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This article brings together four different perspectives which explore the way in which various policy initiatives in recent years have sought to construct young people resident in the United Kingdom within particular policy discourses shaped by neoliberalism. In order to do this it firstly considers the way in which the assumptions of neoliberalism have increasingly been applied by the new Coalition Government to young people and the services provided for them; it then considers the particular role of New Labour in the UK in applying these ideas in practice. Specific examples from the areas of young people’s participation in youth services and higher education policy are then considered.

Abstract Background Echocardiography is a non-invasive method for assessment of the ovine and caprine heart. Complete reference ranges for cardiac dimensions and time indices for both species are not currently available and reliability of these measurements has not been evaluated. The objectives for this study are to report reliability, normal cardiac dimensions and time indices in a large group of adult sheep and goats. Fifty-one adult sheep and forty adult goats were recruited. Full echocardiographic examinations were performed in the standing unsedated animal. All animals underwent echocardiography four times in a 72-hour period. Echocardiography was performed three times by one author and once by another. Images were stored and measured offline. Technique and measurement repeatability and reproducibility and any differences due to animal or day were evaluated. Reference ranges (mean ± 2 standard deviations) were calculated for both species. Results Majority of the images obtained were of good to excellent quality. Image acquisition was straightforward with 5.4% of animals demonstrating a small scanning window. Reliability was excellent for majority of dimensions and time indices. There was less variation in repeatability when compared with reproducibility and differences were greater for technique than for measurements. Dimensions that were less reliable included those for right ventricular diameter and left ventricular free wall. There were many differences in cardiac dimensions between sheep and goats. Conclusions This study has demonstrated that specific reference ranges are required for these two species. Repeatability and reproducibility were excellent for the majority of cardiac dimensions and time indices suggesting that this technique is reliable and valuable for examination of clinical cases over time and for longitudinal research studies.

The present communication presents a new triplex PCR co-amplifying three loci (D3S1358, D8S1179 and D18S51) recommended for STR typing by the European Network of Forensic Science Institutes (ENFSI). Twenty-two different primers were tested to optimise the PCR. Four of the six primer sequences finally chosen were self selected, the fifth was a published one and the sixth derived from a commercially available multiplex kit. Using this PCR-setup, even minimum amounts of genomic DNA are sufficient to analyse the STR loci D3S1358, D8S1179 and D18S51 in parallel. Especially in forensic casework, where DNA is mostly limited and often contaminated with enzyme inhibitors, this new PCR proved to be very advantageous. To demonstrate the reliability, buccal swabs from 2874 persons were typed not only with the new triplex PCR but also with a commercially available multiplex kit.

Background: Competitive PCR of reverse transcribed mRNA sequences is used to quantify transcripts, but the usual approaches are labor-intensive and time-consuming. We describe the non-gel-based quantification of competitive reverse transcription (RT)-PCR products with use of microparticles and flow cytometry., Methods: PCR products of a target sequence and an internal control sequence (competitor) were labeled during PCR using digoxigenin (DIG)- and dinitrophenol (DNP)-labeled primer, respectively, allowing specific binding to microparticles coated with the corresponding antibody. Both amplification products were biotinylated to enable fluorescence labeling with streptavidin-R-phycoerythrin. The mean fluorescence intensity of each microparticle population, corresponding to the amount of bound PCR product, was measured in a flow cytometer. We constructed microparticles coated with antibodies against DIG and DNP to specifically capture PCR products derived from target and competitor sequences, respectively., Results: As required for a reliable competitive PCR assay, nearly identical kinetics were found for the amplification of target and competitor sequences when using only one competitive primer. The method was applied to examine interleukin-8 expression in human lymphocytes after x-irradiation. One hour after irradiation, the concentration of transcripts decreased by half., Conclusions: The flow cytometric assay for the quantification of competitive RT-PCR products avoids additional hybridization steps and antibody labeling. The use of paramagnetic microparticles would also enable the complete automation of this method.

Using repetitive elements as probes, genomic DNA fingerprints of four randomly selected yeast artificial chromosome (YAC) clones (two human and two mouse-derived YAC) were analyzed to determine the mutation level following X-ray exposure. Because the repetitive probes were derived from the mammalian host DNA, most of the fingerprint bands originated from the artificial chromosomes and not from the yeast genome. For none of the YAC clones was the mutation frequency elevated following X-ray exposure. However, for one mouse-derived YAC, the mutation level was unusually high (7%; 42 mutants of 607 clones analyzed), whereas for the other three YACs, the mutation level was nearly 0%. Surprisingly, 40 of the 42 mutations were deletions occurring only at three of the 20 mouse specific fingerprint bands. One of the frequently deleted fragments was cloned, sequenced and mapped to distal mouse chromosome 4, which has been repeatedly reported to be the most unstable region of the whole mouse genome, associated with various tumors. Deletion mapping of six YAC mutants revealed this fragment to be completely deleted in four YACs. In the other two mutants, recombination occurred within the fragment, in each case initiated at the same LINE-1 element. In conclusion, the presented YAC fingerprint is a useful tool for detecting and characterizing unstable regions in mammalian genomes.

Determination of the genotoxic effects of ionizing radiation, especially at low-doses, is of great importance for risk assessment, e.g. in radiological diagnostics. The human-hamster hybrid A(L) cell line has been shown previously to be a well-suited in vitro model for the study of mutations induced by various mutagens. The A(L) cells contain a standard set of hamster chromosomes and a single human chromosome 11, which confers the expression of the human cell surface protein CD59. Using CD59 specific antibodies, cells mutated in the CD59 gene can be detected and quantified by the loss of the cell surface marker. In contrast to previous studies, prior to irradiation we removed spontaneous mutants by magnetic cell separation (MACS) which allows analysis of radiation-induced mutation events only. We exposed A(L) cells to 100kV X-rays at 0.1 to 5Gy. The proportions of X-irradiation-induced CD59(-) mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5Gy the yield of CD59 mutants was a linear function of dose. The molecular analysis of individual CD59-negative clones induced after exposure of 1, 3 and 5Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (< or =1Gy). We conclude that the modified A(L) assay presented here is appropriate for detection and quantification of non-lethal DNA lesions induced by low-dose ionizing radiation.

Background: Reverse transcription-PCR (RT-PCR) is a powerful tool in clinical diagnostics for analyzing even small amounts of RNA, but sensitive assays for quantifying the amplification products are time-consuming or expensive. Here we describe a novel flow cytometry-based assay for rapid and sensitive determination of relative amounts of RT-PCR products., Methods: For flow cytometric quantification, PCR products were labeled with both digoxigenin and biotin during amplification. Subsequently, amplicons were simultaneously bound to anti-digoxigenin microparticles and fluorescently labeled with streptavidin-R-phycoerythrin. Fluorescence intensity per bead was determined by flow cytometry. To study this assay, we examined the expression of the p21(WAF1/CIP1) gene and the proliferating cell nuclear antigen (PCNA) gene in ultraviolet irradiation-exposed human keratinocytes lacking functional p53., Results: Fluorescence was linear with 60-10 000 pg of PCR product. As little as 0.4 fmol (40 pg of a 163-bp amplicon) of PCR product could be distinguished from background. The between-run CV of the fluorescent signal for 10 ng of p21 cDNA was 12% (n = 10). The fluorescence-template curve was sigmoidal. p21(WAF1/CIP1) mRNA was decreased after ultraviolet irradiation of keratinocytes, whereas PCNA mRNA was markedly increased., Conclusion: The flow cytometric assay permits rapid (25 min) and reproducible identification of changes in mRNA abundance.

The incidence of skin cancer is increasing in epidemic proportion. Although solar UV radiation is known to be the major risk factor, much information is lacking about the molecular mechanisms leading to skin cancer. To gain a deeper insight into these mechanisms, we have examined cells of a human keratinocyte cell line (HaCat) after exposure to 0.16 minimal erythema doses of UVB radiation. This dose led to an S-phase delay that was reversible 22 h postirradiation. To examine gene expression 10 h after UV irradiation, a nonradioactive differential display was employed. Three genes were identified as being down-regulated significantly. The first encodes for topoisomerase-IIbeta-binding protein 1 (expression level 5% 6 h after irradiation). This protein is associated with human topoisomerase IIbeta and appears to be necessary for DNA replication during the onset of S phase. The second gene product has previously been reported to be involved in differentiation and is therefore known as differentiation-dependent A4 protein (28% 8 h after irradiation). The third gene is XPO1 (also known as CRM1) (5% 8 h after irradiation), whose protein is involved in nuclear export of mRNA molecules. Differential expression of these genes after UV irradiation has not been reported. Because of their potential involvement in cell cycle control and differentiation, these proteins could be important for understanding the reaction of keratinocytes after exposure to UV radiation.

The performance of rational design to maximize the structural diversity of databases for lead finding and lead refinement was investigated. Rational methods such as maximum dissimilarity methods or hierarchical cluster analysis for designing compound subsets were compared to a random approach to study their efficiency for an enhancement of the diversity of three different databases. All investigations were done based on 2D fingerprints as a validated molecular descriptor. To compare the performance of the rational selection methods to a random approach, we additionally used probability calculations. When using maximum dissimilarity-based selections, a single compound can be a member of different neighborhoods as defined by the similarity threshold value, while in hierarchical clustering each compound is assigned to only a single cluster. Therefore the relationship between the similarity threshold of the maximum diversity selection method and a 2D similarity search threshold was studied. In contrast to hierarchical clustering analysis, maximum dissimilarity selections allow to use a similarity threshold for adding a new compound to an already selected compound list. Reasonable values for this similarity threshold are presented here. More diverse subsets were designed using maximum dissimilarity selections, which cover more biological classes than using random selections. An optimally diverse subset without redundant structures containing only 38% of one original dataset was generated, where no structure is more similar than 0.85 to its nearest neighbor, but all biological classes were represented. When it is acceptable to cover only 90% of all biological targets, 3.5-3.7 times more compounds need to be selected using a random approach than in a rational design approach. Such coverage rate shows the highest efficiency of design techniques compared to a random approach. In those subsets no compound is closer than 0.70 to its nearest neighbor. Furthermore a comparative molecular field analysis (CoMFA) is used to evaluate designed and randomly chosen subsets for a database consisting of inhibitors of the angiotensin-converting enzyme. It was shown that designed subsets using maximum dissimilarity methods lead to more stable quantitative structure-activity relationship (QSAR) models with higher predictive power compared to randomly chosen compounds. This predictive power is especially high when there is no compound in the test dataset with a similarity coefficient less than 0.7 to its nearest neighbor in the training set.