Assays for DNA double-strand break repair by microhomology-based end-joining repair mechanisms.

DNA double stranded breaks (DSBs) are one of the most deleterious types of DNA lesions. The main pathways responsible for repairing these breaks in eukaryotic cells are homologous recombination (HR) and non-homologous end-joining (NHEJ). However, a third group of still poorly characterized DSB repair pathways, collectively termed microhomology-mediated end-joining (MMEJ), relies on short homologies for the end-joining process. Here, we constructed GFP reporter assays to characterize and distinguish MMEJ variant pathways, namely the simple MMEJ and the DNA synthesis-dependent (SD)-MMEJ mechanisms. Transfection of these assay vectors in Chinese hamster ovary (CHO) cells and characterization of the repaired DNA sequences indicated that while simple MMEJ is able to mediate relatively efficient DSB repair if longer microhomologies are present, the majority of DSBs were repaired using the highly error-prone SD-MMEJ pathway. To validate the involvement of DNA synthesis in the repair process, siRNA knock-down of different genes proposed to play a role in MMEJ were performed, revealing that the knock-down of DNA polymerase θ inhibited DNA end resection and repair through simple MMEJ, thus favoring the other repair pathway. Overall, we conclude that this approach provides a convenient assay to study MMEJ-related DNA repair pathways

The role of MAR elements in DNA recombination

The untargeted integration of foreign DNA into the mammalian cell genome, extensively used in gene therapy and biotechnology, remains an incompletely understood process. It is believed to be based on cellular DNA double strand break (DSB) repair machinery and to involve two major steps: i) the formation of long gene arrays (concatemers), and ii) recombination of the resulting concatemer with the genome. The main DSB repair pathways in eukaryotes include non-homologous end-joining (NHEJ), homologous recombination (HR), and microhomology-mediated end-joining (MMEJ). However, it is still not clear, which of these pathways are responsible for transgene integration. Here, we show that NHEJ is not the primary pathway used by mammalian cells in the transgene integration process, while the components of the HR pathway seem to be important for genomic integration but not concatemerization. Instead, concatemer formation appears to be mediated by a subset of the MMEJ pathway, termed synthesis-dependent MMEJ (SD-MMEJ). This mechanism also seems to be preferentially used for plasmid integration into the genome, as confirmed by the analysis of plasmid-to-genome junction sequences, which were found to display an SD-MMEJ pattern. Therefore, we propose the existence of two distinct SD-MMEJ subpathways, relying on different subsets of enzymes. One of these mechanisms appears to be responsible for concatemerization, while the other mechanism, partially dependent in HR enzymes, seems to mediate recombination with the genome. Previous studies performed by our group suggested that matrix attachment regions (MARs), which are epigenetic regulatory DNA elements that participate in the formation of chromatin boundaries and augment transcription, may mediate increased plasmid integration into the genome of CHO cells by stimulating DNA recombination. In the present work, we demonstrate that MAR-mediated plasmid integration results from the enhanced SD-MMEJ pathway. Analysis of transgene integration loci and junction DNA sequences validated the prevalent use of this pathway by the MAR elements to target plasmid DNA into gene-rich areas of the CHO genome. We propose that this finding should in the future help to engineer cells for improved recombinant protein production. In addition to investigating the process of transgene integration, we designed recombination assays to better characterize the components of the MMEJ and SD-MMEJ pathways. We also used CHO cells expressing cycle-sensitive reporter genes to demonstrate a potential role of HR proteins in the cell cycle regulation

Моделювання інвестиційної політики банку методами нечіткої логіки

Effective functioning of the country's economy, the pace and scale of its investment activity, partly extent depends on the state and development of the banking system. Therefore, the question of effective operation of the bank and the management of its capital is relevant to the realities of our time. Researching and solving problems of bank capital management, namely, its investment policy, require using of such economic and mathematical models, that considers both managed and unmanaged (risks, modelling faults, etc.) parameters. Among the models considered in this article, fuzzy logic models cover all of these factors. The considered methods of solving problems of banking capital management under conditions of uncertainty are based, primarily, on statistical models, when the knowledge of the probabilistic characteristics of the main parameters of the model is necessary. In addition, their use requires the fulfilment of strict conditions, which in practice are not fulfilled yet. Fuzzy logic does not replace the classical mathematical models of management; on the contrary, it extends the possibilities of traditional methods. In the course of study the authors have reviewed the investment project estimation task based on NPV with fuzzy logic methods, estimation with alpha-levels method, in particular. Determining the kind of belonging functions (μ – functions) is one of the most important issues in the theory of fuzzy sets. We offer the use of asymmetric membership function formed on the basis of Gauss function with different standard deviation values. To calculate the parameters of the asymmetric Gaussian function, an approximation is constructed that is a triangular membership function. Based on the parameters of the triangular function, the mean-square deviations for the left and right parts of the Gaussian function are calculated. The expected annual profit of the investment project has been calculated and analysed in the application of the triangle and asymmetric Gaussian function. To summarise, the use of the asymmetric Gauss function requires more capital for initial investment, so that the project is profitable. Numerical evaluation of investment project has been done based on triangular membership functions and asymmetric gaussoids are offered. A conclusion about the necessity of thorough membership function selection for investment risk reduction has been made.Проаналізовано моделі, які використовують в банківській діяльності, починаючи від робіт Еджворта до моделей нечіткої логіки. Зазначено їх переваги та недоліки. Обґрунтовано вибір моделі нечіткої логіки як інструменту моделювання. Розглянуто задачу оцінки інвестиційної політики банку, для якої визначено вид функції приналежності, що є одним із найважливіших питань у теорії нечітких множин. За функцію приналежності обрано несиметричну функцію Гауса, що зумовлено специфікою побудови таких функцій економічних показників, для яких у багатьох випадках відомо тільки три значення: найімовірніше, максимально та мінімально можливі. Запропоновано використати несиметричну функцію приналежності, сформовану на основі функції Гауса з різними значеннями середньоквадратичного відхилення. Проведено чисельні експерименти оцінки інвестиційного проекту на основі трикутних функцій приналежності та запропонованих несиметричних гаусоїд. Для розрахунку параметрів несиметричної функції Гауса спочатку побудовано наближення – трикутна функція приналежності. Виходячи з параметрів трикутної функції, розраховано середньоквадратичні відхилення для лівої та правої частин функції Гауса. Показано, що під час використання несиметричної функції Гауса потрібні більші початкові інвестиції для того, щоб проект був прибутковим. Зроблено висновок щодо необхідності ретельного підбору виду функцій приналежності для зменшення інвестиційного ризику

Real space inhomogeneities in high temperature superconductors: the perspective of two-component model

The two-component model of high temperature superconductors in its real space version has been solved using Bogoliubov-de Gennes equations. The disorder in the electron and boson subsystem has been taken into account. It strongly modifies the superconducting properties and leads to local variations of the gap parameter and density of states. The assumption that the impurities mainly modify boson energies offers natural explanation of the puzzling positive correlation between the positions of impurities and the values of the order parameter found in the scanning tunnelling microscopy experiments.Comment: 19 pages, IOPP style include

MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering.

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non-homologous end-joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis-dependent microhomology-mediated end-joining (SD-MMEJ) activities. Genome-wide analysis of the integration loci and junction sequences validated the prevalent use of the SD-MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD-MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384-396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc

Effective action for Superconductors and BCS-Bose crossover

A standard perturbative expansion around the mean-field solution is used to derive the low-energy effective action for superconductors at T=0. Taking into account the density fluctuations at the outset we get the effective action where the density $\rho$ is the conjugated momentum to the phase $\theta$ of the order parameter. In the hydrodynamic regime, the dynamics of the superconductor is described by a time dependent non-linear Schr\"odinger equation (TDNLS) for the field $\Psi(x)=\sqrt{\rho/2} e^{i\theta}$. The evolution of the density fluctuations in the crossover from weak-coupling (BCS) to strong-coupling (Bose condensation of localized pairs) superconductivity is discussed for the attractive Hubbard model. In the bosonic limit, the TDNLS equation reduces to the the Gross-Pitaevskii equation for the order parameter, as in the standard description of superfluidity. The conditions under which a phase-only action can be derived in the presence of a long-range interaction to describe the physics of the superconductivity of ``bad metals'' are discussed.Comment: 13 pages, accepted for publication on Phys. Rev.

Superfluid transition temperature in a trapped gas of Fermi atoms with a Feshbach resonance

We investigate strong coupling effects on the superfluid phase transition in a gas of Fermi atoms with a Feshbach resonance. The Feshbach resonance describes a composite quasi-Boson, which can give rise to an additional pairing interaction between the Fermi atoms. This attractive interaction becomes stronger as the threshold energy of the Feshbach resonance two-particle bound state is lowered. In a recent paper, we showed that in the uniform Fermi gas, this tunable pairing interaction naturally leads to a BCS-BEC crossover of the Nozi`eres and Schmitt-Rink kind, in which the BCS-type superfluid phase transition continuously changes into the BEC-type as the threshold energy is decreased. In this paper, we extend our previous work by including the effect of a harmonic trap potential, treated within the local density approximation (LDA). We also give results for both weak and strong coupling to the Feshbach resonance. We show that the BCS-BEC crossover phenomenon strongly modifies the shape of the atomic density profile at the superfluid phase transition temperature Tc, reflecting the change of the dominant particles going from Fermi atoms to composite Bosons. In the BEC regime, these composite Bosons are shown to first appear well above Tc. We also discuss the "phase diagram" above Tc as a function of the tunable threshold energy. We introduce a characteristic temperature T* describing the effective crossover in the normal phase from a Fermi gas of atoms to a gas of stable molecules.Comment: 43 pages, 13 figures (submitted to PRA

Aharonov-Bohm spectral features and coherence lengths in carbon nanotubes

The electronic properties of carbon nanotubes are investigated in the presence of disorder and a magnetic field parallel or perpendicular to the nanotube axis. In the parallel field geometry, the $\phi_{0}(=hc/e)$-periodic metal-insulator transition (MIT) induced in metallic or semiconducting nanotubes is shown to be related to a chirality-dependent shifting of the energy of the van Hove singularities (VHSs). The effect of disorder on this magnetic field-related mechanism is considered with a discussion of mean free paths, localization lengths and magnetic dephasing rate in the context of recent experiments.Comment: 22 pages, 6 Postscript figures. submitted to Phys. Rev.

Unconventional particle-hole mixing in the systems with strong superconducting fluctuations

Development of the STM and ARPES spectroscopies enabled to reach the resolution level sufficient for detecting the particle-hole entanglement in superconducting materials. On a quantitative level one can characterize such entanglement in terms of the, so called, Bogoliubov angle which determines to what extent the particles and holes constitute the spatially or momentum resolved excitation spectra. In classical superconductors, where the phase transition is related to formation of the Cooper pairs almost simultaneously accompanied by onset of their long-range phase coherence, the Bogoliubov angle is slanted all the way up to the critical temperature Tc. In the high temperature superconductors and in superfluid ultracold fermion atoms near the Feshbach resonance the situation is different because of the preformed pairs which exist above Tc albeit loosing coherence due to the strong quantum fluctuations. We discuss a generic temperature dependence of the Bogoliubov angle in such pseudogap state indicating a novel, non-BCS behavior. For quantitative analysis we use a two-component model describing the pairs coexisting with single fermions and study their mutual feedback effects by the selfconsistent procedure originating from the renormalization group approach.Comment: 4 pages, 4 figure

Density functional calculations of nanoscale conductance

Density functional calculations for the electronic conductance of single molecules are now common. We examine the methodology from a rigorous point of view, discussing where it can be expected to work, and where it should fail. When molecules are weakly coupled to leads, local and gradient-corrected approximations fail, as the Kohn-Sham levels are misaligned. In the weak bias regime, XC corrections to the current are missed by the standard methodology. For finite bias, a new methodology for performing calculations can be rigorously derived using an extension of time-dependent current density functional theory from the Schroedinger equation to a Master equation.Comment: topical review, 28 pages, updated version with some revision