211 research outputs found
Generation of cell lines to complement Adenovirus vectors using recombination-mediated cassette exchange
No full textBackground
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
Recognition of skin malignancy by general practitioners: observational study using data from a population-based randomised controlled trial
No full textSkin malignancy is an important cause of mortality in the United Kingdom and is rising in incidence every year. Most skin cancer presents in primary care, and an important determinant of outcome is initial recognition and management of the lesion. Here we present an observational study of interobserver agreement using data from a population-based randomised controlled trial of minor surgery. Trial participants comprised patients presenting in primary care and needing minor surgery in whom recruiting doctors felt to be able to offer treatment themselves or to be able to refer to a colleague in primary care. They are thus relatively unselected. The skin procedures undertaken in the randomised controlled trial generated 491 lesions with a traceable histology report: 36 lesions (7%) from 33 individuals were malignant or pre-malignant. Chance-corrected agreement (?) between general practitioner (GP) diagnosis of malignancy and histology was 0.45 (0.36–0.54) for lesions and 0.41 (0.32–0.51) for individuals affected with malignancy. Sensitivity of GPs for the detection of malignant lesions was 66.7% (95% confidence interval (CI), 50.3–79.8) for lesions and 63.6% (95% CI, 46.7–77.8) for individuals affected with malignancy. The safety of patients is of paramount importance and it is unsafe to leave the diagnosis and treatment of potential skin malignancy in the hands of doctors who have limited training and experience. However, the capacity to undertake all of the minor surgical demand works demanded in hospitals does not exist. If the capacity to undertake it is present in primary care, then the increased costs associated with enhanced training for general medical practitioners (GPs) must be borne
The human adenovirus 5 L4 promoter is activated by cellular stress response protein p53
No full textDuring adenovirus infection, the emphasis of gene expression switches from early genes to late genes in a highly regulated manner. Two gene products, L4-22K and L4-33K, contribute to this switch by activating the Major Late Transcription Unit (MLTU) and regulating the splicing of its transcript. L4-22K and L4-33K expression is driven initially by a recently described L4 promoter (L4P) embedded within the MLTU that is activated by early and intermediate viral factors: E1A, E4 Orf3 and IVa2. Here we show that this promoter is also significantly activated by the cellular stress response regulator, p53. Exogenous expression of p53 activated L4P in reporter assays whilst depletion of endogenous p53 inhibited the induction of L4P by viral activators. Chromatin immunoprecipitation studies showed that p53 associates with L4P and that during adenovirus type 5 (Ad5) infection this association peaks at 12 h.p.i., coinciding with the phase of the infectious cycle when L4P is active, and is then lost as MLP activation commences. P53 activation of L4P is significant during Ad5 infection since depletion of p53 prior to infection of either immortalised or normal cells led to severely reduced late gene expression. The association of p53 with L4P is transient due to the action of products of L4P activity (L4-22K/33K), which establish a negative feedback loop that ensures the transient activity of L4P at the start of the late phase and contributes to an efficient switch from early to late phase virus gene expression
Discussion and debate: Reflections on Pleistocene Island Occupation
No full textEditorial Note: Within a 48-hour period during January 2014, the JMA co-editors received two papers—those of Curtis Runnels and Thomas P. Leppard printed above—that, quite fortuitously, each addressed the topic of Mediterranean island colonization by archaic hominins, albeit from radically different perspectives. Neither author was aware of the other’s paper, nor has either article subsequently been revised to take account of the other. Realizing the widespread current interest in this subject and the possibility for productive debate prompted by such variant approaches, we commissioned three sets of comments and invited Runnels and Leppard to respond. We are pleased to publish this discussion around questions of great importance for our understanding of the earliest insular prehistory of the Mediterranean, and with significant implications reaching well beyond it.Journal of Mediterranean Archaeology 27.2 (2014) 255-278</p
A prospective randomised comparison of minor surgery in primary and secondary care. The MiSTIC trial
No full textObjective: To determine whether there is equivalence in the competence of GPs and hospital doctors to perform a range of elective minor surgical procedures, in terms of the safety, quality and cost of care.Design: A prospective randomised controlled equivalence trial was undertaken in consenting patients presenting at general practices and needing minor surgery.Setting: The study was conducted in the south of England. Participants: Consenting patients presenting at general practices who needed minor surgery in specified categories for whom the recruiting doctor felt able to offer treatment or to be able to refer to a colleague in primary care.Interventions: On presentation to their GP, patients were randomised to either treatment within primary care or treatment at their local hospital. Evaluation was by assessment of clinical quality and safety of outcome, supplemented by examination of patient satisfaction and cost-effectiveness.Main outcome measures: Two independent observers assessed surgical quality by blinded assessment of wound appearance, between 6 and 8 weeks postsurgery, from photographs of wounds. Other measures included satisfaction with care, safety of surgery in terms of recognition of and appropriate treatment of skin malignancies, and resource use and implications.Results: The 568 patients recruited ( 284 primary care, 284 hospital) were randomised by 82 GPs. In total, 637 skin procedures plus 17 ingrowing toenail procedures were performed ( 313 primary care, 341 hospital) by 65 GPs and 60 hospital doctors. Surgical quality was assessed for 273 ( 87%) primary care and 316 ( 93%) hospital lesions. Mean visual analogue scale score in hospital was significantly higher than that in primary care [ mean difference = 5.46 on 100- point scale; 95% confidence interval ( CI) 0.925 to 9.99], but the clinical importance of the difference was uncertain. Hospital doctors were better at achieving complete excision of malignancies, with a difference that approached statistical significance [ 7/ 16 GP ( 44%) versus 15/20 hospital ( 75%), chi(2) = 3.65, p = 0.056]. The proportion of patients with post-operative complications was similar in both groups. The mean cost for hospital-based minor surgery was pound 1222.24 and for primary care pound 449.74. Using postoperative complications as an outcome, both effectiveness and costs of the alternative interventions are uncertain. Using completeness of excision of malignancy as an outcome, hospital minor surgery becomes more cost-effective. The 705 skin procedures undertaken in this trial generated 491 lesions with a traceable histology report: 36 lesions ( 7%) from 33 individuals were malignant or premalignant. Chance-corrected agreement ( kappa) between GP diagnosis of malignancy and histology was 0.45 ( 95% CI 0.36 to 0.54) for lesions and 0.41 ( 95% CI 0.32 to 0.51) for individuals affected by malignancy. Sensitivity of GPs for detection of malignant lesions was 66.7% ( 95% CI 50.3 to 79.8) for lesions and 63.6% ( 95% CI 46.7 to 77.8) for individuals affected by malignancy.Conclusions: The quality of minor surgery carried out in general practice is not as high as that carried out in hospital, using surgical quality as the primary outcome, although the difference is not large. Patients are more satisfied if their procedure is performed in primary care, largely because of convenience. However, there are clear deficiencies in GPs' ability to recognise malignant lesions, and there may be differences in completeness of excision when compared with hospital doctors. The safety of patients is of paramount importance and this study does not demonstrate that minor surgery carried out in primary care is safe as it is currently practised. There are several alternative models of minor surgery provision worthy of consideration, including ones based in primary care that require all excised tissue to be sent for histological examination, or that require further training of GPs to undertake the necessary work. The results of this study suggest that a hospital-based service is more cost-effective. It must be concluded that it is unsafe to leave minor surgery in the hands of doctors who have never been trained to do it. Further work is required to determine GPs' management of a range of skin conditions (including potentially life-threatening malignancies), rather than just their recognition of them. Further economic modelling work is required to look at the potential costs of training sufficient numbers of GPs and GPs with special interests to meet the demand for minor surgery safely in primary care, and of the alternative of transferring minor surgery large-scale to the hospital sector. Different models of provision need thorough testing before widespread introduction
Te Deum
No full text1.Te Deum : four double choir and double orchestra [C-dúr Te Deum : szólistákra, kettős karra és kettős zenekarra] /Marc-Antoine Charpentier ; edited by Denise Launay +2.Laetatus sum : Psalm 121 /Francesco Cavalli ; edited by Raymond Leppard +3.Magnificat /Francesco Cavalli ; edited by Raymond Leppar
World University Service of Canada International Seminar Recipient, 1991
No full textb&w photographExcellent condition(L-R) John Garroway, BComm '73, Alumni Association Executive, Chair of Awards Committee presents Denise Leppard, Saint Mary's Student and Recipient of W.U.S.C. International Seminar with a cheque for $500 to assist with her expenses in Botswana.This photo is part of the Alumni Office fonds. Additional photos availabl
"The joy of meaning and design wrenched out of chaos": The Modernismpostmodernism Continuum of James Joyce's Ulysses and Don DeLillo's The Names
No full textA modern author allows language to approach the play of postmodernism. A postmodern author allows language to return to a modern sense of innocence. James Joyce and Don DeLillo, the authors in question, have both allowed language to develop a continuum of sorts between modernist and postmodernist language: giving language the freedom of postmodernism and the authorial presence of modernism.
Joyce, with Molly's chapter, moves toward a postmodern use of language. The final chapter of Ulysses, employs the conventions of dream-visions and, precisely because the chapter may be termed a 'dream,' Molly's soliloquy becomes the reality of the novel which leaves the preceding male chapters to be interpreted as a perception of reality. Molly's chapter foresees the coming of postmodernism (namely in Finnegan's Wake) by abruptly changing narrative voice from the orderly thought process of males navigating a city to the stream-of-consciousness dream-vision of a female stationed in bed. The chapter prophesies the transition from modernism to postmodernism and the transition of importance from the author to the language.
DeLillo, within the text of The Names, has demonstrated his refusal to allow language sole power over the text by inserting his voice into the text in order to carry a conversation with the reader concerning authorship, reading, critics, and language. With this demand that his voice be found within the text, DeLillo returns to a sort of modernist ideal of the author as god of the text. However, just as Joyce does not fully give over the text to language, DeLillo does not take complete control of his text from language. With Tap's chapter, DeLillo returns to a modern use of the innocence of language. The reader experiences an abrupt change of narrative voice from that of the male narrator/author to that of a child, a boy, Tap. Here we do not have a soliloquy but a short story that Tap has written. DeLillo, by including the child's voice, the child's writing, and by situating it in a place of importance as the final chapter, moves toward modernist thinking in that language needs to return to innocence
Leppard, John Edward (Death, 1895-10-10)
No full textAddress: 913 Plum St.Age at death: 4mo.20da.Pg 101/1895/149/MW S/City/Dr.J.J. Malony/J. B. Habig/St.Joseph's NewOriginal record filed in drawer labeled 'Leonhard-Lewis, P'
Glacier Acceleration and Thinning after Ice Shelf Collapse in the Larsen B Embayment, Antarctica
No full textIce velocities derived from five Landsat 7 images acquired between January 2000 and February 2003 show a two- to six-fold increase in centerline speed of four glaciers flowing into the now-collapsed section of the Larsen B Ice Shelf. Satellite laser altimetry from ICEsat indicates the surface of Hektoria Glacier lowered by up to 38 +/- 6 m a six-month period beginning one year after the break-up in March 2002. Smaller elevation losses are observed for Crane and Jorum glaciers over a later 5-month period. Two glaciers south of the collapse area, Flask and Leppard, show little change in speed or elevation. Seasonal variations in speed preceding the large post-collapse velocity increases suggest that both summer melt percolation and changes in the stress field due to shelf removal play a major role in glacier dynamics
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