Dissipative particle dynamics simulation of the relaxation behaviour of a triblock copolymer supramolecular network

Since block copolymers self-assemble into various nanostructures and these are widely used in soft materials such as cosmetics and paints, these continue to be the subjects of fundamental studies that progress new technologies. ABA triblock copolymers self-organise into supramolecular networks in which crosslinked structures change upon heating and other external stimulation. Supramolecular networks that consist of ABA triblock copolymers have three components: bridges, loops and dangles. These supramolecular networks have structural regions (clusters) in which end blocks of polymer chains are aggregated and connected by the internal blocks of the polymer chains. Despite of the importance of relaxation behaviour during the measurement and control of polymer materials, the molecular-level behaviour of these systems has not been addressed. We observed pull-out phenomenon that the ends of these polymers detach from clusters, and estimated characteristic detachment times by counting the number of detachments and compared it with the time of longest relaxation in that system.     

Understanding the Tendency of Amorphous Solid Dispersions to Undergo Amorphous–Amorphous Phase Separation in the Presence of Absorbed Moisture

Formulation of an amorphous solid dispersion (ASD) is one of the methods commonly considered to increase the bioavailability of a poorly water-soluble small-molecule active pharmaceutical ingredient (API). However, many factors have to be considered in designing an API–polymer system, including any potential changes to the physical stability of the API. In this study, the tendency of ASD systems containing a poorly water-soluble API and a polymer to undergo amorphous–amorphous phase separation was evaluated following exposure to moisture at increasing relative humidity. Infrared spectroscopy was used as the primary method to investigate the phase behavior of the systems. In general, it was observed that stronger drug–polymer interactions, low-ASD hygroscopicity, and a less hydrophobic API led to the formation of systems resistant to moisture-induced amorphous–amorphous phase separation. Orthogonal partial least squares analysis provided further insight into the systems, confirming the importance of the aforementioned properties. In order to design a more physically stable ASD that is resistant to moisture-induced amorphous–amorphous phase separation, it is important to consider the interplay between these properties.     

Optimization of bovine serum albumin sorption and recovery by hydrogels

Aqueous two-phase systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. Partitioning of proteins in such systems provides a powerful method for separating and purifying mixtures of biomolecules by extraction. If one of the phase forming polymers is a crosslinked gel, then the solution-controlled gel sorption may be considered as a modification of aqueous two-phase extraction. Since PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex) are common chromatographic media, we choose a PEG/dextran gel system as a model system in this study. The partitioning behavior of pure bovine serum albumin (BSA) in PEG/dextran gel systems is investigated to see the effects of variations in PEG and NaCl concentrations on the partition coefficient K. By making use of the Box-Wilson experimental design, K is shown to be maximized at 9.8 (%, w/w) PEG and 0.2 M NaCl concentrations, respectively, as 182.     

A novel nanocomposite film prepared from crosslinked cellulosic whiskers

Cellulose whiskers are increasingly being used as a reinforcing phase in polymer systems and their use is a growing area of importance in bionanocomposite research. Although the reinforcing effect of cellulose whiskers has been studied in various polymers, the impact of crosslinking cellulose whiskers has not been explored so far. This work deals with the development of novel cellulose nanocomposites, wherein the cellulose nanowhiskers are crosslinked with poly(methyl vinyl ether-co-maleic acid) and poly(ethylene glycol). The morphology of the nanocomposite was studied using atomic force microscopy (AFM), which revealed a network structure embedded in a continuous phase. The water sorption studies demonstrated that the crosslinked nanocomposites are capable of absorbing up to ～900% water and have potential to be used as hydrogels.     

An Epistemology on the Nature of Polymers

Liquids have neither a periodic structure nor the completely random character of gases therefore the detailed study of their x-ray scattering diagram encounters many difficulties. The idea of periodic regularity in molecules of liquid polymers has been an attractive proposition for the simple interpretation of liquid polymer x-ray diagrams. The categorisation of polymer substances as being between a crystal phase with a perfect order and an amorphous disordered state is an over simplification of the complex reality. For obtaining structural information, during the early stages of the application of x-ray diffraction, a near crystalline model of the molecular arrangements in liquids was utilised. However, the results from these investigations led to just an approximation of the crystalline state. Our studies have analysed the real image of Fourier space of liquid polymers, for the first time, using anomalous diffractometry. The findings show the precise atomic structure of liquid polymers when transformed, by cooling, to solid polymers. We demonstrate that there is an intermediate ordered structure, characterised by the real full image of Fourier space. This prominent state of matter, an intermediate ordered structure, is defined by a regular unit cell with a five-fold symmetry. These structural atomic studies contribute to a more detailed understanding of the properties of polymers than the traditional studies of the degree of crystallinity.     

Kinetic heterogeneity of F-actin polymers. Further evidence that the elongation reaction may occur through condensation of the actin filaments with small aggregates.

We have shown that F-actin, polymerized in 50 mM-KCl at 20 degrees C and pH 8.0, can be resolved by centrifugation into two polymer populations, which differ morphologically as well as kinetically. The first population represents about 10% of the overall polymer and is composed of small amorphous aggregates. It rapidly exchanges the bound nucleotide with free ATP in the medium, either directly or through the monomers. The second population is composed of long actin filaments. These are labelled by free ATP in the medium only through condensation with labelled small amorphous aggregates.     

Counter-ion dynamics in crosslinked poly(styrene sulfonate) systems studied by NMR

The field dependence of the longitudinal and transverse nuclear magnetic relaxation rates of 23Na+ in aqueous crosslinked Na-poly(styrene sulfonate) (PSS) systems (ion exchange resins) has been obtained as a function of the degree of crosslinking. The relaxation is considerably enhanced relative to solutions of non-crosslinked NaPSS at equal ionizable group concentration. This is due to the dynamic constraints of the polymer chains, which render the averaging of the counter-ion chain interaction less efficient. The field dependence of the relaxation rates in the crosslinked NaPSS systems reveals two processes that are out of the extreme narrowing limit. This is in contrast to the relaxation behavior found in non-crosslinked NaPSS systems. To characterize these processes their correlation times were combined with constants of selfdiffusion to estimate the distances diffused by an ion in order to average the electric field gradient at its nucleus. These two distances are interpreted as characteristic length scales in the network. At all degrees of crosslinking it was found that the smallest of these length scales is roughly equal to the distance between two neighbouring crosslinks. The largest characteristic distance extends over several crosslinks and reflects inhomogeneities in the crosslink concentration. These conclusions were also reached from similar experiments on 7Li+ in LiPSS systems.     

Bioresponsive systems based on crosslinked polysaccharide hydrogels

Detection of bacterial and fungal contamination is of extreme importance in the fields of medical products or food packaging. Here a diagnostic tool based on pectinase and cellulase triggered release of a dye from a crosslinked polysaccharide matrix was developed. The hydrogel-based bioresponsive matrix consisted of carboxymethylcellulose (CMC) as a substrate for cellulases and polygalacturonate (PGA) as substrate for pectinases. To improve the stability of the hydrogels, methacrylic groups were inserted as crosslinking molecules. For polymerisation, two different methods were used, namely UV and thermal crosslinking. Controlled release triggered by extracellular enzymes of potentially pathogenic/contaminating microorganisms was investigated by the incorporation of Alizarin into the hydrogels. UV polymerised hydrogels turned out to be more suitable than thermal crosslinked polymers. Integration of such polymer based bioresponsive systems in medical surfaces or package systems could therefore act as an in situ monitoring system for detecting the presence of bacteria or fungi like e.g. Aspergillus species.     

Esterification crosslinking structures of rayon fibers with 1,2,3,4-butanetetracarboxylic acid and their water-responsive properties

1,2,3,4-Butanetetracarboxylic acid (BTCA) was applied to crosslink amorphous regions of cotton-type rayon fibers via anhydride-mediated esterification for the purpose of enhanced mechanical properties. Crosslinking was conducted under a series of curing temperature with the presence of sodium hypophosphite (SHP) as the effective catalyst for anhydride formation. The conversion rate based on Fourier transform infrared (FTIR) spectra was in good agreement with that from the direct calculation based on titrated carboxyl groups and ester linkages. Crosslinking degree increased linearly with the curing temperature. The optimal tensile properties were derived from the curing temperature of 180 °C with an intermediate crosslinking degree. Moreover, the crosslinked structures of the rayon fibers had a great impact on absorption of liquid and gaseous water. The liquid water retention capacity diminished substantially with the crosslinking degree, while interaction between moisture and the fibers revealed that sorption–desorption cycles and concomitant hystereses were scarcely affected by the crosslinked fiber structures.     

Evidence for lysogeny and viral resistance in the cyanobacteriumPhormidium uncinatum

Escherichia coli B/5 12-h cultures were exposed to filter-sterilized acid mine water (AMW), fixed in situ, and examined for morphological changes by transmission electron microscopy, scanning electron microscopy, and x-ray spectrometry. Thin sections showed that layers of the Gram-negative envelope were altered and often lacking. Additionally, polar regions of the cell were frequently devoid of cytoplasm. AMW-exposed cells were distorted and had an amorphous substance associated with them. Spectra obtained by x-ray spectrometry suggested that this amorphous substance was cytoplasm rather than a mineral precipitate from AMW. Morphometric analyses of control and AMW-exposed populations showed significant differences in mean volume, length, and width of cells stressed in AMW; this indicates that smaller cells were selectively destroyed by the action of AMW. We concluded that loss of cytoplasm and cell lysis were the consequence of AMW damage to the bacterial envelope.     

Macromolecule-small molecule interactions; introduction of additional binding sites in polyethyleneimine by disulfide cross-linkages

Polyethyleneimine and its acyl derivatives have been thiolated with thiobutyrolactone and the SH groups introduced have been crosslinked in the presence of and in the absence of methyl orange, respectively. After crosslinking of the polymers, the bound methyl orange was removed. The resultant two kinds of crosslinked polymers have been examined for their ability to bind methyl orange. The polymer crosslinked in the presence of methyl orange shows more binding sites and stronger binding than does the polymer crosslinked in the absence of methyl orange. It seems, therefore, that the conformation of the polymer may be molded to provide sites that can accommodate a specific small molecule.     

Characterization of thin gelatin hydrogel membranes with balloon properties for dynamic tissue engineering

Cell or tissue stretching and strain are present in any in vivo environment, but is difficult to reproduce in vitro. Here, we describe a simple method for casting a thin (about 500 μm) and soft (about 0.3 kPa) hydrogel of gelatin and a method for characterizing the mechanical properties of the hydrogel simply by changing pressure with a water column. The gelatin is crosslinked with mTransglutaminase and the area of the resulting hydrogel can be increased up 13-fold by increasing the radial water pressure. This is far beyond physiological stretches observed in vivo. Actuating the hydrogel with a radial force achieves both information about stiffness, stretchability, and contractability, which are relevant properties for tissue engineering purposes. Cells could be stretched and contracted using the gelatin membrane. Gelatin is a commonly used polymer for hydrogels in tissue engineering, and the discovered reversible stretching is particularly interesting for organ modeling applications.     

Birth and growth kinetics of brome mosaic virus microcrystals

The early steps of crystal nucleation and growth in Brome Mosa?c virus and polyethylene glycol mixtures were analyzed using time-resolved x-ray scattering (at the European Synchrotron Radiation Facility, Grenoble, France). The system was chosen as a crystallization model since the phase diagram of the macromolecule/polymer mixture was known to present, at high polymer concentration, a solid, precipitated phase made of the synchronized formation of a large number of microcrystals. The precipitation and crystallization of the samples was induced by the controlled mixing of virus and polymer using a stopped-flow device. Appearance and growth of Bragg diffraction peaks were used to follow the crystal nucleation and growth as a function of time, virus and polymer concentration, and polymer size. In all samples, the crystallization starts after a few seconds and proceeds for approximately 1-20 min until there is almost no virus left in the solution. The crystalline system was found to be face-centered cubic, with a unit cell size of 391 angstroms. The data analysis allowed us to show the presence of viruses in only two states, in solution or in crystals, revealing that the formation of periodic order proceeds without any detectable intermediate amorphous state.     

Spatial distribution of mammalian cells dictated by material surface chemistry

Anisotropic cell culture surfaces patterned with amino and alkylsilanes can guide cell distribution and provide an approach to study important processes involved in tissue engineering, such as cell attachment and locomotion. By combining photolithographic and silane coupling techniques, glass coverslips were patterned with either n-octadecyldimethylchlorosilane (ODDMS) or dimethyldichlorosilane (DMS), and N-(2-aminoethyl)-3-aminopropyl-trimethoxysilane (EDS). The alkylsilanes, theoretically, have similar methyl and methylene groups exposed at the surface but different structures, with DMS being amorphous and ODDMS ordered. Neuroblastoma cells, osteosarcoma cells, and fibroblasts plated on surfaces patterned with EDS/ODDMS and EDS/DMS specifically localized on the EDS regions, but distributed randomly on ODDMS/DMS patterned surfaces. The preferential assembly of cells onto EDS regions did not depend on the structure of the adjacent alkylsilane regions and was a time-dependent process. Angle dependent x-ray photoelectron spectroscopy (XPS) and contact angle measurements indicated that EDS was immobilized on glass as a fractional hydrophilic monolayer, and ODDMS and DMS were bound as patchy amorphous hydrophobic multilayers. Neither surface coverage nor thickness of the overlayer seemed to be as important as surface chemistry, or charge, in guiding mammalian cell distribution. These results are consistent with the concept that mammalian cells attach to and are guided by positively charged surfaces.     

Structure of the Escherichia coli 16 S ribosomal RNA. Psoralen crosslinks and N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine crosslinks detected by electron microscopy

Escherichia coli 16 S ribosomal RNA in reconstitution buffer has been photochemically crosslinked with aminomethyltrimethylpsoralen and chemically crosslinked with N-acetyl-N'-(p-glyoxylylbenzoyl)cystamine. The positions of crosslinking have been detected by viewing the molecules in the electron microscope. DNA restriction fragments that contain psoralen mono-adducts were hybridized and crosslinked to the samples so that the orientations of the crosslinked molecules were seen directly. A two-dimensional histogram method has been used to classify the different types of looped crosslinked molecules. These methods allow the identification of 13 distinct types of loops in the photochemically crosslinked molecules and 31 distinct types of loops in the chemically crosslinked molecules. The psoralen experiments are a reinvestigation of some of our earlier results. Some of the crosslinks were previously reported in the incorrect orientation; with the corrected orientation, seven of the psoralen crosslinks can now be correlated with complementarities in the proposed secondary-structure models. However, there are still six other psoralen crosslinks that indicate additional contacts not found in the current models. The chemical crosslinks indicate pairs of single-stranded regions that must be close in the folded molecule. Many of these crosslinks occur between regions that are distant in the secondary structure; these crosslinks indicate part of the three-dimensional form of the folded molecule.     

Identification of proximate regions in a complex of retinal guanylyl cyclase 1 and guanylyl cyclase-activating protein-1 by a novel mass spectrometry-based method

A key challenge in studying protein/protein interactions is to accurately identify contact surfaces, i.e. regions of two proteins that are in direct physical contact. Aside from x-ray crystallography and NMR spectroscopy few methods are available that address this problem. Although x-ray crystallography often provides detailed information about contact surfaces, it is limited to situations when a co-crystal of proteins is available. NMR circumvents this requirement but is limited to small protein complexes. Other methods, for instance protection from proteolysis, are less direct and therefore less informative. Here we describe a new method that identifies candidate contact surfaces in protein complexes. The complexes are first stabilized by cross-linking. They are then digested with a protease, and the cross-linked fragments are analyzed by mass spectrometry. We applied this method, referred to as COSUMAS (contact surfaces by mass spectrometry), to two proteins, retinal guanylyl cyclase 1 (RetGC1) and guanylyl cyclase-activating protein-1 (GCAP-1), that regulate cGMP synthesis in photoreceptors. Two regions in GCAP-1 and three in RetGC1 were identified as possible contact sites. The two regions of RetGC1 that are in the vicinities of Cys(741) and Cys(780) map to a kinase homology domain in RetGC1. Their identities as contact sites were independently evaluated by peptide inhibition analysis. Peptides with sequences from these regions block GCAP-1-mediated regulation of guanylyl cyclase at both high and low Ca2+ concentrations. The two regions of GCAP-1 cross-linked to these peptides were in the vicinities of Cys(17) and Cys(105) of GCAP-1. Peptides with sequences derived from these regions inhibit guanylyl cyclase activity directly. These results support a model in which GCAP-1 binds constitutively to RetGC1 and regulates cyclase activity by structural changes caused by the binding or dissociation of Ca2+.     

多肽固相序列分析载体的合成及应用

用合成的聚合物作载体,与模型15肽(纯品)偶联,使用Edman降解法进行多肽序列分析。比较了各种类型载体对肽的偶联效果。实验结果表明合成的大孔型四次乙基五胺类树脂载体已经达到国外同类产品水平。     

Separation of epidermal cells by density gradient centrifugation on a continuous colloidal silica (Percoll) gradient

A new method designed for the specific isolation and characterization of ligand-receptor complexes using a heterobifunctional crosslinking agent and immunoprecipitation is described. The complexes are first covalently crosslinked by photoactivation of the crosslinking agent. After lysis of the cells, the crosslinked complexes are immunoprecipitated using an antiserum directed against the crosslinking agent. With this method, ligand-receptor complexes formed in only minute amounts become available for further investigation. By using this anticrosslinker antiserum, different receptor systems can be investigated without raising new receptor- or ligand-specific antibodies for each system. As a test system, a radioiodinated lectin was used as ligand molecule and erythrocyte membranes acted as receptor carriers.     

Electric dichroism of poly(riboadenylic acid)

Electric dichroism measurements on poly(A) in low-ionic-strength solution demonstrate that below a molecular weight of 130,000 the double-stranded polymer is hydrodynamically rigid and above that molecular weight becomes increasingly flexible. At 500,000 it is considerably more flexible than DNA of the same molecular weight, with a mean end-to-end distance of about 1150 Å compared to approximately 1600 Å for DNA. The fully extended length for both DNA and poly(A) of this molecular weight is about 2750 Å. It is further shown that the orientation of these polyelectrolytes in an electric field is consistent with theoretical treatments of the counter-ion distribution and a preliminary model based on the additivity of classical valence charge anisotropy and counter-ion polarization is postulated for the orientation mechanism. Single-stranded pol (A) is shown not only to retain its base stacking in the presence of the electric field but to extend the persistent regions of stacked bases so that it attains a rodlike structure very similar to the one in the double-stranded polymer is found to be less than that expected from consideration of the x-ray structure. An explanation for this result is sought in the electric asymmetry of the helical polymer.