Effect of Chromatographic Conditions on Supercoiled Plasmid DNA Stability and Bioactivity

Funding: This work was supported by FEDER funds through the POCI—COMPETE 2020 Operational Programme Competitiveness and Internationalization in Axis I—Strengthening research, technological development, and innovation (Project POCI-01-0145-FEDER-007491), and National Funds by FCT Foundation for Science and Technology (Project UID/Multi /00709/2013). This work was also developed within the scope of the project CICECO-Aveiro Institute of Materials, FCT Ref. UID/CTM/50011/2019, financed by national funds through the FCT/MCTES. G.M. Azevedo acknowledges the support and fellowship of Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/203482/2014-0). J.F.A. Valente acknowledges the PhD fellowship (Ref SFRH/BD/96809/2013) from FCT.Acknowledgments: The authors would like to thank Thomas Roberts for providing the pcDNA3–FLAG–p53 construct through Addgene, ref: 10838.The dysfunction of the tumor suppressor gene TP53 has been associated with the pathogenesis of the majority of the cases of cancer reported to date, leading the cell to acquire different features known as the cancer hallmarks. In normal situations, the protein p53 protects the cells against tumorigenesis. By detecting metabolic stress or DNA damage in response to stress, p53 can lead the cell to senescence, autophagy, cell cycle arrest, DNA repair, and apoptosis. Thus, in the case of p53 mutations, it is reasonable to assume that the reestablishment of its function, may restrain the proliferation of cancer cells. The concept of cancer gene therapy can be based on this assumption, and suitable biotechnological approaches must be explored to assure the preparation of gene-based biopharmaceuticals. Although numerous procedures have already been established to purify supercoiled plasmid DNA (sc pDNA), the therapeutic application is highly dependent on the biopharmaceutical’s activity, which can be affected by the chromatographic conditions used. Thus, the present work aims at comparing quality and in vitro activity of the supercoiled (sc) isoform of the p53 encoding plasmid purified by three different amino acids-based chromatographic strategies, involving histidine–agarose, arginine–macroporous, and histidine–monolith supports. The B-DNA topology was maintained in all purified pDNA samples, but their bioactivity, related to the induction of protein p53 expression and apoptosis in cancer cells, was higher with arginine–macroporous support, followed by histidine–monolith and histidine–agarose. Despite the purity degree of 92% and recovery yield of 43% obtained with arginine–macroporous, the sc pDNA sample led to a higher expression level of the therapeutic p53 protein (58%) and, consequently, induced a slightly higher apoptotic effect (27%) compared with sc pDNA samples obtained with histidine–monolithic support (26%) and histidine–agarose support (24%). This behavior can be related to the mild chromatographic conditions used with arginine–macroporous support, which includes the use of low salt concentrations, at neutral pH and lower temperatures, when compared to the high ionic strength of ammonium sulfate and acidic pH used with histidine-based supports. These results can contribute to field of biopharmaceutical preparation, emphasizing the need to control several experimental conditions while adapting and selecting the methodologies that enable the use of milder conditions as this can have a significant impact on pDNA stability and biological activity.info:eu-repo/semantics/publishedVersio

Electron spin as a spectrometer of nuclear spin noise and other fluctuations

This chapter describes the relationship between low frequency noise and coherence decay of localized spins in semiconductors. Section 2 establishes a direct relationship between an arbitrary noise spectral function and spin coherence as measured by a number of pulse spin resonance sequences. Section 3 describes the electron-nuclear spin Hamiltonian, including isotropic and anisotropic hyperfine interactions, inter-nuclear dipolar interactions, and the effective Hamiltonian for nuclear-nuclear coupling mediated by the electron spin hyperfine interaction. Section 4 describes a microscopic calculation of the nuclear spin noise spectrum arising due to nuclear spin dipolar flip-flops with quasiparticle broadening included. Section 5 compares our explicit numerical results to electron spin echo decay experiments for phosphorus doped silicon in natural and nuclear spin enriched samples.Comment: Book chapter in "Electron spin resonance and related phenomena in low dimensional structures", edited by Marco Fanciulli. To be published by Springer-Verlag in the TAP series. 35 pages, 9 figure

Theory of nuclear induced spectral diffusion: Spin decoherence of phosphorus donors in Si and GaAs quantum dots

We propose a model for spectral diffusion of localized spins in semiconductors due to the dipolar fluctuations of lattice nuclear spins. Each nuclear spin flip-flop is assumed to be independent, the rate for this process being calculated by a method of moments. Our calculated spin decoherence time $T_{M}=0.64$ ms for donor electron spins in Si:P is a factor of two longer than spin echo decay measurements. For $^{31}$P nuclear spins we show that spectral diffusion is well into the motional narrowing regime. The calculation for GaAs quantum dots gives $T_{M}=10-50$ $\mu$s depending on the quantum dot size. Our theory indicates that nuclear induced spectral diffusion should not be a serious problem in developing spin-based semiconductor quantum computer architectures.Comment: 15 pages, 9 figures. Accepted for publication in Phys. Rev.

Targeting Tyrosine Phosphatases by 3-Bromopyruvate Overcomes Hyperactivation of Platelets from Gastrointestinal Cancer Patients

Venous thromboembolism (VTE) is one of the most common causes of cancer related mortality. It has been speculated that hypercoagulation in cancer patients is triggered by direct or indirect contact of platelets with tumor cells, however the underlying molecular mechanisms involved are currently unknown. Unraveling these mechanisms may provide potential avenues for preventing platelet-tumor cell aggregation. Here, we investigated the role of protein tyrosine phosphatases in the functionality of platelets in both healthy individuals and patients with gastrointestinal cancer, and determined their use as a target to inhibit platelet hyperactivity. This is the first study to demonstrate that platelet agonists selectively activate low molecular weight protein tyrosine phosphatase (LMWPTP) and PTP1B, resulting in activation of Src, a tyrosine kinase known to contribute to several platelet functions. Furthermore, we demonstrate that these phosphatases are a target for 3-bromopyruvate (3-BP), a lactic acid analog currently investigated for its use in the treatment of various metabolic tumors. Our data indicate that 3-BP reduces Src activity, platelet aggregation, expression of platelet activation makers and platelet-tumor cell interaction. Thus,

Search for direct production of charginos and neutralinos in events with three leptons and missing transverse momentum in √s = 7 TeV pp collisions with the ATLAS detector

A search for the direct production of charginos and neutralinos in final states with three electrons or muons and missing transverse momentum is presented. The analysis is based on 4.7 fb−1 of proton–proton collision data delivered by the Large Hadron Collider and recorded with the ATLAS detector. Observations are consistent with Standard Model expectations in three signal regions that are either depleted or enriched in Z-boson decays. Upper limits at 95% confidence level are set in R-parity conserving phenomenological minimal supersymmetric models and in simplified models, significantly extending previous results

Jet size dependence of single jet suppression in lead-lead collisions at sqrt(s(NN)) = 2.76 TeV with the ATLAS detector at the LHC

Measurements of inclusive jet suppression in heavy ion collisions at the LHC provide direct sensitivity to the physics of jet quenching. In a sample of lead-lead collisions at sqrt(s) = 2.76 TeV corresponding to an integrated luminosity of approximately 7 inverse microbarns, ATLAS has measured jets with a calorimeter over the pseudorapidity interval |eta| < 2.1 and over the transverse momentum range 38 < pT < 210 GeV. Jets were reconstructed using the anti-kt algorithm with values for the distance parameter that determines the nominal jet radius of R = 0.2, 0.3, 0.4 and 0.5. The centrality dependence of the jet yield is characterized by the jet "central-to-peripheral ratio," Rcp. Jet production is found to be suppressed by approximately a factor of two in the 10% most central collisions relative to peripheral collisions. Rcp varies smoothly with centrality as characterized by the number of participating nucleons. The observed suppression is only weakly dependent on jet radius and transverse momentum. These results provide the first direct measurement of inclusive jet suppression in heavy ion collisions and complement previous measurements of dijet transverse energy imbalance at the LHC.Comment: 15 pages plus author list (30 pages total), 8 figures, 2 tables, submitted to Physics Letters B. All figures including auxiliary figures are available at http://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/HION-2011-02