Soluble Prokaryotic Overexpression and Purification of Human GM-CSF Using the Protein Disulfide Isomerase b′a′ Domain

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a member of the colony-stimulating factor (CSF) family, which functions to enhance the proliferation and differentiation of hematopoietic stem cells and other hematopoietic lineages such as neutrophils, dendritic cells, or macrophages. These proteins have thus generated considerable interest in clinical therapy research. A current obstacle to the prokaryotic production of human GM-CSF (hGM-CSF) is its low solubility when overexpressed and subsequent complex refolding processes. In our present study, the solubility of hGM-CSF was examined when combined with three N-terminal fusion tags in five E. coli strains at three different expression temperatures. In the five E. coli strains BL21 (DE3), ClearColi BL21 (DE3), LOBSTR, SHuffle T7 and Origami2 (DE3), the hexahistidine-tagged hGM-CSF showed the best expression but was insoluble in all cases at each examined temperature. Tagging with the maltose-binding protein (MBP) and the b′a′ domain of protein disulfide isomerase (PDIb′a′) greatly improved the soluble overexpression of hGM-CSF at 30 °C and 18 °C. The solubility was not improved using the Origami2 (DE3) and SHuffle T7 strains that have been engineered for disulfide bond formation. Two conventional chromatographic steps were used to purify hGM-CSF from the overexpressed PDIb′a′-hGM-CSF produced in ClearColi BL21 (DE3). In the experiment, 0.65 mg of hGM-CSF was isolated from a 0.5 L flask culture of these E. coli and showed a 98% purity by SDS-PAGE analysis and silver staining. The bioactivity of this purified hGM-CSF was measured at an EC₅₀ of 16.4 ± 2 pM by a CCK8 assay in TF-1 human erythroleukemia cells.     

Simulated Microgravity Inhibits the Proliferation of Chang Liver Cells by Attenuation of the Major Cell Cycle Regulators and Cytoskeletal Proteins

Simulated microgravity (SMG) induced the changes in cell proliferation and cytoskeleton organization, which plays an important factor in various cellular processes. The inhibition in cell cycle progression has been considered to be one of the main causes of proliferation inhibition in cells under SMG, but their mechanisms are still not fully understood. This study aimed to evaluate the effects of SMG on the proliferative ability and cytoskeleton changes of Chang Liver Cells (CCL-13). CCL-13 cells were induced SMG by 3D clinostat for 72 h, while the control group were treated in normal gravity at the same time. The results showed that SMG reduced CCL-13 cell proliferation by an increase in the number of CCL-13 cells in G0/G1 phase. This cell cycle phase arrest of CCL-13 cells was due to a downregulation of cell cycle-related proteins, such as cyclin A1 and A2, cyclin D1, and cyclin-dependent kinase 6 (Cdk6). SMG-exposed CCL-13 cells also exhibited a downregulation of α-tubulin 3 and β-actin which induced the cytoskeleton reorganization. These results suggested that the inhibited proliferation of SMG-exposed CCL-13 cells could be associate with the attenuation of major cell cycle regulators and main cytoskeletal proteins.     

Grinding-hardening with liquid nitrogen: Mechanisms and technology

This paper studies an innovative development of a steel grinding–hardening technology using an inert cryogen—liquid nitrogen. It was found that phase transformations took place during grinding with the application of liquid nitrogen and resulted in hardened surface layer in a ground component. The layer had a fine laths martensite structure which gave rise to a remarkably high hardness. It was also shown that the treatment can produce superior surface integrity, with compressive surface residual stresses and without surface oxidation. Due to the inert nature of the liquid nitrogen, the grinding process becomes environmentally conscious.     

Characterization of molecular orientation in injection–stretch–blow‐molded poly(ethylene terephthalate) bottles by means of external reflection infrared spectroscopy

The molecular orientation at the outer surface of injection–stretch–blow‐molded bottles made from poly(ethylene terephthalate) was characterized and quantified by means of front‐surface reflection infrared spectroscopy based on a method developed previously. Results were obtained for two different bottle shapes (cylindrical and rectangular) molded at different injection mold temperatures (16, 38, and 60°C). For the cylindrical bottles, the preferred molecular chain orientation was found to be in the axial direction, with the Hermans orientation function near 0.3 for all three mold temperatures. For the less symmetrical rectangular bottles, a significant difference was observed between the large and small faces. For the large face, the orientation was mainly in the hoop direction; the Hermans orientation function was in the range of 0.3–0.5 and was essentially the same at all mold temperatures and positions along the bottle height. For the small face, on the other hand, the preferred orientation changed from the hoop direction near the bottom to the axial direction near the top, and the variation was more pronounced at lower mold temperatures. The utility of the front‐surface reflection technique was clearly demonstrated. It was also applied, with the use of an infrared microscope, to examine the orientation gradient across the wall thickness. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 1319–1327, 2007     

Microstructure and morphology of amine-cured epoxy coatings before and after outdoor exposures—An AFM study

Atomic force microscopy (AFM) has been used to study the morphology and microstructure of an amine-cured epoxy before and after outdoor exposure. Measurements were made from samples prepared in an essentially CO₂-free, H₂O-free glove box and from samples prepared in ambient conditions. For those prepared in a CO₂-free glove box, AFM imaging was conducted on (1) an unexposed air/coating surface, (2) an unexposed coating bulk, (3) an unexposed coating/substrate interface, and (4) a field exposed air/coating surface. For samples prepared in ambient conditions, only the unexposed air/coating surface was investigated. The same regions of the exposed samples were scanned periodically by the AFM to monitor changes in the surface morphology of the coating as UV exposure progressed. Small angle neutron scattering and Fourier transform infrared spectroscopy (FTIR) studies were performed to verify the microstructure and to follow chemical changes during outdoor exposure, respectively. The results have shown that amine blushing, which occurs only under ambient conditions, had a significant effect on the surface morphology and microstructure of the epoxy. The surface morphology of the samples prepared under CO₂-free, dry conditions was generally smooth and homogeneous. However, the interface and the bulk samples clearly revealed a two-phase structure consisting of bright nodular domains and dark interstitial regions, indicating an inhomogeneous microstructure. Such heterogeneous structure of the bulk was in good agreement with results obtained by small angle neutron scattering of unexposed samples and by AFM phase imaging of the degraded sample surface. The relationship between submicrometer physical changes and molecular chemical degradation is discussed. Presented at the 82nd Annual Meeting of the Federation of Societies for Coatings Technology, October 27–29, 2004, in Chicago, IL.     

A unified method for optimal arbitrary pole placement

We consider the classic problem of pole placement by state feedback. We offer an eigenstructure assignment algorithm to obtain a novel parametric form for the pole-placing feedback matrix that can deliver any set of desired closed-loop eigenvalues, with any desired multiplicities. This parametric formula is then exploited to introduce an unconstrained nonlinear optimisation algorithm to obtain a feedback matrix that delivers the desired pole placement with optimal robustness and minimum gain. Lastly we compare the performance of our method against several others from the recent literature.     

Use of laser scanning confocal microscopy for characterizing changes in film thickness and local surface morphology of UV-exposed polymer coatings

Laser scanning confocal microscopy (LSCM) has been used to characterize the changes in film thickness and local surface morphology of polymer coatings during the UV degradation process. With the noninvasive feature of LSCM, one can obtain thickness information directly and nondestructively at various exposure times without destroying the specimens or deriving the thickness values from IR measurement by assuming uniform film ablation. Two acrylic polymer coatings were chosen for the study, and the physical and chemical changes of the two systems at various exposure times were measured and analyzed. Those measurable physical changes caused by UV exposure include film ablation, formation of pits and other surface defects, and increases in surface roughness. It was found in both coatings that changes in measured film thickness by LSCM were not correlated linearly to the predicted thickness loss using the changes in the CH band obtained by the Fourier Transform Infrared (FTIR) spectroscopy measurements in the later degradation stages. This result suggested it was not a uniform film ablation process during the UV degradation. At later stages, where surface deformation became severe, surface roughness and profile information using LSCM were also proven to be useful for analyzing the surface degradation process Presented at the 81st Annual Meeting of the Federation of Societies for Coatings Technology, November 13–14, 2004 in Philadelphia, PA.     

Process conditions affect pigment quality and yield in extracts of purple sunflower hulls

Extract of purple sunflower hulls is a potential red food colorant; however, suitable process conditions must still be identified. Selected process variables were studied using bench-scale units to prepare, clarify, concentrate and spray dry extracts. Concentration by evaporation at 32°C and addition of maltodextrin to 15% (wt/dry wt) prior to drying largely eliminated pigment degradation during those steps. Relative to water extracts, extracts prepared with 5 to 15% ethanol in water generally yielded more pigment with similar levels of degradation and loss during subsequent processing. Use of 5% ethanol/2% citric acid reduced yield by about half relative to water, but gave a powder with a lower degradation index and wetting time. Percentage recovery of pigment during concentration and drying was not greatly affected by solvent type or extraction temperature.     

Peer-to-peer bichromatic reverse nearest neighbours in mobile ad-hoc networks

The increasing use of mobile communications has raised many issues of decision support and resource allocation. A crucial problem is how to solve queries of Reverse Nearest Neighbour (RNN). An RNN query returns all objects that consider the query object as their nearest neighbour. Existing methods mostly rely on a centralised base station. However, mobile P2P systems offer many benefits, including self-organisation, fault-tolerance and load-balancing. In this study, we propose and evaluate 3 distinct P2P algorithms focusing on bichromatic RNN queries, in which mobile query peers and static objects of interest are of two different categories, based on a time-out mechanism and a boundary polygon around the mobile query peers. The Brute-Force Search Algorithm provides a naive approach to exploit shared information among peers whereas two other Boundary Search Algorithms filter a number of peers involved in query processing. The algorithms are evaluated in the MiXiM simulation framework with both real and synthetic datasets. The results show the practical feasibility of the P2P approach for solving bichromatic RNN queries for mobile networks.     

Improved oxidative stability of sunflower oil in the presence of an oxygen-scavenging film

The oxidative stability of sunflower oil (SFO) was measured during storage at 23 and 37°C in the presence of a novel oxygen-scavenging film that contained polyfuryloxirane (PFO). Commercially refined and deodorized SFO was stored in a lighted room in sealed transparent packages containing either PFO film or an antioxidant, 0.02% butylated hydroxytoluene (BHT). Oxidative stability was evaluated by determination of peroxide values and gas-chromatographic measurement of headspace hexanal. SFO stored in the presence of the oxygen-scavenging film was more stable than oil stored without the film, or than film stored with 0.02% BHT. The PFO film scavenges oxygen through energy-transfer sensitization of singlet oxygen. The film is doped with eosin and the naturally-occurring dye, curcumin, which absorb over a wide range of visible wavelengths. Curcumin transfers its absorbed energy to eosin, which sensitizes the production of singlet oxygen. The singlet oxygen is scavenged by PFO. The use of two dyes increases the efficiency of the sensitization process, reducing the illumination time and intensity required for effective oxygen scavenging.