Integrated process for purification of plasmid DNA using aqueous two-phase systems combined with membrane filtration and lid bead chromatography

An integrated process for purifying a 6.1 kilo base pair (kbp) plasmid from a clarified Escherichia coli cell lysate based on an ultra/diafiltration step combined with polymer/polymer aqueous two-phase system and a new type of chromatography is described. The process starts with a volume reduction (ultrafiltration) and buffer exchange (diafiltration) of the clarified lysate using a hollow fibre membrane system. The concentrated and desalted plasmid solution is then extracted in a thermoseparating aqueous two-phase system, where the contaminants (RNA and proteins) to a large extent are removed. While the buffer exchange (diafiltration) is necessary in order to extract the plasmid DNA exclusively to the top phase, experiments showed that the ultrafiltration step increased the productivity of the aqueous two-phase system by a factor of more than 10. The thermoseparated water phase was then subjected to a polishing step using lid bead chromatography. Lid beads are a new type of restricted access chromatography beads, here with a positively charged inner core that adsorbed the remaining RNA while its inert surface layer prevented adsorption of the plasmid DNA thus passing in the flow-through of the column. Differently-sized plasmid DNA in the range of 2.7-20.5 kbp were also partitioned in the aqueous two-phase system. Within this size range, all plasmid DNA was exclusively extracted to the top phase. The complete process is free of additives and easy scalable for use in large scale production of plasmid DNA. The overall process yield for plasmid DNA was 69%.     

Multiblock copolymer synthesis via controlled radical polymerization in aqueous dispersions. Part 1: Synthesis of S-tert-alkyl-N,N-alkoxycarbonylalkyldithiocarbamates

In a novel two- or three-step synthetic route, S-(1,4-phenylenebis(propane-2,2-diyl)) bis(N-methyldithiocarbamate) is reacted at low temperature with various alkyl chloroformates to form various S-tert-alkyl-N,N-alkoxycarbonylmethyl-dithiocarbamate RAFT agents. Also an alternative and novel synthetic route towards S-(1,4-phenylenebis(propane-2,2-diyl)) bis(N-methyldithiocarbamate), is proposed.     

Differential renormalization of van der Waals spin models

An exact renormalization group equation in differential form is derived for spin systems with general many-body interactions in the van der Waals limit. This equation is solved and the free energy is determined as an integral along the renormalization trajectory in the interaction space. It is shown that the transformation can always be modified in such a way that an undetermined integration constant for the free energy vanishes exactly, also below the critical temperature. We also demonstrate how the invariance of the free energy under a parameterdependent equivalence transformation can provide information about the critical behavior of the system. In this alternative approach dangerous irrelevant variables play an essential role.On leave of absence from Istituto di Fisica e Unità GNSM del CNR, Università di Padova, Padova, Italy.     

Emulsion polymerization of methyl methacrylate in the presence of novel addition–fragmentation chain‐transfer reactive surfactant (transurf)

Reactive surfactants allow the surfactant molecules to become covalently bound to the particles and thus provide added stability to the colloid particles, longer shelf life, better shear resistance, and polymer particles that can be redispersed. This article reports for the first time the use of a novel addition–fragmentation chain‐transfer reactive surfactant (transurf) in ab initio emulsion polymerizations of methyl methacrylate at 70 °C. It was found that the rate was lowered and average particle diameter nearly doubled when the transurf was used as compared with the SDS experiments (control). In addition the molecular weight distribution was very broad but had a lower Mn than observed in the sodium dodecyl sulfate experiment. Unfortunately because of the formation of many water‐soluble oligomers, the amount of transurf incorporated could not be obtained accurately. However, it was estimated theoretically that only a very small amount of transurf would be consumed, but for an alternative method to increase the incorporation of transurf into the particles, the ratio of monomer to transurf must be kept as low as possible. The best way to achieve this would be to carry the experiments out under starved‐feed conditions. © 2001 John Wiley & Sons, Inc. J Polym Sci Part A: Polym Chem 39: 2813–2820, 2001     

CFB cyclones at high temperature: Operational results and design assessment

Pressure drop and cut size measurements are reported for a full scale cyclone operating within a 58 MWth CFB-combustor unit at 775 ℃.
The paper reviews the vast number of equations to calculate the pressure drop and separation efficiency of cyclones, generally for operation at ambient temperature and at low Cs [〈0.5]. None of the literature correlations predicts the pressure drop with a fair accuracy within the range of experimental operating conditions. The cut size d50 can be estimated using direct empirical methods or using the Stokes number, Stk50. Both methods were used to compare measured and predicted values of d50. With the exception of Muschelknautz and Krambrock, none of the equations made accurate predictions.
Finally, an alternative method to determine the friction factor of the pressure drop equation （Euler number, Eu） and of the cut size is proposed. The Eu number is determined from the geometry of common cyclones, and the derived value of Stk50 defines more accurate cut sizes. The remaining discrepancy of less than 5%, when compared with the measured values, is tentatively explained in terms of a reduced cyclone diameter due to the solids layer formed near its wall. Further measurements, mostly using positron emission particle tracking, elucidate the particle motion in the cyclone and both tracking results and the influence of the particle movement on Eu and Stk50 will be discussed in a follow-up paper.     

On the convergence properties of the orthogonal similarity transformations to tridiagonal and semiseparable (plus diagonal) form

In this paper, we will compare the convergence properties of three basic reduction methods, by placing them in a general framework. It covers the reduction to tridiagonal, semiseparable and semiseparable plus diagonal form. These reductions are often used as the first step in the computation of the eigenvalues and/or eigenvectors of arbitrary matrices. In this way, the calculation of the eigenvalues using, for example, the QR-algorithm reduces in complexity. First we will investigate the convergence properties of these three reduction algorithms. It will be shown that for the partially reduced matrices at step k of any of these reduction algorithms, the lower right k × k (already reduced) sub-block will have the Lanczos–Ritz values, w.r.t. a certain starting vector. It will also be shown that the reductions to semiseparable and to semiseparable plus diagonal form have an extra convergence behavior a special type of subspace iteration is performed on the lower right k × k submatrix, which contains these Ritz-values. Secondly we look in more detail at the behavior of the involved subspace iteration. It will be shown that the reduction method can be interpreted as a nested type of multi-shift iteration. Theoretical results will be presented, making it possible to predict the convergence behavior of these reduction algorithms. Also a theoretical bound on the convergence rate is presented. Finally we illustrate by means of numerical examples, how it is possible to tune the convergence behavior such that it can become a powerful tool for certain applications.     

Fast computation of eigenvalues of companion,comrade, and related matrices

The class of eigenvalue problems for upper Hessenberg matrices of banded-plus-spike form includes companion and comrade matrices as special cases. For this class of matrices a factored form is developed in which the matrix is represented as a product of essentially 2×2 matrices and a banded upper-triangular matrix. A non-unitary analogue of Francis’s implicitly-shifted QR algorithm that preserves the factored form and consequently computes the eigenvalues in O(n ²) time and O(n) space is developed. Inexpensive a posteriori tests for stability and accuracy are performed as part of the algorithm. The results of numerical experiments are mixed but promising in certain areas. The single-shift version of the code applied to companion matrices is much faster than the nearest competitor.     

Geometric means of structured matrices

The geometric mean of positive definite matrices is usually identified with the Karcher mean, which possesses all properties—generalized from the scalar case—a geometric mean is expected to satisfy. Unfortunately, the Karcher mean is typically not structure preserving, and destroys, e.g., Toeplitz and band structures, which emerge in many applications. For this reason, the Karcher mean is not always recommended for modeling averages of structured matrices. In this article a new definition of a geometric mean for structured matrices is introduced, its properties are outlined, algorithms for its computation, and numerical experiments are provided. In the Toeplitz case an existing mean based on the Kähler metric is analyzed for comparison.     

Extended Hamiltonian Hessenberg Matrices arise in Projection based Model Order Reduction

We show that extended Hamiltonian Hessenberg matrices arise naturally in projection-based model order reduction. Therefore we reduce a large dynamical system by projecting it on an extended Krylov subspace. The eigenvalues of the reduced order model can then be computed directly by applying the extended Hamiltonian QR algorithm. (© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim)