Vector and Cell Line Engineering Technologies Toward Recombinant Protein Expression in Mammalian Cell Lines

The rapid growth of global biopharmaceutical market in the recent years has been a good indication of its significance in biotechnology industry. During a long period of time in recombinant protein production from 1980s, optimizations in both upstream and downstream processes were launched. In this regard, one of the most promising strategies is expression vector engineering technology based on incorporation of DNA opening elements found in the chromatin border regions of vectors as well as targeting gene integration. Along with these approaches, cell line engineering has revealed convenient outcomes in isolating high-producing clones. According to the fact that more than 50% of the approved therapeutic proteins is being manufactured in mammalian cell lines, in this review, we focus on several approaches and developments in vector and cell line engineering technologies in mammalian cell culture.     

A weight-function approach for determining watershed initial conditions for combustion waves

In many generic combustion models, one finds that a combustionwave will develop with a specific wave speed. However, thereare possible initial temperature profiles which do not evolveinto such waves, but rather die out to the ambient temperature.There can exist, in some models, a clear distinction betweenthose initial conditions that do evolve into combustion wavesand those that do not; this is sometimes referred to as thewatershed initial condition. When fuel consumption is consideredto be negligible, analytical methods can be used to obtain theexact watershed. In this paper, we consider the problem of determiningpseudo-watersheds and ascertaining the relationship betweenthese pseudo-watersheds and the exact watersheds. In the processa novel weight-function approach for infinite spatial domainsis developed.     

Synthesis and antisense properties of fluoro cyclohexenyl nucleic acid (F-CeNA), a nuclease stable mimic of 2'-fluoro RNA

We report the design and synthesis of 2'-fluoro cyclohexenyl nucleic acid (F-CeNA) pyrimidine phosphoramidites and the synthesis and biophysical, structural, and biological evaluation of modified oligonucleotides. The synthesis of the nucleoside phosphoramidites was accomplished in multigram quantities starting from commercially available methyl-D-mannose pyranoside. Installation of the fluorine atom was accomplished using nonafluorobutanesulfonyl fluoride, and the cyclohexenyl ring system was assembled by means of a palladium-catalyzed Ferrier rearrangement. Installation of the nucleobase was carried out under Mitsunobu conditions followed by standard protecting group manipulations to provide the desired pyrimidine phosphoramidites. Biophysical evaluation indicated that F-CeNA shows behavior similar to that of a 2'-modified nucleotide, and duplexes with RNA showed slightly lower duplex thermostability as compared to that of the more rigid 3'-fluoro hexitol nucleic acid (FHNA). However, F-CeNA modified oligonucleotides were significantly more stable against digestion by snake venom phosphodiesterases (SVPD) as compared to unmodified DNA, 2'-fluoro RNA (FRNA), 2'-methoxyethyl RNA (MOE), and FHNA modified oligonucleotides. Examination of crystal structures of a modified DNA heptamer duplex d(GCG)-T*-d(GCG):d(CGCACGC) by X-ray crystallography indicated that the cyclohexenyl ring system exhibits both the (3)H(2) and (2)H(3) conformations, similar to the C3'-endo/C2'-endo conformation equilibrium seen in natural furanose nucleosides. In the (2)H(3) conformation, the equatorial fluorine engages in a relatively close contact with C8 (2.94 ?) of the 3'-adjacent dG nucleotide that may represent a pseudo hydrogen bond. In contrast, the cyclohexenyl ring of F-CeNA was found to exist exclusively in the (3)H(2) (C3'-endo like) conformation in the crystal structure of the modified A-form DNA decamer duplex [d(GCGTA)-T*-d(ACGC)](2.) In an animal experiment, a 16-mer F-CeNA gapmer ASO showed similar RNA affinity but significantly improved activity compared to that of a sequence matched MOE ASO, thus establishing F-CeNA as a useful modification for antisense applications.     

Locating Cantori for Symmetric Tokamap and Symmetric Ergodic Magnetic Limiter Map Using Mean-Energy Error Criterion

We use a method based on the conservation of energy, the mean-energy error criterion, to approximately locate the place of a cantorus by locating the series of its convergents. The mean-energy error curve has nearly stationary parts in the vicinity of elliptic (minimax) orbits, the so-called magnetic islands. Stable minimax orbits converge to orbits homoclinic to a cantorus. By tracing the island series, we limit the cantorus to a narrow region. A near-critical perturbation parameter is used so that, while the cantorus may be destabilized, its high-order minimax orbits remain intact. As illustrations, we consider two symplectic maps, systematically derived from the Hamilton–Jacobi equation and Jacobi’s theorem, in the context of the magnetically confined plasmas in a tokamak: a symmetric tokamap realistically reproduces the main features of a tokamak, and a symmetric ergodic magnetic limiter (EML) map is defined to describe the action of EML rings on the magnetic field lines in the tokamak.     

Synthesis of cytotoxic cyanobactin,Wewakazole B

We report herein the synthesis of cytotoxic cyanobactin, Wewakazole B through an efficient solution-phase approach. The key steps of the synthesis are the macrocyclic lactamization of linear dodecapeptide and construction of two hexapeptides with three different substituted oxazole rings.     

Effect of water phase change on temperature distribution in proton exchange membrane fuel cells

A two dimensional, two-phase non-isothermal electrochemical-transport using a fully coupled numerical model is developed to investigate heat transfer and water phase change effects on temperature distribution in a PEM fuel cell. The multiphase mixture is used formulation for the two phase transport process and developed model is treated as a single domain. This process leads to a single set of conservation equations consisting of continuity, momentum, species, potential and energy for all regions of cell. The results indicate that heat release due to condensation of water vapor affects the temperature distribution. When the relative humidity of the cathode is low, phase change would have a small effect on the maximum temperature that appears at the cell inlet, but it has higher effect on temperature variation further down stream towards the exit of cathode channel and its GDL. Under full-humidity conditions, the cell temperature at all regions of cell increases due to the phase change that starts to appear at the inlet, but the maximum effect of phase change occurs further up stream in cathode channel and its GDL. Also, vapor-phase diffusion which provides a new mechanism for heat removal from the cell, affects the cell temperature distribution.     

Study of the numerical artifacts in differential analysis of highly conducting gratings

Li's Fourier factorization rule [J. Opt. Soc. Am. A13, 1870 (1996)] was recently shown to be problematic to apply to highly conducting metallic gratings. We provide further information about the applicability of different differential methods and are concerned with the relation of observed numerical artifacts, the total number of retained space harmonics, the presence of both positive and negative permittivity inside the groove region, and the validity of Li's inverse rule. Two different cases corresponding to lossless and low-loss binary metallic gratings are considered, and it is shown that an increase in the number of retained space harmonics can relieve the presence of numerical artifacts.     

Structure of Liquid‐Sheet Sprays

Spray characteristics and their spatial distribution have been investigated experimentally for sprays generated by the breakup of thin liquid sheets in co‐flowing air streams. The spray characteristics such as droplet mean and fluctuation velocity and Sauter mean diameter have been measured by using phase Doppler anemometry under various liquid and air flow conditions at the nozzle exit. The results show that at a given spray cross section the droplet axial mean velocity has a maximum value at the spray center, and decreases towards the edge of the spray; whereas the Sauter mean diameter has a minimum value at the center and increases monotonically towards the spray periphery. Data analysis indicates that sufficiently downstream of the nozzle exit the droplet mean velocity attains a jet‐like self‐similar distribution in the transverse direction, and such universal distribution is also observed for the turbulent fluctuation velocity and turbulent intensity, although it is achieved further downstream compared to the mean velocity profile. The Sauter mean diameter at the spray center has a complex variation in the downstream direction due to secondary atomization at high air velocity near the nozzle exit and droplet entrainment, migration and possible coalescence farther downstream.     

Simultaneous determination of carbazole-based explosives in environmental waters by dispersive liquid—liquid microextraction coupled to HPLC with UV-Vis detection

Dispersive liquid-liquid microextraction as a rapid, simple and efficient method coupled with high performance liquid chromatography-UV-Vis detection was used for sample preparation and subsequent determination of carbazole, tri nitro carbazole (TrNC) and tetra nitro carbazole in water samples. The influence of several important variables on the extraction efficiency has been evaluated. The methods works best with chloroform as an extractant and acetonitrile as the dispersive solvent. Under optimum conditions, the calibration curve is linear in the range from 0.007 to 1.75?μg?mL^?1 for TNC, 0.006 to 1.52?μg?mL^?1 for TrNC, and 0.008–2.10?μg?mL^?1 for carbazole. The limits of detection (LODs; at a signal-to-noise ratio of 3), range from 1.7 to 1.1?ng?mL^?1, for TNC, TrNC and carbazole. Also, the relative standard deviations (RSD, n?=?6) for the extraction of TNC (at 174?ng?mL^?1), TrNC (at 151?ng?mL^?1) and carbazole (at 84?ng?mL^?1) vary between 4.1 and 5.2%. The enrichment factors range from 179 to 186. The method was successfully applied to the determination of TNC, TrNC and carbazole in environmental samples.