, Tessa; Luxenhofer, Robert (2020)
    Amphiphilic block copolymers that undergo (reversible) physical gelation in aqueous media are of great interest in ditIerent areas including drug delivery, tissue engineering, regenerative medicine, and biofabrication. We investigated a small library of ABA-type triblock copolymers comprising poly(2-methyl-2-oxazoline) as the hydrophilic shell A and different aromatic poly(2-oxazoline)s and poly(2-oxazine)s cores B in an aqueous solution at different concentrations and temperatures. Interestingly, aqueous solutions of poly(2-methyl-2-oxazoline)-block-poly(2-phenyl-2-oxazine)-block-poly(2-methyl-2-oxazoline) (PMeOx-b-PPheOzi-b-PMeOx) undergo inverse thermogelation below a critical temperature by forming a reversible nanoscale wormlike network. The viscoelastic properties of the resulting gel can be conveniently tailored by the concentration and the polymer composition. Storage moduli of up to 110 kPa could be obtained while the material retains shear-thinning and rapid self-healing properties. We demonstrate three-dimensional (3D) printing of excellently defined and shape-persistent 24-layered scaffolds at different aqueous concentrations to highlight its application potential, e.g., in the research area of biofabrication. A macroporous microstructure, which is stable throughout the printing process, could be confirmed via cryo-scanning electron microscopy (SEM) analysis. The absence of cytotoxicity even at very high concentrations opens a wide range of different applications for this first-in-class material in the field of biomaterials.
  • Yuen, Michaele; Sandaradura, Sarah A.; Dowling, James J.; Kostyukova, Alla S.; Moroz, Natalia; Quinlan, Kate G.; Lehtokari, Vilma-Lotta; Ravenscroft, Gianina; Todd, Emily J.; Ceyhan-Birsoy, Ozge; Gokhin, David S.; Maluenda, Jerome; Lek, Monkol; Nolent, Flora; Pappas, Christopher T.; Novak, Stefanie M.; D'Amico, Adele; Malfatti, Edoardo; Thomas, Brett P.; Gabriel, Stacey B.; Gupta, Namrata; Daly, Mark J.; Ilkovski, Biljana; Houweling, Peter J.; Davidson, Ann E.; Swanson, Lindsay C.; Brownstein, Catherine A.; Gupta, Vandana A.; Medne, Livija; Shannon, Patrick; Martin, Nicole; Bick, David P.; Flisberg, Anders; Holmberg, Eva; Van den Bergh, Peter; Lapunzina, Pablo; Waddell, Leigh B.; Sioboda, Darcee D.; Bertini, Enrico; Chitayat, David; Telfer, William R.; Laquerriere, Annie; Gregorio, Carol C.; Ottenheijm, Coen A. C.; Boennemann, Carsten G.; Pelin, Katarina; Beggs, Alan H.; Hayashi, Yukiko K.; Romero, Norma B.; Laing, Nigel G.; Nishino, Ichizo; Wallgren-Pettersson, Carina; Melki, Judith; Fowler, Velia M.; MacArthur, Daniel G.; North, Kathryn N.; Clarke, Nigel F. (2014)
  • Lahtinen, Maarit; Heinonen, Petri; Oivanen, Mikko; Karhunen, Pirkko; Kruus, Kristiina; Sipilä, Jussi (2013)
  • Ghafourisaleh, Saba; Popov, Georgi; Leskela, Markku; Putkonen, Matti; Ritala, Mikko (2021)
    Because of its high conductivity and intrinsic stability, poly(3,4-ethylenedioxythiophene (PEDOT) has gained great attention both in academic research and industry over the years. In this study, we used the oxidative molecular layer deposition (oMLD) technique to deposit PEDOT from 3,4-ethylenedioxythiophene (EDOT) and a new inorganic oxidizing agent, rhenium pentachloride (ReCl5). We extensively characterized the properties of the films by scanning electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy (XPS), energy-dispersive X-ray spectroscopy (EDS), Raman, and conductivity measurements. The oMLD of polymers is based on the sequential adsorption of the monomer and its oxidation-induced polymerization. However, oMLD has been scarcely used because of the challenge of finding a suitable combination of volatile, reactive, and stable organic monomers applicable at high temperatures. ReCl5 showed promising properties in oMLD because it has high thermal stability and high oxidizing ability for EDOT. PEDOT films were deposited at temperatures of 125-200 degrees C. EDS and XPS measurements showed that the as-deposited films contained residues of rhenium and chlorine, which could be removed by rinsing the films with deionized water. The polymer films were transparent in the visible region and showed relatively high electrical conductivities within the 2-2000 S cm(-1) range.
  • Otsuka, Issei; Zhang, Xuewei; Winnik, Francoise M. (2019)
    Aqueous dispersions of nanogels that respond to switches in environmental pH and/or temperature by changes in their hydrodynamic radius (R-h) and/or zeta-potential are prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization-induced thermal (70 degrees C) self-assembly (PITSA) of N-isopropylacrylamide (NIPAM) in the presence of a poly(methacrylic acid) (PMAA)-substituted macromolecular chain transfer agent and a cross-linker. Photochromic spiropyran (SP) moieties are coupled to the carboxylic acid groups of the nanogels. Upon UV irradiation, the neutral SP isomerizes to the zwitterionic merocyanine (ME) form. Upon UV light irradiation, microgels formed by assembly of SP nanogels undergo a collective motion toward the UV-light source.
  • Senju, Yosuke; Lappalainen, Pekka (2019)
    The actin cytoskeleton is indispensable for several cellular processes, including migration, morphogenesis, polarized growth, endocytosis, and phagocytosis. The organization and dynamics of the actin cytoskeleton in these processes are regulated by Rho family small GTPases and kinase-phosphatase pathways. Moreover, membrane phospholipids, especially the phosphatidylinositol phosphates have emerged as important regulators of actin dynamics. From these, PI(4,5)P-2 is the most abundant at the plasma membrane, and directly regulates the activities and subcellular localizations of numerous actin-binding proteins. Here, we discuss recent studies demonstrating that actin-binding proteins interact with PI(4,5)P-2-rich membranes through drastically different affinities and dynamics correlating with their roles in cytoskeletal dynamics. Moreover, by using mesenchymal cell migration and clathrin-mediated endocytosis as examples, we present a model for how interplay between PI(4,5)P-2 and actin-binding proteins control the actin cytoskeleton in cells.
  • Kirila, Tatyana; Smirnova, Anna; Aseyev, Vladimir; Tenkovtsev, Andrey; Tenhu, Heikki; Filippov, Alexander (2021)
    The behavior of star-shaped six-arm poly-2-alkyl-2-oxazines and poly-2-alkyl-2-oxazolines in aqueous solutions on heating was studied by light scattering, turbidimetry and microcalorimetry. The core of stars was hexaaza [2(6)] orthoparacyclophane and the arms were poly-2-ethyl-2-oxazine, poly-2-isopropyl-2-oxazine, poly-2-ethyl-2-oxazoline, and poly-2-isopropyl-2-oxazoline. The arm structure affects the properties of polymers already at low temperatures. Molecules and aggregates were present in solutions of poly-2-alkyl-2-oxazines, while aggregates of two types were observed in the case of poly-2-alkyl-2-oxazolines. On heating below the phase separation temperature, the characteristics of the investigated solutions did not depend practically on temperature. An increase in the dehydration degree of poly-2-alkyl-2-oxazines and poly-2-alkyl-2-oxazolines led to the formation of intermolecular hydrogen bonds, and aggregation was the dominant process near the phase separation temperature. It was shown that the characteristics of the phase transition in solutions of the studied polymer stars are determined primarily by the arm structure, while the influence of the molar mass is not so significant. In comparison with literature data, the role of the hydrophobic core structure in the formation of the properties of star-shaped polymers was analyzed.
  • Fliervoet, Lies A. L.; Lisitsyna, Ekaterina S.; Durandin, Nikita A.; Kotsis, Ilias; Maas-Bakker, Roel F. M.; Yliperttula, Marjo; Hennink, Wim E.; Vuorimaa-Laukkanen, Elina; Vermonden, Tina (2020)
    Combining multiple stimuli-responsive functionalities into the polymer design is an attractive approach to improve nucleic acid delivery. However, more in-depth fundamental understanding how the multiple functionalities in the polymer structures are influencing polyplex formation and stability is essential for the rational development of such delivery systems. Therefore, in this study the structure and dynamics of thermosensitive polyplexes were investigated by tracking the behavior of labeled plasmid DNA (pDNA) and polymer with time-resolved fluorescence spectroscopy using fluorescence resonance energy transfer (FRET). The successful synthesis of a heterofunctional poly(ethylene glycol) (PEG) macroinitiator containing both an atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain-transfer (RAFT) initiator is reported. The use of this novel PEG macroinitiator allows for the controlled polymerization of cationic and thermosensitive linear triblock copolymers and labeling of the chain-end with a fluorescent dye by maleimide-thiol chemistry. The polymers consisted of a thermosensitive poly(N-isopropylacrylamide) (PNIPAM, N), hydrophilic PEG (P), and cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA, D) block, further referred to as NPD. Polymer block D chain-ends were labeled with Cy3, while pDNA was labeled with FITC. The thermosensitive NPD polymers were used to prepare pDNA polyplexes, and the effect of the N/P charge ratio, temperature, and composition of the triblock copolymer on the polyplex properties were investigated, taking nonthermosensitive PD polymers as the control. FRET was observed both at 4 and 37 degrees C, indicating that the introduction of the thermosensitive PNIPAM block did not compromise the polyplex structure even above the polymer's cloud point. Furthermore, FRET results showed that the NPD- and PD-based polyplexes have a less dense core compared to polyplexes based on cationic homopolymers (such as PEI) as reported before. The polyplexes showed to have a dynamic character meaning that the polymer chains can exchange between the polyplex core and shell. Mobility of the polymers allow their uniform redistribution within the polyplex and this feature has been reported to be favorable in the context of pDNA release and subsequent improved transfection efficiency, compared to nondynamic formulations.
  • Immonen, Mika; Chandrakusuma, Angga; Sibakov, Juhani; Poikelispaa, Minna; Sontag-Strohm, Tuula (2021)
    Grain protein fractions have great potential as ingredients that contain high amounts of valuable nutritional components. The aim of this study was to study the rheological behavior of destarched oat and pea proteins and their blends in extrusion-like conditions with a closed cavity rheometer. Additionally, the possibility of producing fibrous structures with high-moisture extrusion from a blend of destarched oat and pea protein was investigated. In the temperature sweep measurement (60-160 degrees C) of the destarched oat protein concentrate and pea protein isolate blend, three denaturation and polymerization sections were observed. In addition, polymerization as a function of time was recorded in the time sweep measurements. The melting temperature of grain proteins was an important factor when producing texturized structures with a high-moisture extrusion. The formation of fibrillar structures was investigated with high-moisture extrusion from the destarched oat and pea protein blend at temperatures ranging from 140 to 170 degrees C. The protein-protein interactions were significantly influenced in the extruded samples. This was due to a decrease in the amount of extractable protein in selective buffers. In particular, there was a decrease in non-covalent and covalent bonds due to the formation of insoluble protein complexes.
  • Semenyuk, Pavel I.; Kurochkina, Lidia P.; Mäkinen, Lauri; Muronetz, Vladimir I.; Hietala, Sami (2021)
    A prospective technology for reversible enzyme complexation accompanied with its inactivation and protection followed by reactivation after a fast thermocontrolled release has been demonstrated. A thermoresponsive polymer with upper critical solution temperature, poly(N-acryloyl glycinamide) (PNAGA), which is soluble in water at elevated temperatures but phase separates at low temperatures, has been shown to bind lysozyme, chosen as a model enzyme, at a low temperature (10 & DEG;C and lower) but not at room temperature (around 25 & DEG;C). The cooling of the mixture of PNAGA and lysozyme solutions from room temperature resulted in the capturing of the protein and the formation of stable complexes; heating it back up was accompanied by dissolving the complexes and the release of the bound lysozyme. Captured by the polymer, lysozyme was inactive, but a temperature-mediated release from the complexes was accompanied by its reactivation. Complexation also partially protected lysozyme from proteolytic degradation by proteinase K, which is useful for biotechnological applications. The obtained results are relevant for important medicinal tasks associated with drug delivery such as the delivery and controlled release of enzyme-based drugs.
  • Salminen, Linda; Karjalainen, Erno; Aseyev, Vladimir; Tenhu, Heikki (2021)
    This article introduces butyl acrylate-based materials that are toughened with dynamic crosslinkers. These dynamic crosslinkers are salts where both the anion and cation polymerize. The ion pairs between the polymerized anions and cations form dynamic crosslinks that break and reform under deformation. Chemical crosslinker was used to bring shape stability. The extent of dynamic and chemical crosslinking was related to the mechanical and thermal properties of the materials. Furthermore, the dependence of the material properties on different dynamic crosslinkers-tributyl-(4-vinylbenzyl)ammonium sulfopropyl acrylate (C4ASA) and trihexyl-(4-vinylbenzyl)ammonium sulfopropyl acrylate (C6ASA)-was studied. The materials' mechanical and thermal properties were characterized by means of tensile tests, dynamic mechanical analysis, differential scanning calorimetry, and thermogravimetric analysis. The dynamic crosslinks strengthened the materials considerably. Chemical crosslinks decreased the elasticity of the materials but did not significantly affect their strength. Comparison of the two ionic crosslinkers revealed that changing the crosslinker from C4ASA to C6ASA results in more elastic, but slightly weaker materials. In conclusion, dynamic crosslinks provide substantial enhancement of mechanical properties of the materials. This is a unique approach that is utilizable for a wide variety of polymer materials.