Chromatographic Processing Scheme for Continuous Harvesting of rec-Prourokinase from Serum Free Medium Cell Cultures

Continuous perfusion cell culture, using albumin containing medium, offers the potential advantages of higher recombinant Prourokinase (r-ProUK) yields, higher initial product purity and increased throughput compared to batch culture technology using medium supplemented with fetal bovine serum. We have characterized the production of r-ProUK in medium supplemented with a lipid rich bovine serum albumin (Albumax) in a perfusion system. The results of these studies showed that it was necessary to modify the r-ProUK batch recovery scheme to process r-ProUK from a perfusion system. To accommodate large volumes of perfusate harvested over a ten to fourteen day production cycle, cation exchange and hydrophobic interaction chromatography (HIC) resins were identified that had increased product binding capacity, better flow characteristics and wider pH ranges which allowed caustic cleaning. The mobile phase composition, pH and ionic strength were modified to improve r-ProUK yields from the identified resins, and procedures were developed to eliminate r-ProUK degradation products. Strategies were defined for processing continuous harvest, which contained four to seven times the amount of r-ProUK of batch harvests.     

Ruby fluorescence pressure scale： Revisited

Effect of non-hydrostatic stress on X-ray diffraction in a diamond anvil cell (DAC) is studied. The pressure gradient in the sample chamber leads to the broadening of the diffraction peaks, which increase with the hkl index of the crystal. It is found that the difference between the determined d-spacing compressive ratio d/d0 and the real d-spacing compressive ratio dr/d0 is determined by the yield stress of the pressure transmitting media (if used) and the shear modulus of the sample. On the basis of the corrected experiment data of Mao et al. (MXB86), which was used to calibrate the most widely used ruby fluorescence scale, a new relationship of ruby fluorescence pressure scale is corrected, i.e., P = (1904/9.827)[(1+λ/λ0)9.827-1].     

Application of pulsed-magnetic field enhances non-viral gene delivery in primary cells from different origins

Primary cell lines are more difficult to transfect when compared to immortalized/transformed cell lines, and hence new techniques are required to enhance the transfection efficiency in these cells. We isolated and established primary cultures of synoviocytes, chondrocytes, osteoblasts, melanocytes, macrophages, lung fibroblasts, and embryonic fibroblasts. These cells differed in several properties, and hence were a good representative sample of cells that would be targeted for expression and delivery of therapeutic genes in vivo. The efficiency of gene delivery in all these cells was enhanced using polyethylenimine-coated polyMAG magnetic nanoparticles, and the rates (17–84.2%) surpassed those previously achieved using other methods, especially in cells that are difficult to transfect. The application of permanent and pulsating magnetic fields significantly enhanced the transfection efficiencies in synoviocytes, chondrocytes, osteoblasts, melanocytes and lung fibroblasts, within 5 min of exposure to these magnetic fields. This is an added advantage for future in vivo applications, where rapid gene delivery is required before systemic clearance or filtration of the gene vectors occurs.     

Application of nanodiamonds in human body fluid analysis by matrix-assisted laser desorption/ionization mass spectrometry

Direct mass spectrometric analysis of complex biological samples is very important and challenging. In this paper, nanodiamonds have been successfully used in matrix-assisted laser desorption/ionization mass spectrometric analysis of human serum and urine. As a practical tool and platform, it can be widely used in the field of humoral proteomics, and it plays a very promising role in clinical diagnosis, including identification of novel disease-associated biomarkers.     

Design and fabrication of a dielectrophoresis-based cell-positioning and cell-culture device for construction of cell networks

In this paper, we describe the design and fabrication of a dielectrophoresis (DEP)-based cell-positioning and cell-culture device for the construction of cell networks. This device enables both individual cell positioning and cell culture. Titanium electrodes were fabricated by deposition. Furthermore, microchambers and microchannels composed of SU-8, which is a negative photoresist, were used to carry out cell culture and enable cell differentiation. Using our device, N1E-115 cells were individually positioned in the microchambers, and the positioning yield was 45%. After positioning, the cells could be continuously cultured in the microchambers. Furthermore, the cells differentiated, and their neurites extended through the microchannels after cultivation for several days. These results indicate that our device greatly increases the prospects for individual cell positioning and can be used to construct cell networks that have several applications in the medical field, for example, in drug screening.     

Polyhedral approximation and practical convex hull algorithm for certain classes of voxel sets

In this paper we introduce an algorithm for the creation of polyhedral approximations for certain kinds of digital objects in a three-dimensional space. The objects are sets of voxels represented as strongly connected subsets of an abstract cell complex. The proposed algorithm generates the convex hull of a given object and modifies the hull afterwards by recursive repetitions of generating convex hulls of subsets of the given voxel set or subsets of the background voxels. The result of this method is a polyhedron which separates object voxels from background voxels. The objects processed by this algorithm and also the background voxel components inside the convex hull of the objects are restricted to have genus 0. The second aim of this paper is to present some practical improvements to the discussed convex hull algorithm to reduce computation time.     

Numerical simulation of the rheological properties of granular composites by using a structural approach

Composites with an elastomeric matrix containing rigid particles of diameter 10–1000 μm are studied. One of possible mechanisms of the rheological behavior of such filled systems, related to the origination and growth of vacuoles near the rigid inclusions in a viscous matrix, is considered. For simulating the mechanism of formation of rheological properties of the filled elastomers, we use a structural cell in the form of an elastomeric cylinder, whose height and diameter are equal in magnitude, with a rigid spherical inclusion at its center. Deformation of the cells is examined with the observance of boundary conditions providing the preservation of their close packing. The inclusion is assumed to be rigid, and the matrix properties are described by equations of the linear hereditary viscoelasticity theory. The formation of vacuoles is described by using the approach suggesting that an initial debonding begins to propagate when the energy accumulated in the extended matrix reaches a value sufficient to create a new interface. The heterogeneity of the composite is simulated by taking into account the variability of the local filler concentration. Creep curves obtained for composite cells with different content of the solid phase are presented. Comparisons between the numerical and experimental results show a satisfactory agreement. Translated from Mekhanika Kompozitnykh Materialov, Vol. 44, No. 6, pp. 895–906, November–December, 2008.     

水／离子液体两相体系中出芽短梗霉催化4-氯-乙酰乙酸乙酯不对称还原合成（S）-4-氯-3-羟基-丁酸乙酯

考察了水/离子液体两相体系中出芽短梗霉(Aureobasidium pullulansCGMCC1244)催化4-氯乙酰乙酸乙酯(COBE)不对称还原生成光学活性(S)-4-氯-3-羟基丁酸乙酯((S)-CHBE)的性能,并对反应条件如摇床转速、相体积比、温度、初始底物浓度和pH值等进行了优化.结果表明,在水/1-丁基-3-甲基咪唑六氟磷酸盐体系中,出芽短梗霉催化COBE不对称还原生成(S)-CHBE,在30℃,pH6·6,摇床转速180r/min和不对称反应8h条件下,反应物的转化率、产物ee值和浓度分别达到95·6%,98·5%和47·1g/L.在控制pH值为6·6的情况下,通过分添加底物可有效提高产物(S)-CHBE浓度至75·1g/L.     

Defining and optimizing algorithms for neighbouring particle identification in SPH fluid simulations

Lagrangian particle methods such as smoothed particle hydrodynamics (SPH) are very demanding in terms of computing time for large domains. Since the numerical integration of the governing equations is only carried out for each particle on a restricted number of neighbouring ones located inside a cut‐off radius r_c, a substantial part of the computational burden depends on the actual search procedure; it is therefore vital that efficient methods are adopted for such a search. The cut‐off radius is indeed much lower than the typical domain's size; hence, the number of neighbouring particles is only a little fraction of the total number. Straightforward determination of which particles are inside the interaction range requires the computation of all pair‐wise distances, a procedure whose computational time would be unpractical or totally impossible for large problems. Two main strategies have been developed in the past in order to reduce the unnecessary computation of distances: the first based on dynamically storing each particle's neighbourhood list (Verlet list) and the second based on a framework of fixed cells. The paper presents the results of a numerical sensitivity study on the efficiency of the two procedures as a function of such parameters as the Verlet size and the cell dimensions. An insight is given into the relative computational burden; a discussion of the relative merits of the different approaches is also given and some suggestions are provided on the computational and data structure of the neighbourhood search part of SPH codes. Copyright © 2008 John Wiley & Sons, Ltd.