Impact of repeated micro and macro blood sampling on clinical chemistry and haematology in rats for toxicokinetic studies.

Non-clinical rodent safety studies are essential in the development of new medicines to assess for potential adverse effects. Typically, toxicokinetic samples are collected from a satellite group. AstraZeneca implemented repeated microsampling of main study animals as standard in the one-month small molecule regulatory toxicology studies. A retrospective analysis of the clinical chemistry and haematology data collected in 52 independent studies from the adult rat controls explored the impact of micro and macro sampling of main study animals. For the majority of variables, the blood sampling technique had no significant impact on the mean or range. For microsampling, a few variables had statistically significant effects on the mean signal but these were considered to have limited biological relevance and would therefore not introduce a meaningful bias to any toxicological evaluation. The macrosampling had the expected effects on the red cell parameters of haemoglobin, haematocrit and red blood count due to the larger blood volume draw. In contrast, microsampling showed no such changes. In conclusion, this large-scale retrospective analysis supports the use of microsampling, for toxicokinetics, of main study animals and enables us to conduct rodent toxicology studies without satellite animals and further reduce the number of animals used in toxicological assessments.

Response to mTOR inhibition: activity of eIF4E predicts sensitivity in cell lines and acquired changes in eIF4E regulation in breast cancer.

BACKGROUND: Inhibitors of the kinase mTOR, such as rapamycin and everolimus, have been used as cancer therapeutics with limited success since some tumours are resistant. Efforts to establish predictive markers to allow selection of patients with tumours likely to respond have centred on determining phosphorylation states of mTOR or its targets 4E-BP1 and S6K in cancer cells. In an alternative approach we estimated eIF4E activity, a key effector of mTOR function, and tested the hypothesis that eIF4E activity predicts sensitivity to mTOR inhibition in cell lines and in breast tumours. RESULTS: We found a greater than three fold difference in sensitivity of representative colon, lung and breast cell lines to rapamycin. Using an assay to quantify influences of eIF4E on the translational efficiency specified by structured 5'UTRs, we showed that this estimate of eIF4E activity was a significant predictor of rapamycin sensitivity, with higher eIF4E activities indicative of enhanced sensitivity. Surprisingly, non-transformed cell lines were not less sensitive to rapamycin and did not have lower eIF4E activities than cancer lines, suggesting the mTOR/4E-BP1/eIF4E axis is deregulated in these non-transformed cells. In the context of clinical breast cancers, we estimated eIF4E activity by analysing expression of eIF4E and its functional regulators within tumour cells and combining these scores to reflect inhibitory and activating influences on eIF4E. Estimates of eIF4E activity in cancer biopsies taken at diagnosis did not predict sensitivity to 11-14 days of pre-operative everolimus treatment, as assessed by change in tumour cell proliferation from diagnosis to surgical excision. However, higher pre-treatment eIF4E activity was significantly associated with dramatic post-treatment changes in expression of eIF4E and 4E-binding proteins, suggesting that eIF4E is further deregulated in these tumours in response to mTOR inhibition. CONCLUSIONS: Estimates of eIF4E activity predict sensitivity to mTOR inhibition in cell lines but breast tumours with high estimated eIF4E activity gain changes in eIF4E regulation in order to enhance resistance.

Phosphorylation of estrogen receptor beta at serine 105 is associated with good prognosis in breast cancer.

Estrogen receptor (ER) action is modulated by posttranslational modifications. Although ERalpha phosphorylation correlates with patient outcome, ERbeta is similarly phosphorylated but its significance in breast cancer has not been addressed. We investigated whether ERbeta that is phosphorylated at serine 105 (S105-ERbeta) is expressed in breast cancer and assessed potential clinical implications of this phosphorylation. Following antibody validation, S105-ERbeta expression was studied in tissue microarrays comprising 108 tamoxifen-resistant and 351 tamoxifen-sensitive cases and analyzed against clinical data. S105-ERbeta regulation in vitro was assessed by Western blot, flow cytometry, and immunofluorescence. Nuclear S105-ERbeta was observed in breast carcinoma and was associated with better survival (Allred score > or =3), even in tamoxifen-resistant cases, and additionally correlated with ERbeta1 and ERbeta2 expression. Distinct S105-ERbeta nuclear speckles were seen in some higher grade tumors. S105-ERbeta levels increased in MCF-7 cells in response to 17beta-estradiol, the ERbeta-specific agonist diarylpropionitrile, and the partial ERbeta-agonist genistein. S105-ERbeta nuclear speckles were also seen in MCF-7 cells and markedly increased in size and number at 24 hours following 17beta-estradiol and, in particular diarylpropionitrile, treatment. These speckles were coexpressed with ERbeta1 and ERbeta2. Presence of S105-ERbeta in breast cancer and association with improved survival, even in endocrine resistant breast tumors suggest S105-ERbeta might be a useful additional prognostic marker in this disease.

Expression of oestrogen receptor beta isoforms is regulated by transcriptional and post-transcriptional mechanisms.

Although ERs (oestrogen receptors) mediate breast tumour behaviour, the precise role of ERbeta remains unclear. This is mainly because analyses have been complicated by the presence in breast tissue of three ERbeta protein variants (ERbeta1, ERbeta2 and ERbeta5) that derive from differential 3' splicing. We have recently identified the first known mechanisms responsible for the differential control of isoform expression, involving regulation of translation via 5'-UTRs (untranslated regions). In the present study, we have uncovered further complexity involving the influence of multiple promoters and cross-talk between 5'- and 3'-UTRs. We demonstrate that full-length ERbeta mRNAs are transcribed from three separate promoters; two promoters are well-established within the literature, whereas the third represents a novel finding. Each promoter produces transcripts with distinct 5'-UTRs. The differential 3' splicing that produces transcripts coding for the ERbeta isoforms also defines isoform-specific 3'-UTRs. We identified exact 3'-UTR sequences for each isoform, and have shown that alternative polyadenylation sites are used in a cell-type specific manner to produce transcripts with 3'-UTRs of different lengths. Critically, we show that 5'- and 3'-UTRs combine to specify the efficiencies with which individual transcripts are translated, with 3'-UTR length having a key influence. In addition, we demonstrate how 17beta-oestradiol, a key driver of breast cancer development, affects the regulation of ERbeta expression at both transcriptional and translational levels.

Education, training, and professional issues of radiographers in six European countries: a comparative review.

Radiographers constitute an important part of a multidisciplinary radiation-based imaging and therapy chain. However, is there a common framework for assuring high education, training, and subsequent practice of profession among European countries? A study was conducted, based on a questionnaire that consisted of three parts, concerning education and training (Part A), national registry (Part B), and professional issues (Part C). Analysis of the collected data suggested that a common policy is generally followed in the countries investigated; however, differences were not negligible. A common framework of educational programmes among European countries could form the basis for overall standardisation at national and international level.

The Reliability of Big "Patient Satisfaction" Data.

Big data in healthcare can bring significant clinical and cost benefits. Of equal but often overlooked importance is the role of patient satisfaction data in improving the quality of healthcare service and treatment, where satisfaction is measured through feedback by patients on their meetings with medical specialists and experts. One of the major problems in analyzing patient feedback data is the nonstandard research designs often used for gathering such data: the designs can be uncrossed, unbalanced, and fully nested. Traditional measures of data reliability are more difficult to calculate for such data. Also, patient data can contain significant proportions of missing values that further complicate the calculation of reliability. This paper describes a reliability approach that is robust in the face of nonstandard research designs and missing values for use with large-scale patient survey data. The dataset contains nearly 85,000 patient responses to over 2,000 healthcare practitioners in five different subtypes over a 15-year period in the United Kingdom. Reliability measures are calculated to provide benchmarks involving minimum numbers of patients and practitioners for deeper drill-down analysis. The paper concludes with a demonstration of how regression models generated from big patient feedback data can be assessed in terms of reliability at the total data level as well as drill-down levels.

Regulation of prion gene expression by transcription factors SP1 and metal transcription factor-1.

Prion diseases are associated with the conformational conversion of the host-encoded cellular prion protein into an abnormal pathogenic isoform. Reduction in prion protein levels has potential as a therapeutic approach in treating these diseases. Key targets for this goal are factors that affect the regulation of the prion protein gene. Recent in vivo and in vitro studies have suggested a role for prion protein in copper homeostasis. Copper can also induce prion gene expression in rat neurons. However, the mechanism involved in this regulation remains to be determined. We hypothesized that transcription factors SP1 and metal transcription factor-1 (MTF-1) may be involved in copper-mediated regulation of human prion gene. To test the hypothesis, we utilized human fibroblasts that are deleted or overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. Menkes deletion fibroblasts have high intracellular copper, whereas Menkes overexpressed fibroblasts have severely depleted intracellular copper. We have utilized this system previously to demonstrate copper-dependent regulation of the Alzheimer amyloid precursor protein. Here we demonstrate that copper depletion in MNK overexpressed fibroblasts decreases cellular prion protein and PRNP gene levels. Conversely, expression of transcription factors SP1 and/or MTF-1 significantly increases prion protein levels and up-regulates prion gene expression in copper-replete MNK deletion cells. Furthermore, siRNA "knockdown" of SP1 or MTF-1 in MNK deletion cells decreases prion protein levels and down-regulates prion gene expression. These data support a novel mechanism whereby SP1 and MTF-1 act as copper-sensing transcriptional activators to regulate human prion gene expression and further support a role for the prion protein to function in copper homeostasis. Expression of the prion protein is a vital component for the propagation of prion diseases; thus SP1 and MTF-1 represent new targets in the development of key therapeutics toward modulating the expression of the cellular prion protein and ultimately the prevention of prion disease.