An investigation on E. coli host strain influences and strategies to improve supercoiled plasmid DNA production for gene therapy and vaccination

The growing demand for quick and effective methods of producing large amounts of plasmid DNA for human therapy and vaccination has increased the practical challenges associated with process optimisation to improve supercoiled plasmid DNA yields obtained through current methods. The supercoiled isoform of DNA is the preferred form for use in gene therapy and vaccination as this isoform is known to produce higher levels of in vitro and in vivo transgene expression than other forms of plasmid DNA. This study was designed to investigate whether different strategies can be implemented early on in a process to improve supercoiled plasmid DNA yields obtained upstream, with the view to aid and/or ease further downstream stages. The main theme investigated is the influence of the host strain on supercoiled plasmid DNA production. Seventeen strains of Escherichia coli and three different plasmids were investigated at shake flask scale, before two strains were selected for scale up to 7L fermentation scale. The results obtained indicated that the host strain plasmid combination can heavily influence both the quantity and quality of plasmid DNA obtained and this behaviour cannot simply be determined by looking at the host strain genotype. Fermentation runs on the two strains selected for scale up (BL21 DE3 gWiz and HB101 gWiz) demonstrated that these two strains scale up very well, maintaining high specific pDNA yields (1.5mg/L/OD for BL21 DE3 gWiz) and high SC-DNA yields (98% for HB101 gWiz). Temperature amplification studies using strains harbouring pUC18 have shown that although most strain-plasmid combinations yielded more plasmid at a higher temperature of 40°C, the extent of this increase is highly influenced by the host strain. Indeed in some cases, such as for the strains ABLE K, W3110, W1485, a higher plasmid yield was obtained at 37°C. However, as the growth rates of these cultures were not measured, the extent of the accumulation of plasmid DNA due to the effects of the growth rate and/or temperature during the exponential phase of growth is unknown at this time. Similarities to what has been reported as temperature induced runaway plasmid replication have been observed in this study, although no experiments were conducted to confirm whether these observations were indeed as result of runaway replication as defined in the literature. Potential alternative strategies investigated included implementing anaerobiosis to test if these conditions can improve supercoiled plasmid DNA production at fermentation scale, and whether a ‘Quiescent cell expression system’ (a state where chromosomal replication and expression is temporarily shut down but residual proteins remain metabolically active) can be implemented to improve plasmid DNA yields by redirecting resources away from biomass production. The results suggest that under the conditions set in this study, these strategies do not increase plasmid DNA production or the percentage of supercoiled plasmid obtained. In conclusion, the results from this investigation have demonstrated that a highly effective and influential strategy for improving the quality and quantity of plasmid DNA obtained is the initial choice of the host strainplasmid combination. Further improvements can then be obtained through the application of other reported fermentation strategies

The K\"ahler-Ricci flow on surfaces of positive Kodaira dimension

The existence of K\"ahler-Einstein metrics on a compact K\"ahler manifold has been the subject of intensive study over the last few decades, following Yau's solution to Calabi's conjecture. The Ricci flow, introduced by Richard Hamilton has become one of the most powerful tools in geometric analysis. We study the K\"ahler-Ricci flow on minimal surfaces of Kodaira dimension one and show that the flow collapses and converges to a unique canonical metric on its canonical model. Such a canonical is a generalized K\"ahler-Einstein metric. Combining the results of Cao, Tsuji, Tian and Zhang, we give a metric classification for K\"aher surfaces with a numerical effective canonical line bundle by the K\"ahler-Ricci flow. In general, we propose a program of finding canonical metrics on canonical models of projective varieties of positive Kodaira dimension

A Spectral Bernstein Theorem

We study the spectrum of the Laplace operator of a complete minimal properly immersed hypersurface $M$ in $\R^{n+1}$. (1) Under a volume growth condition on extrinsic balls and a condition on the unit normal at infinity, we prove that $M$ has only essential spectrum consisting of the half line $[0, +\infty)$. This is the case when $\lim_{\tilde{r}\to +\infty}\tilde{r}\kappa_i=0$, where $\tilde{r}$ is the extrinsic distance to a point of $M$ and $\kappa_i$ are the principal curvatures. (2) If the $\kappa_i$ satisfy the decay conditions $|\kappa_i|\leq 1/\tilde{r}$, and strict inequality is achieved at some point $y\in M$, then there are no eigenvalues. We apply these results to minimal graphic and multigraphic hypersurfaces.Comment: 16 pages. v2. Final version: minor revisions, we add Proposition 3.2. Accepted for publication in the Annali di Matematica Pura ed Applicata, on the 05/03/201

K\"{a}hler-Einstein metrics on strictly pseudoconvex domains

The metrics of S. Y. Cheng and S.-T. Yau are considered on a strictly pseudoconvex domains in a complex manifold. Such a manifold carries a complete K\"{a}hler-Einstein metric if and only if its canonical bundle is positive. We consider the restricted case in which the CR structure on $\partial M$ is normal. In this case M must be a domain in a resolution of the Sasaki cone over $\partial M$. We give a condition on a normal CR manifold which it cannot satisfy if it is a CR infinity of a K\"{a}hler-Einstein manifold. We are able to mostly determine those normal CR 3-manifolds which can be CR infinities. Many examples are given of K\"{a}hler-Einstein strictly pseudoconvex manifolds on bundles and resolutions.Comment: 30 pages, 1 figure, couple corrections, improved a couple example

Liouville theorems for harmonic maps

We prove several Liouville theorems for harmonic maps between certain classes of Riemannian manifolds. In particular, the results can be applied to harmonic maps from the Euclidean space ( R m , g 0 ) to a large class of Riemannian manifolds. Our assumptions on the harmonic maps concern the asymptotic behavior of the maps at ∞.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46575/1/222_2005_Article_BF02100594.pd

Positivity of relative canonical bundles and applications

Given a family $f:\mathcal X \to S$ of canonically polarized manifolds, the unique K\"ahler-Einstein metrics on the fibers induce a hermitian metric on the relative canonical bundle $\mathcal K_{\mathcal X/S}$. We use a global elliptic equation to show that this metric is strictly positive on $\mathcal X$, unless the family is infinitesimally trivial. For degenerating families we show that the curvature form on the total space can be extended as a (semi-)positive closed current. By fiber integration it follows that the generalized Weil-Petersson form on the base possesses an extension as a positive current. We prove an extension theorem for hermitian line bundles, whose curvature forms have this property. This theorem can be applied to a determinant line bundle associated to the relative canonical bundle on the total space. As an application the quasi-projectivity of the moduli space $\mathcal M_{\text{can}}$ of canonically polarized varieties follows. The direct images $R^{n-p}f_*\Omega^p_{\mathcal X/S}(\mathcal K_{\mathcal X/S}^{\otimes m})$, $m > 0$, carry natural hermitian metrics. We prove an explicit formula for the curvature tensor of these direct images. We apply it to the morphisms $S^p \mathcal T_S \to R^pf_*\Lambda^p\mathcal T_{\mathcal X/S}$ that are induced by the Kodaira-Spencer map and obtain a differential geometric proof for hyperbolicity properties of $\mathcal M_{\text{can}}$.Comment: Supercedes arXiv:0808.3259v4 and arXiv:1002.4858v2. To appear in Invent. mat

Differential inequalities on complete Riemannian manifolds and applications

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/46226/1/208_2005_Article_BF01455859.pd

School-based prevention for adolescent Internet addiction: prevention is the key. A systematic literature review

Adolescents’ media use represents a normative need for information, communication, recreation and functionality, yet problematic Internet use has increased. Given the arguably alarming prevalence rates worldwide and the increasingly problematic use of gaming and social media, the need for an integration of prevention efforts appears to be timely. The aim of this systematic literature review is (i) to identify school-based prevention programmes or protocols for Internet Addiction targeting adolescents within the school context and to examine the programmes’ effectiveness, and (ii) to highlight strengths, limitations, and best practices to inform the design of new initiatives, by capitalizing on these studies’ recommendations. The findings of the reviewed studies to date presented mixed outcomes and are in need of further empirical evidence. The current review identified the following needs to be addressed in future designs to: (i) define the clinical status of Internet Addiction more precisely, (ii) use more current psychometrically robust assessment tools for the measurement of effectiveness (based on the most recent empirical developments), (iii) reconsider the main outcome of Internet time reduction as it appears to be problematic, (iv) build methodologically sound evidence-based prevention programmes, (v) focus on skill enhancement and the use of protective and harm-reducing factors, and (vi) include IA as one of the risk behaviours in multi-risk behaviour interventions. These appear to be crucial factors in addressing future research designs and the formulation of new prevention initiatives. Validated findings could then inform promising strategies for IA and gaming prevention in public policy and education

Operation and performance of the ATLAS semiconductor tracker

The semiconductor tracker is a silicon microstrip detector forming part of the inner tracking system of the ATLAS experiment at the LHC. The operation and performance of the semiconductor tracker during the first years of LHC running are described. More than 99% of the detector modules were operational during this period, with an average intrinsic hit efficiency of (99.74±0.04)%. The evolution of the noise occupancy is discussed, and measurements of the Lorentz angle, δ-ray production and energy loss presented. The alignment of the detector is found to be stable at the few-micron level over long periods of time. Radiation damage measurements, which include the evolution of detector leakage currents, are found to be consistent with predictions and are used in the verification of radiation background simulations

Measurements of fiducial cross-sections for t\bart production with one or two additional b-jets in pp collisions at √s =8 TeVusing the ATLAS detector

Fiducial cross-sections for $t\bar{t}$ production with one or two additional $b$-jets are reported, using an integrated luminosity of 20.3 fb$^{-1}$ of proton--proton collisions at a centre-of-mass energy of 8 TeV at the Large Hadron Collider, collected with the ATLAS detector. The cross-section times branching ratio for $t\bar{t}$ events with at least one additional $b$-jet is measured to be 950 $\pm$ 70 (stat.) $^{+240}_{-190}$ (syst.) fb in the lepton-plus-jets channel and 50 $\pm$ 10 (stat.) $^{+15}_{-10}$ (syst.) fb in the $e \mu$ channel. The cross-section times branching ratio for events with at least two additional $b$-jets is measured to be 19.3 $\pm$ 3.5 (stat.) $\pm$ 5.7 (syst.) fb in the dilepton channel ($e \mu$,\,$\mu\mu$, and \,$ee$) using a method based on tight selection criteria, and 13.5 $\pm$ 3.3 (stat.) $\pm$ 3.6 (syst.) fb using a looser selection that allows the background normalisation to be extracted from data. The latter method also measures a value of 1.30 $\pm$ 0.33 (stat.) $\pm$ 0.28 (syst.)\% for the ratio of $t\bar{t}$ production with two additional $b$-jets to $t\bar{t}$ production with any two additional jets. All measurements are in good agreement with recent theory predictions.Comment: 41 pages plus author list + cover page (58 total), 9 Figures, 16 tables, submitted to EPJC, all figures including auxiliary figures are available at https://atlas.web.cern.ch/Atlas/GROUPS/PHYSICS/PAPERS/TOPQ-2014-10