Pectic Galactan Polysaccharide-Based Gene Delivery System for Targeting Neuroinflammation

Treating neuroinflammation-related injuries and disorders through manipulation of neuroinflammation functions is being heralded as a new therapeutic strategy. In this study, a novel pectic galactan (PG) polysaccharide based gene therapy approach is developed for targeting reactive gliosis in neuroinflammation. Galectin-3 (Gal-3) is a cell protein with a high affinity to β-galactoside sugars and is highly expressed in reactive gliosis. Since PG carries galactans, it can target reactive gliosis via specific carbohydrate interaction between galactan and Gal-3 on the cell membrane, and therefore can be utilized as a carrier for delivering genes to these cells. The carrier is synthesized by modifying quaternary ammonium groups on the PG. The resulting quaternized PG (QPG) is found to form complexes with plasmid DNA with a mean diameter of 100 nm and have the characteristics required for targeted gene therapy. The complexes efficiently condense large amounts of plasmid per particle and successfully bind to Gal-3. The in vivo study shows that the complexes are biocompatible and safe for administration and can selectively transfect reactive glial cells of an induced cortical lesion. The results confirm that this PG-based delivery system is a promising platform for targeting Gal-3 overexpressing neuroinflammation cells for treating neuroinflammation-related injuries and neurodegenerative diseases.     

rwg: An assessment of within-group interrater agreement.

F. L. Schmidt and J. E. Hunter (1989) critiqued the within-group interrater reliability statistic (rwg) described by L. R. James et al (1984). S. W. Kozlowski and K. Hattrup (1992) responded to the Schmidt and Hunter critique and argued that rwg is a suitable index of interrater agreement. This article focuses on the interpretation of rwg as a measure of agreement among judges' ratings of a single target. A new derivation of rwg is given that underscores this interpretation. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease

Cavities in the hydrophobic core of the neutral protease ofBacillus stearothermophilus were analyzed using a threedimensionalmodel that was inferred from the crystal structure of thermolysin,the highly homologous neutral protease of B.thermoproteolyticus(85% sequence identity). Site–directed mutagenesis wasused to fill some of these cavities, thereby improving hydrophobicpacking in the protein interior. The mutations had small effectson the thermostability, even after drastic changes, such asLeu284Trp and Met168Trp. The effects on T50, the temperatureat which 50% of the enzyme is irreversibly inactivated in 30min, ranged from 0.0 to +0.4°C. These results can be explainedby assuming that the mutations have positive and negative structuraleffects of approximately the same magnitude. Alternatively,it could be envisaged that the local unfolding steps, whichrender the enzyme susceptible towards autolysis and which arerate limiting in the process of thermal inactivation, are onlyslightly affected by alterations in the hydrophobic core.     

Effect of nonionic detergents on lipoxygenase catalysis

In many studies on lipoxygenase catalysis, nonionic detergents are used to obtain an optically transparent solution of the fatty acid substrate. In order to resolve some controversies that exist with regard to the interpretation of kinetic data obtained with solutions containing nonionic detergents, a systematic investigation was undertaken into the effects of Lubrol, Tween-20 and Triton X-100 (0–0.8 g/L) on the kinetics of linoleate (2.5–110 μM) dioxygenation, catalyzed by lipoxygenase-1 or lipoxygenase-2 from soybean, at pH 9 or 10, at 25°C. Under most conditions, it was found that the detergents slowed down the reaction. However, at high linoleate concentrations, where substrate inhibition of lipoxygenase is significant, small amounts of detergent increased the dioxygenation rate. In a quantitative analysis of the results, a kinetic model in which the incorporation of linoleate in the detergent micelles is formulated as a simple reversible equilibrium, and in which both lipoxygenase-1 and-2 interact with free linoleate, but not with linoleate incorporated in the micelles, appeared to be sufficient to predict experimental results over a wide range of experimental conditions. According to this model, the changes in the dioxygenation kinetics caused by the presence of nonionic detergents are similar (but not equal) to those caused by competitive inhibitors. The conclusions that monomeric, nonmicellar linoleate is the preferred substrate for lipoxygenase and that the observed inhibition and stimulation are solely due to changes in the effective linoleate concentration strongly corroborate the earlier observations by Galpin and Allen [Biochim. Biophys. Acta 488 (1977), 392–401].     

Liposomes in chemo- and immunotherapy of cancer

In this paper, we report on the in vivo behavior of liposomes as a function of their size and composition. It is emphasized that by varying these parameters we can influence not only the rate of blood elimination but also the intrahepatic destination of the liposomes. Thus, we show that small liposomes with diameters well below 100 nm can reach and be internalized by the parenchymal cells of the liver, i.e. the hepatocytes. The rate and the extent at which this occurs depends on the liposomal composition. With respect to the application of liposomes as a drug carrier system in anticancer therapy, we put emphasis on the liver macrophage, i.e. the Kupffer cell, as a target cell. Large liposomes with diameters well over 100 nm exclusively are taken up by these cells as far as hepatic uptake is concerned. By encapsulation within liposomes, a drug may be delivered specifically to these macrophages; this will prevent its rapid excretion from the body and/or undesired accumulation in other cell types. Two examples of the way in which this condition may be exploited are presented. First, we demonstrate the formation of intracellular depots in the macrophages of the cytostatic drug 5-fluorodeoxyuridine (FUdR), thus preventing the rapid metabolism of the drug by the hepatocytes and allowing its sustained release from the macrophages and subsequent uptake by adjacent metastatic tumor cells. Second, we show that the liposome-encapsulated immunomodulator muramyl dipeptide is capable of activating liver macrophages both in vitro and in vivo to a tumor-specific cytotoxic state, and this can result in substantial reduction of metastatic growth in the livers of mice inoculated in the spleen with colon adenocarcinoma cells.     

Designing amino acid sequences to fold with good hydrophobic cores

We present two methods for designing amino acid sequences ofproteins that will fold to have good hydrophobic cores. Giventhe coordinates of the desired target protein or polymer structure,the methods generate sequences of hydrophobic (H) and polar(P) monomers that are intended to fold to these structures.One method designs hydrophobic inside, polar outside; the otherminimizes an energy function in a sequence evolution process.The sequences generated by these methods agree at the levelof 60–80% of the sequence positions in 20 proteins inthe Protein Data Bank. A major challenge in protein design isto create sequences that can fold uniquely, i.e. to a singleconformation rather than to many. While an earlier lattice-basedsequence evolution method was shown not to design unique folders,our method generates unique folders in lattice model tests.These methods may also be useful in designing other types offoldable polymer not based on amino acids     

Contribution of the C-terminal amino acid to the stability of Bacillus subtilis neutral protease

The role of the C-terminal Leu300 in maintaining thermal stabilityof the neutral protease of Bacillus subtilis was investigated.From model building studies based on the three dimensional structureof thermolysin, the neutral protease of B.thermoproteolyticus,it was conduded that this residue is located in a hydrophobicpocket composed of residues located in the C-terminal and themiddle domain. To test the hypothesis that Leu300, by contributingto a stabilizing interaction between these domains, is importantfor enzyme stability, several neutral protease mutants wereconstructed and characterized. The thermostability of the enzymewas lowered by deleting Leu300 or by replacing this residueby a smaller (Ala), a polar (Asn) or a sterically unfavourable(He) amino acid. Thermostabiity was increased upon replacingLeu300 by Phe. These results are in agreement with model-buildingstudies. The effects on thermostability observed after mutatingthe corresponding Val318 in the thermostable neutral proteaseof B.stearothermophilus were less pronounced.     

Creative practices in the design studio culture: collaboration and communication

For the purpose of developing collaborative support in design studio environments, we have carried out ethnographic fieldwork in professional and academic product design studios. Our intention was to understand design practices beyond the productivity point of view and take into account the experiential, inspirational and aesthetical aspects of design practices. Using examples from our fieldwork, we develop our results around three broad themes by which design professionals support communication and collaboration: (1) use of artefacts, (2) use of space and (3) designerly practices. We use the results of our fieldwork for drawing implications for designing technologies for the design studio culture.     

Multifunctional Gadolinium‐Doped Mesoporous TiO2 Nanobeads: Photoluminescence,Enhanced Spin Relaxation,and Reactive Oxygen Species Photogeneration,Beneficial for Cancer Diagnosis and Treatment

Materials with controllable multifunctional abilities for optical imaging (OI) and magnetic resonant imaging (MRI) that also can be used in photodynamic therapy are very interesting for future applications. Mesoporous TiO₂ sub‐micrometer particles are doped with gadolinium to improve photoluminescence functionality and spin relaxation for MRI, with the added benefit of enhanced generation of reactive oxygen species (ROS). The Gd‐doped TiO₂ exhibits red emission at 637 nm that is beneficial for OI and significantly improves MRI relaxation times, with a beneficial decrease in spin–lattice and spin–spin relaxation times. Density functional theory calculations show that Gd³⁺ ions introduce impurity energy levels inside the bandgap of anatase TiO₂, and also create dipoles that are beneficial for charge separation and decreased electron–hole recombination in the doped lattice. The Gd‐doped TiO₂ nanobeads (NBs) show enhanced ability for ROS monitored via ^?OH radical photogeneration, in comparison with undoped TiO₂ nanobeads and TiO₂ P25, for Gd‐doping up to 10%. Cellular internalization and biocompatibility of TiO₂@x Gd NBs are tested in vitro on MG‐63 human osteosarcoma cells, showing full biocompatibility. After photoactivation of the particles, anticancer trace by means of ROS photogeneration is observed just after 3 min irradiation.