Repeat prescribing of non-steroidal anti-inflammatory drugs excluding aspirin:how careful are we?

About 5% of all National Health Service prescriptions in Britain and a quarter of reports of suspected adverse reactions are accounted for by non-steroidal anti-inflammatory drugs. Their prescription was investigated in two computerised group practices serving 11850 patients. Altogether 198 patients receiving repeat prescriptions of non-steroidal anti-inflammatory drugs were identified and relevant clinical details extracted from their notes. Of these patients, 119 were over 65 years old; 172 were receiving one of six different non-steroidal anti-inflammatory drugs; and 76 were taking drugs that can interact with non-steroidal anti-inflammatory drugs. Ninety one patients had one or more medical conditions that may be aggravated by non-steroidal anti-inflammatory drugs, and 36 had experienced side effects important enough for their treatment to be changed. A questionnaire to assess opinions and knowledge of non-steroidal anti-inflammatory drugs was given to 42 general practitioners and 26 rheumatologists. Although the two groups showed a comparable knowledge of the properties and costs of non-steroidal anti-inflammatory drugs, they differed significantly in their views on the circumstances under which these drugs should be used. Clear guidelines on the prescription of these drugs would indicate when careful monitoring is essential for patients to benefit from them safely.

Ordering effects and choice set awareness in repeat-response stated preference studies

We present an experiment designed to investigate the presence and nature of ordering effects within repeat-response stated preference (SP) studies. Our experiment takes the form of a large sample, full-factorial, discrete choice SP exercise investigating preferences for tap water quality improvements. Our study simultaneously investigates a variety of different forms of position-dependent and precedent-dependent ordering effect in preferences for attributes and options and in response randomness. We also examine whether advanced disclosure of the choice tasks impacts on the probability of exhibiting ordering effects of those different types. We analyze our data both non-parametrically and parametrically and find robust evidence for ordering effects. We also find that the patterns of order effect in respondents' preferences are significantly changed but not eradicated by the advanced disclosure of choice tasks a finding that offers insights into the choice behaviors underpinning order effects. © 2011 Elsevier Inc.

Regulation of apoptotic protease activating factor-1 oligomerization and apoptosis by the WD-40 repeat region

Apoptotic protease activating factor-1 (Apaf-1) has been identified as a proximal activator of caspase-9 in cell death pathways that trigger mitochondrial damage and cytochrome c release. The mechanism of Apaf-1 action is unclear but has been proposed to involve the clustering of caspase-9 molecules, thereby facilitating autoprocessing of adjacent zymogens. Here we show that Apaf-1 can dimerize via the CED-4 homologous and linker domains of the molecule providing a means by which Apaf-1 can promote the clustering of caspase-9 and facilitate its activation. Apaf-1 dimerization was repressed by the C-terminal half of the molecule, which contains multiple WD-40 repeats, but this repression was overcome in the presence of cytochrome c and dATP. Removal of the WD-40 repeat region resulted in a constitutively active Apaf-1 that exhibited greater cytotoxicity in transient transfection assays when compared with full-length Apaf-1. These data suggest a mechanism for Apaf-1 function and reveal an important regulatory role for the WD-40 repeat region.

StaRProtein: a web server for prediction of stability of repeat proteins

Repeat proteins have become increasingly important due to their capability to bind to almost any proteins and the potential as alternative therapy to monoclonal antibodies. In the past decade repeat proteins have been designed to mediate specific protein-protein interactions. The tetratricopeptide and ankyrin repeat proteins are two classes of helical repeat proteins that form different binding pockets to accommodate various partners. It is important to understand the factors that define folding and stability of repeat proteins in order to prioritize the most stable designed repeat proteins to further explore their potential binding affinities. Here we developed distance-dependant statistical potentials using two classes of alpha-helical repeat proteins, tetratricopeptide and ankyrin repeat proteins respectively, and evaluated their efficiency in predicting the stability of repeat proteins. We demonstrated that the repeat-specific statistical potentials based on these two classes of repeat proteins showed paramount accuracy compared with non-specific statistical potentials in: 1) discriminate correct vs. incorrect models 2) rank the stability of designed repeat proteins. In particular, the statistical scores correlate closely with the equilibrium unfolding free energies of repeat proteins and therefore would serve as a novel tool in quickly prioritizing the designed repeat proteins with high stability. StaRProtein web server was developed for predicting the stability of repeat proteins.

A coiled-coil-repeat protein 'Ccrp' in Bdellovibrio bacteriovorus prevents cellular indentation, but is not essential for vibroid cell morphology

Bdellovibrio bacteriovorus are small, vibroid, predatory bacteria that grow within the periplasmic space of a host Gram-negative bacterium. The intermediate-filament (IF)-like protein crescentin is a member of a broad class of IF-like, coiled-coil-repeat-proteins (CCRPs), discovered in Caulobacter crescentus, where it contributes to the vibroid cell shape. The B. bacteriovorus genome has a single ccrp gene encoding a protein with an unusually long, stutter-free, coiled-coil prediction; the inactivation of this did not alter the vibriod cell shape, but caused cell deformations, visualized as chiselled insets or dents, near the cell poles and a general 'creased' appearance, under the negative staining preparation used for electron microscopy, but not in unstained, frozen, hydrated cells. Bdellovibrio bacteriovorus expressing 'teal' fluorescent protein (mTFP), as a C-terminal tag on the wild-type Ccrp protein, did not deform under negative staining, suggesting that the function was not impaired. Localization of fluorescent Ccrp-mTFP showed some bias to the cell poles, independent of the cytoskeleton, as demonstrated by the addition of the MreB-specific inhibitor A22. We suggest that the Ccrp protein in B. bacteriovorus contributes as an underlying scaffold, similar to that described for the CCRP protein FilP in Streptomyces coelicolor, preventing cellular indentation, but not contributing to the vibroid shape of the B. bacteriovorus cells.

PCR amplification of short tandem repeat sequences allows serial studies of chimaerism/engraftment following BMT in rodents

Animal models of bone marrow transplantation (BMT) allow evaluation of new experimental treatment strategies. One potential strategy involves the treatment of donor marrow with ultra-violet B light to allow transplantation across histocompatibility boundaries without an increase in graft rejection or graft-versus-host disease. A major requirement for a new experimental protocol, particularly if it involves manipulation of the donor marrow, is that the manipulated marrow gives rise to long-term multilineage engraftment. DNA based methodologies are now routinely used by many centres to evaluate engraftment and degree of chimaerism post-BMT in humans. We report the adaptation of this methodology to the serial study of engraftment in rodents. Conditions have been defined which allow analysis of serial tail vein samples using PCR of short tandem repeat sequences (STR-PCR). These markers have been used to evaluate the contribution of ultraviolet B treated marrow to engraftment following BMT in rodents without compromising the health of the animals under study. Chimaerism data from sequential tail vein samples and bone marrow from selected sacrificed animals showed excellent correlation, thus confirming the validity of this approach in analysing haemopoietic tissue. Thus the use of this assay may facilitate experimental studies in animal BMT.

Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction

The influence of mixed hematopoietic chimerism (MC) after allogeneic bone marrow transplantation remains unknown. Increasingly sensitive detection methods have shown that MC occurs frequently. We report a highly sensitive novel method to assess MC based on the polymerase chain reaction (PCR). Simple dinucleotide repeat sequences called microsatellites have been found to vary in their repeat number between individuals. We use this variation to type donor-recipient pairs following allogeneic BMT. A panel of seven microsatellites was used to distinguish between donor and recipient cells of 32 transplants. Informative microsatellites were subsequently used to assess MC after BMT in this group of patients. Seventeen of the 32 transplants involved a donor of opposite sex; hence, cytogenetics and Y chromosome-specific PCR were also used as an index of chimerism in these patients. MC was detected in bone marrow aspirates and peripheral blood in 18 of 32 patients (56%) by PCR. In several cases, only stored slide material was available for analysis but PCR of microsatellites or Y chromosomal material could be used successfully to assess the origin of cells in this archival material. Cytogenetic analysis was possible in 17 patients and MC was detected in three patients. Twelve patients received T-cell-depleted marrow and showed a high incidence of MC as revealed by PCR (greater than 80%). Twenty patients received unmanipulated marrow, and while the incidence of MC was lower (44%), this was a high percentage when compared with other studies. Once MC was detected, the percentages of recipient cells tended to increase. However, in patients exhibiting MC who subsequently relapsed, this increase was relatively sudden. The overall level of recipient cells in the group of MC patients who subsequently relapsed was higher than in those who exhibited stable MC. Thus, while the occurrence of MC was not indicative of a poor prognosis per se, sudden increases in the proportions of recipient cells may be a prelude to graft rejection or relapse.

Association of Functional Heme Oxygenase-1 Gene Promoter Polymorphism with Renal Transplantation Outcomes

Heme oxygenase-1 (HO-1) is a cytoprotective molecule and increased expression in experimental transplant models correlates with reduced graft injury. A functional dinucleotide repeat (GT)n polymorphism, within the HO-1 promoter, regulates gene expression; a short number of repeats (S-allele

Genetically increased risk of sleep disruption in alzheimer's disease

STUDY OBJECTIVES: To investigate the role of a monoamine A oxidase promoter polymorphism in sleep disruption in Alzheimer's disease (AD). DESIGN: A case-control association analysis. SETTING: Sleep disturbance in AD is common, is extremely stressful for caregivers, and increases the risk of institutionalisation. It remains unclear why only some patients develop sleep disturbance; neuropathologic changes of AD are not typically seen in the areas of the brain responsible for sleep. We hypothesized that the risk of sleep disturbance is, at least in part, influenced by the availability of serotonin used for melatonin synthesis secondary to polymorphic variation at the enzyme monoamine oxidase A (MAO-A). PATIENTS: Patients with AD diagnosed according to standard criteria. INTERVENTIONS: Data were collected using the Sleep domain of the Neuropsychiatric Inventory with Caregiver Distress. Patients' cognition and function were assessed using the Mini-Mental State Examination and the Functional Assessment Staging. Genotyping of apolipoprotein E (APOE) and of the 30 bp variable number tandem repeat of the MAO-A promoter was by standard methods. MEASUREMENTS AND RESULTS: Of 426 patients surveyed, 54% experienced sleep disturbance. We found that the high-activity 4-repeat allele of the MAO-A VNTR promoter polymorphism confers increased susceptibility to sleep disturbance (p = .008). A quantitative sleep disturbance score was significantly higher in the patients possessing MAO-A 4-repeat allele genotypes. APOE had no influence on the development of an altered sleep phenotype. CONCLUSIONS: We conclude that sleep disturbance in AD is common and distressing and is associated with genetic variation at MAO-A.

Cloning of maximakinin precursor cDNAs from Chinese toad, Bombina maxima, venom.

Using a novel technique that we have developed for cloning of amphibian skin secretion peptide cDNAs from lyophilized samples, we report here that maximakinin (DLPKINRKGP-bradykinin) is encoded by two different cDNAs, named BMK-1 and BMK-2, containing either four tandem repeat sequences or a single copy. The open reading frames of both precursor cDNAs were found to be 152 and 116 amino acid residues, respectively. These data provide evidence that the structural diversity of peptides in amphibian skin secretions arising from molecular evolutionary events, can be mediated by parallel diversity in encoding mRNAs that in itself may reflect serial gene duplications.

Development of microsatellites for the peat moss Sphagnum capillifolium using ISSR cloning

Sphagnum mosses are major components of peat bogs but populations of many species are under threat due to habitat fragmentation resulting from the cutting of peat for fuel. We have used an intersimple sequence repeat (ISSR)-based cloning method to develop nine polymorphic nuclear microsatellites for the peat moss species Sphagnum capillifolium. Between three and seven alleles per locus were detected in a sample of 48 haploid gametophytes and levels of gene diversity ranged from 0.5391 to 0.7960. These represent the first microsatellite markers developed for this important genus and most also exhibited cross-species amplification across a range of common Sphagnum species.

Universal primers for the amplification of chloroplast microsatellites in grasses (Poaceae)

Attempts to design truly universal primers to amplify chloroplast microsatellites have met with limited success due to nonconservation of repeat loci across widely divergent taxa. We have used the complete chloroplast genome sequences of rice, maize and wheat to design five pairs of primers that amplify homologous mononucleotide repeats across the Poaceae (grasses). Sequencing confirmed conservation of repeat motifs across subfamilies and a preliminary study in Anthoxanthum odoratum revealed polymorphism at two loci with a haplotype diversity value of 0.495. These primers provide a valuable tool to study cytoplasmic diversity in this extensively studied and economically important range of taxa.

High-resolution organellar genome analysis of Triticum and Aegilops sheds new light on cytoplasm evolution in wheat

We have utilised polymorphic chloroplast microsatellites to analyse cytoplasmic relationships between accessions in the genera Triticum and Aegilops. Sequencing of PCR products revealed point mutations and insertions/deletions in addition to the standard repeat length expansion/contraction which most likely represent ancient synapomorphies. Phylogenetic analyses revealed three distinct groups of accessions. One of these contained all the non-Aegilops speltoides S-type cytoplasm species, another comprised almost exclusively A, C, D, M, N, T and U cytoplasm-type accessions and the third contained the polyploid Triticum species and all the Ae. speltoides accessions, further confirming that Ae. speltoides or a closely related but now extinct species was the original B-genome donor of cultivated polyploid wheat. Successive decreases in levels of genetic diversity due to domestication were also observed. Finally, we highlight the importance of elucidating longer-term evolutionary processes operating at microsatellite repeat loci.