Generation of cell lines to complement Adenovirus vectors using recombination-mediated cassette exchange

Background Adenovirus serotype 5 (Ad5) has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies. Results We have used LoxP/Cre recombination mediated cassette exchange (RMCE) to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses. Conclusions RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression

Tracking daily fatigue fluctuations in multiple sclerosis : ecological momentary assessment provides unique insights

The preparation of this manuscript was supported by a UK Economic and Social Research Council (ESRC) PhD studentship (ES/1026266/1) awarded to DP. The study was funded by the Psychology Unit at the University of Southampton. The authors declare that they have no conflict of interest. The authors thank all participants of this study. Open access via Springer Compact Agreement.Peer reviewedPublisher PD

The Lima Theatre, 1966-67

The Lima Theatre, 1966-6

Allosteric activation of the nitric oxide receptor soluble guanylate cyclase mapped by cryo-electron microscopy.

Soluble guanylate cyclase (sGC) is the primary receptor for nitric oxide (NO) in mammalian nitric oxide signaling. We determined structures of full-length Manduca sexta sGC in both inactive and active states using cryo-electron microscopy. NO and the sGC-specific stimulator YC-1 induce a 71° rotation of the heme-binding β H-NOX and PAS domains. Repositioning of the β H-NOX domain leads to a straightening of the coiled-coil domains, which, in turn, use the motion to move the catalytic domains into an active conformation. YC-1 binds directly between the β H-NOX domain and the two CC domains. The structural elongation of the particle observed in cryo-EM was corroborated in solution using small angle X-ray scattering (SAXS). These structures delineate the endpoints of the allosteric transition responsible for the major cyclic GMP-dependent physiological effects of NO

Intrusive Teaching: The Strain of Care Labor, Identity and the Emerging Majority in Higher Education

United States publicly-funded higher education systems are experiencing increasing pressures. In response, higher education institutions have broadened their appeal to students less likely to attend college as part of their fiscal strategies. This growing student population consists of first-generation students and individuals from marginalized backgrounds who often enter college underprepared, and higher education must retain these emerging-majority students to ensure fiscal stability. When enrollment and retention are viewed from a business model, faculty duties expand into triage care and student emotional support. This qualitative investigation of faculty in a publicly-funded state university explores intrusive teaching practices marked by monitoring and intervening in their students’ emotional and social issues

Canon, criterion and circularity: An analysis of the epistemology of canonical theism

In recent years, William J. Abraham has suggested the creation of a new subdiscipline for examining the epistemology of theology. This article provides an overview of this proposal, highlighting some of the philosophical concepts, such as ‘Aristotelian epistemic fit’ and particularism, that Abraham drew upon when formulating this approach. It then proceeds to an examination of Abraham’s application of these ideas to his preferred theological scheme, canonical theism. Limitations and challenges to Abraham’s position are discussed as well as ways in which weaknesses in his approach might be addressed

Newman, Wesley and the logic of unity: An inductive approach to ecumenism

It is a privilege to be invited to contribute to the Festschrift dedicated to Professor Johan Buitendag, Emeritus Dean, Faculty of Theology and Religion at the University of Pretoria, South Africa. While his own work often examined the relationship between theology and natural science, he was also passionate about ecumenism and, in that spirit, the present essay utilised what might be described as an inductive approach to an important ecumenical question, the unity between Methodists and Catholics. Ecumenical dialogue is often undertaken from a theological and dogmatic perspective. This essay took a different approach in that it sought to inductively examine what might be described as the inner logic of two ecclesial systems (Methodist and Catholic). In doing so, it was premised upon an Aristotelian philosophical principle, epistemic fit, in that it presupposed that both these ecclesial bodies possess an intrinsic logic that can be brought to the surface in order to exhibit parallel contours in the wider ecclesial terrain. Contribution: This article fits well within the scope of HTS Teologiese Studies/Theological Studies in that it contributed to a new subdiscipline in epistemology, which explores the conceptual and epistemological dimensions of ecclesiology. Using the concept of epistemic fit, the article offered an inductive approach to some of the practical issues that emerge in ecumenical dialogue

Non-perturbative thermal flows and resummations

We construct a functional renormalisation group for thermal fluctuations. Thermal resummations are naturally built in, and the infrared problem of thermal fluctuations is well under control. The viability of the approach is exemplified for thermal scalar field theories. In gauge theories the present setting allows for the construction of a gauge-invariant thermal renormalisation group.Comment: 16 pages, eq (38) added to match published versio