A rapid, efficient, and economical inverse polymerase chain reaction-based method for generating a site saturation mutant library

With the development of deep sequencing methodologies, it has become important to construct site saturation mutant (SSM) libraries in which every nucleotide/codon in a gene is individually randomized. We describe methodologies for the rapid, efficient, and economical construction of such libraries using inverse polymerase chain reaction (PCR). We show that if the degenerate codon is in the middle of the mutagenic primer, there is an inherent PCR bias due to the thermodynamic mismatch penalty, which decreases the proportion of unique mutants. Introducing a nucleotide bias in the primer can alleviate the problem. Alternatively, if the degenerate codon is placed at the 5' end, there is no PCR bias, which results in a higher proportion of unique mutants. This also facilitates detection of deletion mutants resulting from errors during primer synthesis. This method can be used to rapidly generate SSM libraries for any gene or nucleotide sequence, which can subsequently be screened and analyzed by deep sequencing. (C) 2013 Elsevier Inc. All rights reserved.

Analysis of a Genomic DNA Expression Library of Mycobacterium tuberculosis Using Tuberculosis Patient Sera: Evidence for Modulation of Host Immune Response

DNA obtained from a human sputum isolate of Mycobacterium tuberculosis, NTI-64719, which showed extensive dissemination in the guinea pig model resulting in a high score for virulence was used to construct an expression library in the lambda ZAP vector. The size of DNA inserts in the library ranged from 1 to 3 kb, and recombinants represented 60% of the total plaques obtained. When probed with pooled serum from chronically infected tuberculosis patients, the library yielded 176 recombinants with a range of signal intensities. Among these, 93 recombinants were classified into 12 groups on the basis of DNA hybridization experiments, The polypeptides synthesized by the recombinants were predominantly LacZ fusion proteins, Serum obtained from patients who were clinically diagnosed to be in the early phase of M. tuberculosis infection was used to probe the 176 recombinants obtained. interestingly, some recombinants that gave very strong signals in the original screen did not react with early-phase serum; conversely, others whose signals were extremely weak in the original screen gave very intense signals with serum from recently infected patients, This indicates the differential nature of either the expression of these antigens or the immune response elicited by them as a function of disease progression.

Resonances, scattering theory, and rigged Hilbert spaces

The problem of decaying states and resonances is examined within the framework of scattering theory in a rigged Hilbert space formalism. The stationary free,''in,'' and ''out'' eigenvectors of formal scattering theory, which have a rigorous setting in rigged Hilbert space, are considered to be analytic functions of the energy eigenvalue. The value of these analytic functions at any point of regularity, real or complex, is an eigenvector with eigenvalue equal to the position of the point. The poles of the eigenvector families give origin to other eigenvectors of the Hamiltonian: the singularities of the ''out'' eigenvector family are the same as those of the continued S matrix, so that resonances are seen as eigenvectors of the Hamiltonian with eigenvalue equal to their location in the complex energy plane. Cauchy theorem then provides for expansions in terms of ''complete'' sets of eigenvectors with complex eigenvalues of the Hamiltonian. Applying such expansions to the survival amplitude of a decaying state, one finds that resonances give discrete contributions with purely exponential time behavior; the background is of course present, but explicitly separated. The resolvent of the Hamiltonian, restricted to the nuclear space appearing in the rigged Hilbert space, can be continued across the absolutely continuous spectrum; the singularities of the continuation are the same as those of the ''out'' eigenvectors. The free, ''in'' and ''out'' eigenvectors with complex eigenvalues and those corresponding to resonances can be approximated by physical vectors in the Hilbert space, as plane waves can. The need for having some further physical information in addition to the specification of the total Hamiltonian is apparent in the proposed framework. The formalism is applied to the Lee–Friedrichs model and to the scattering of a spinless particle by a local central potential. Journal of Mathematical Physics is copyrighted by The American Institute of Physics.

Multipliers of Sobolev spaces

Let Wm,p denote the Sobolev space of functions on Image n whose distributional derivatives of order up to m lie in Lp(Image n) for 1 less-than-or-equals, slant p less-than-or-equals, slant ∞. When 1 < p < ∞, it is known that the multipliers on Wm,p are the same as those on Lp. This result is true for p = 1 only if n = 1. For, we prove that the integrable distributions of order less-than-or-equals, slant1 whose first order derivatives are also integrable of order less-than-or-equals, slant1, belong to the class of multipliers on Wm,1 and there are such distributions which are not bounded measures. These distributions are also multipliers on Lp, for 1 < p < ∞. Moreover, they form exactly the multiplier space of a certain Segal algebra. We have also proved that the multipliers on Wm,l are necessarily integrable distributions of order less-than-or-equals, slant1 or less-than-or-equals, slant2 accordingly as m is odd or even. We have obtained the multipliers from L1(Image n) into Wm,p, 1 less-than-or-equals, slant p less-than-or-equals, slant ∞, and the multiplier space of Wm,1 is realised as a dual space of certain continuous functions on Image n which vanish at infinity.

A Team of Continuous-Action Learning Automata for Noise-Tolerant Learning of Half-Spaces

Learning automata are adaptive decision making devices that are found useful in a variety of machine learning and pattern recognition applications. Although most learning automata methods deal with the case of finitely many actions for the automaton, there are also models of continuous-action-set learning automata (CALA). A team of such CALA can be useful in stochastic optimization problems where one has access only to noise-corrupted values of the objective function. In this paper, we present a novel formulation for noise-tolerant learning of linear classifiers using a CALA team. We consider the general case of nonuniform noise, where the probability that the class label of an example is wrong may be a function of the feature vector of the example. The objective is to learn the underlying separating hyperplane given only such noisy examples. We present an algorithm employing a team of CALA and prove, under some conditions on the class conditional densities, that the algorithm achieves noise-tolerant learning as long as the probability of wrong label for any example is less than 0.5. We also present some empirical results to illustrate the effectiveness of the algorithm.

Holomorphic Sobolev spaces, Hermite and special Hermite semigroups and a Paley-Wiener theorem for the windowed Fourier transform

The images of Hermite and Laguerre-Sobolev spaces under the Hermite and special Hermite semigroups (respectively) are characterized. These are used to characterize the image of Schwartz class of rapidly decreasing functions f on R-n and C-n under these semigroups. The image of the space of tempered distributions is also considered and a Paley-Wiener theorem for the windowed (short-time) Fourier transform is proved.

Open-access more harm than good in developing world

The traditional 'publish for free and pay to read' business model adopted by publishers of academic journals can lead to disparity in access to scholarly literature, exacerbated by rising journal costs and shrinking library budgets. However, although the 'pay to publish and read for free' business model of open-access publishing has helped to create a level playing field for readers, it does more harm than good in the developing world.

Proximinality in Banach spaces

In this paper, we study approximatively τ-compact and τ-strongly Chebyshev sets, where τ is the norm or the weak topology. We show that the metric projection onto τ-strongly Chebyshev sets are norm-τ continuous. We characterize approximatively τ-compact and τ-strongly Chebyshev hyperplanes and use them to characterize factor reflexive proximinal subspaces in τ-almost locally uniformly rotund spaces. We also prove some stability results on approximatively τ-compact and τ-strongly Chebyshev subspaces.

Semigroups on Frechet spaces and equations with infinite delays

In this paper, we show existence and uniqueness of a solution to a functional differential equation with infinite delay. We choose an appropriate Frechet space so as to cover a large class of functions to be used as initial functions to obtain existence and uniqueness of solutions.

Semigroups on Frechet spaces and equations with infinite delays

In this paper, we show existence and uniqueness of a solution to a functional differential equation with infinite delay. We choose an appropriate Frechet space so as to cover a large class of functions to be used as initial functions to obtain existence and uniqueness of solutions.

Identification and characterization of a library of microheterogeneous cyclohexadepsipeptides from the fungus Isaria

Ten new cyclic hexadepsipeptides, six isariins and four isaridins, from the fungus Isaria have been identified and characterized by high-performance liquid chromatography, coupled to tandem electrospray ionization mass spectrometry (LC-ESIMS/MS). The isariins possess a beta-hydroxy acid residue and five alpha-amino acids, while isaridins contain a beta-amino acid, an alpha-hydroxy acid, and four alpha-amino acids. One- and two-dimensional NMR spectroscopy confirmed the chemical identity of some of the isariin fractions. Mass spectral fragmentation patterns of [M + H](+) ions reveal clear diagnostic fragment ions for the isariins and isaridins. Previously described cyclic depsipeptides, isarfelins from Isaria felina (Guo, Y. X.; Liu, Q. H.; Ng, T. B.; Wang H. X. Peptides 2005, 26, 2384), are now reassigned as members of the isaridin family. Examination of isaridin sequences revealed significant similarities with cyclic hexadepsipeptides such as destruxins and roseotoxins. The structure of an isariin (isariin A) investigated by NMR spectroscopy indicated the presence of a hybrid alpha beta C-11 turn, formed by the beta-hydroxy acid and glycine residues and a (D)Leu-(L)Ala type II' beta-turn. Additionally, the inhibitory effect of isariins and an isaridin on the intra-erythrocytic growth of Plasmodium falciparum is presented.