489 research outputs found

    Critical evaluation of HPV16 gene copy number quantification by SYBR green PCR

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    Background: Human papilloma virus (HPV) load and physical status are considered useful parameters for clinical evaluation of cervical squamous cell neoplasia. However, the errors implicit in HPV gene quantification by PCR are not well documented. We have undertaken the first rigorous evaluation of the errors that can be expected when using SYBR green qPCR for quantification of HPV type 16 gene copy numbers. We assessed a modified method, in which external calibration curves were generated from a single construct containing HPV16 E2, HPV16 E6 and the host gene hydroxymethylbilane synthase in a 1: 1: 1 ratio.Results: When testing dilutions of mixed HPV/host DNA in replicate runs, we observed errors in quantifying E2 and E6 amplicons of 5 - 40%, with greatest error at the lowest DNA template concentration (3 ng/mu l). Errors in determining viral copy numbers per diploid genome were 13 - 53%. Nevertheless, in cervical keratinocyte cell lines we observed reasonable agreement between viral loads determined by qPCR and Southern blotting. The mean E2/E6 ratio in episome-only cells was 1.04, but with a range of 0.76 - 1.32. In three integrant-only lines the mean E2/E6 ratios were 0.20, 0.72 and 2.61 (values confirmed by gene-specific Southern blotting). When E2/E6 ratios in fourteen HPV16-positive cervical carcinomas were analysed, conclusions regarding viral physical state could only be made in three cases, where the E2/E6 ratio was <= 0.06.Conclusion: Run-to-run variation in SYBR green qPCR produces unavoidable inaccuracies that should be allowed for when quantifying HPV gene copy number. While E6 copy numbers can be considered to provide a useable indication of viral loads, the E2/E6 ratio is of limited value. Previous studies may have overestimated the frequency of mixed episomal/integrant HPV infections

    'The heart of what we do': policies on teaching, learning and assessment in the learning and skills sector

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    One of the stated aims of government policy in England is to put teaching, training,and learning at the heart of the learning and skills system. This paper provides a critical review of policies on teaching, learning and assessment in the learning and skills sector over the past five years. It draws upon data collected and analysed in the early stages of an ESRC-funded Teaching and Learning Research Programme project. Using evidence from policy sources, we argue that despite policy rhetoric about devolution of responsibility to the 'front line', the dominant 'images' that government has of putting teaching, learning and assessment at the heart of the Learning and Skills Sector involves a narrow concept of learning and skills; an idealisation of learner agency lacking an appreciation of the pivotal role of the learner/tutor relationship and a top-down view of change in which central government agencies are relied on to secure education standards

    Lack of Evidence for an Association between Iridovirus and Colony Collapse Disorder

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    Colony collapse disorder (CCD) is characterized by the unexplained losses of large numbers of adult worker bees (Apis mellifera) from apparently healthy colonies. Although infections, toxins, and other stressors have been associated with the onset of CCD, the pathogenesis of this disorder remains obscure. Recently, a proteomics study implicated a double-stranded DNA virus, invertebrate iridescent virus (Family Iridoviridae) along with a microsporidium (Nosema sp.) as the cause of CCD. We tested the validity of this relationship using two independent methods: (i) we surveyed healthy and CCD colonies from the United States and Israel for the presence of members of the Iridovirus genus and (ii) we reanalyzed metagenomics data previously generated from RNA pools of CCD colonies for the presence of Iridovirus-like sequences. Neither analysis revealed any evidence to suggest the presence of an Iridovirus in healthy or CCD colonies
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