Development of a Reagentless Biosensor for Inorganic Phosphate, Applicable over a Wide Concentration Range.

A fluorescent reagentless biosensor for inorganic phosphate (Pi), based on the E. coli PstS phosphate binding protein, was redesigned to allow measurements of higher Pi concentrations and at low, substoichiometric concentrations of biosensor. This was achieved by weakening Pi binding of the previous biosensor, and different approaches are described that could enable this change in properties. The readout, providing response to the Pi concentration, is delivered by tetramethylrhodamine fluorescence. In addition to two cysteine mutations for rhodamine labeling at positions 17 and 197, the final variant had an I76G mutation in the hinge region between the two lobes that make up the protein. Upon Pi binding, the lobes rotate on this hinge and the mutation on the hinge lowers affinity ∼200-fold, with a dissociation constant now in the tens to hundreds micromolar range, depending on solution conditions. The signal change on Pi binding was up to 9-fold, depending on pH. The suitability of the biosensor for steady-state ATPase assays was demonstrated with low biosensor usage and its advantage in ability to cope with Pi contamination.

A gene-driven approach to the identification of ENU mutants in the mouse.

The construction of parallel archives of DNA and sperm from mice mutagenized with ethylnitrosurea (ENU) represents a potentially powerful and rapid approach for identifying point mutations in any gene in the mouse genome. We provide support for this approach and report the identification of mutations in the gene (Gjb2) encoding connexin 26, using archives established from the UK ENU mutagenesis program.

Integrating pharmacology and clinical pharmacology in pharmaceutical companies.

Integration of clinical and preclinical pharmacology in pharmaceutical companies could be improved by several key recommendations: Companies should ensure that there is an adequate pool of trained clinical pharmacologists and preclinical pharmacologists. Training should include topics that allow clinical pharmacologists to be cognizant of the methods, issues and challenges faced by the preclinical pharmacologists and vice versa. Companies should incentivize such integration internally by aligning objectives and metrics/incentives. In academic medicine and the NHS there should be support for involvement of clinical pharmacologists in basic academic research and industrial R & D and new ways of facilitating and incentivizing preclinical pharmacologists and clinical pharmacologists to move between these various environments should be sought.

SB-742457 and donepezil in Alzheimer disease: a randomized, placebo-controlled study.

OBJECTIVE: To estimate the treatment effects of SB-742457 and donepezil in Alzheimer disease (AD) in a contemporary clinical trial. METHOD: Randomized, controlled, parallel-group, exploratory study with a 4-week, single-blind, placebo run-in phase and 24-week, double-blind treatment phase. Primary endpoints were Clinician's Interview-Based Impression of Change with caregiver input (CIBIC+) and the Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-Cog). RESULTS: One hundred ninety eight subjects with mild-to-moderate probable AD (MMSE scores 12-26) were randomized; 196 were included in the intent-to-treat population (placebo, n = 61; SB-742457 35 mg/day, n = 68; donepezil 10 mg/day, n = 67), and 161 completed. Drug-placebo treatment differences in CIBIC+ score at week 24 were -0.17 (90% confidence interval [CI]: -0.50, 0.16) for SB-742457 and -0.28 (90% CI: -0.61, 0.05) for donepezil. Drug-placebo treatment differences (90% CI) in change from baseline ADAS-Cog score at Week 24 were -0.4 (-2.2, 1.4) for SB-742457 and -1.2 (-3.0, 0.6) for donepezil. All treatments were generally safe and well tolerated. CONCLUSIONS: In this exploratory study, SB-742457 and donepezil were associated with improvements in global function. Treatment effect on cognition for both SB-742457 and donepezil was smaller than those previously observed in previous clinical studies with donepezil.

Fluorescent single-stranded DNA binding protein as a probe for sensitive, real-time assays of helicase activity.

The formation and maintenance of single-stranded DNA (ssDNA) are essential parts of many processes involving DNA. For example, strand separation of double-stranded DNA (dsDNA) is catalyzed by helicases, and this exposure of the bases on the DNA allows further processing, such as replication, recombination, or repair. Assays of helicase activity and probes for their mechanism are essential for understanding related biological processes. Here we describe the development and use of a fluorescent probe to measure ssDNA formation specifically and in real time, with high sensitivity and time resolution. The reagentless biosensor is based on the ssDNA binding protein (SSB) from Escherichia coli, labeled at a specific site with a coumarin fluorophore. Its use in the study of DNA manipulations involving ssDNA intermediates is demonstrated in assays for DNA unwinding, catalyzed by DNA helicases.

Gender equality in university sportspeople's drinking.

INTRODUCTION AND AIMS: In large population-based alcohol studies males are shown consistently to drink more, and more hazardously, than females. However, research from some countries suggests that gender differences in drinking are converging, with females drinking more than in the past. Large population-based research may miss gender-based changes in drinking behaviours that occur in sub-populations most at risk of hazardous drinking. We examine gender differences in a sub-population where hazardous drinking is common and endorsed, namely university sportspeople. DESIGN AND METHODS: The Alcohol Use Disorders Identification Test (AUDIT) and a drinking motives measure were used to assess hazardous drinking behaviours and drinking motives in 631 university sportspeople (females = 331, 52%). RESULTS: There were no gender differences in AUDIT scores. However, drinking motives differed between genders, with coping motives being a significant predictor of hazardous drinking in females but not males. Hazardous drinking, including binge drinking (46.3%) and frequent binge drinking (35%), in New Zealand university sportspeople is high for both males and females. DISCUSSION AND CONCLUSIONS: New Zealand university sportspeople are one population where gender differences in drinking are not apparent and run counter to European population based research and research in US sporting populations. Gender role equality in the university systems, and endorsement of drinking in sporting culture, may account for the lack of gender differences in this New Zealand sporting population. Future research on gender differences in drinking should examine sub-populations where gender role differentiation is low, and socio-cultural/structural factors supporting gender equality are high.

Clinical trial design and dissemination: comprehensive analysis of clinicaltrials.gov and PubMed data since 2005.

OBJECTIVE: To investigate the distribution, design characteristics, and dissemination of clinical trials by funding organisation and medical specialty. DESIGN: Cross sectional descriptive analysis. DATA SOURCES: Trial protocol information from clinicaltrials.gov, metadata of journal articles in which trial results were published (PubMed), and quality metrics of associated journals from SCImago Journal and Country Rank database. SELECTION CRITERIA: All 45 620 clinical trials evaluating small molecule therapeutics, biological drugs, adjuvants, and vaccines, completed after January 2006 and before July 2015, including randomised controlled trials and non-randomised studies across all clinical phases. RESULTS: Industry was more likely than non-profit funders to fund large international randomised controlled trials, although methodological differences have been decreasing with time. Among 27 835 completed efficacy trials (phase II-IV), 15 084 (54.2%) had disclosed their findings publicly. Industry was more likely than non-profit trial funders to disseminate trial results (59.3% (10 444/17 627) v 45.3% (4555/10 066)), and large drug companies had higher disclosure rates than small ones (66.7% (7681/11 508) v 45.2% (2763/6119)). Trials funded by the National Institutes of Health (NIH) were disseminated more often than those of other non-profit institutions (60.0% (1451/2417) v 40.6% (3104/7649)). Results of studies funded by large drug companies and NIH were more likely to appear on clinicaltrials.gov than were those from non-profit funders, which were published mainly as journal articles. Trials reporting the use of randomisation were more likely than non-randomised studies to be published in a journal article (6895/19 711 (34.9%) v 1408/7748 (18.2%)), and journal publication rates varied across disease areas, ranging from 42% for autoimmune diseases to 20% for oncology. CONCLUSIONS: Trial design and dissemination of results vary substantially depending on the type and size of funding institution as well as the disease area under study.

Exploring the motivations behind misreporting self-measured blood glucose in adolescents with type 1 diabetes - a qualitative study.

BACKGROUND: Despite advances in diabetes management, the reporting and self-monitoring of blood glucose (SMBG) remains fundamental. While previous work has established that the misreporting of SMBG to family and medical professionals is surprisingly common, the motivations behind this behaviour have never been examined. We aimed to investigate the motivations behind misreporting of SMBG in adolescents with type 1 diabetes (T1DM). METHODS: Fifteen semi-structured interviews were conducted with adolescents (aged 12-19 inclusive) with T1DM recruited through diabetes clinics across the Otago/Southland region of New Zealand from November 2015 to January 2016. These were transcribed and content analysis performed to identify themes and subthemes in misreporting behaviour. RESULTS: The mean age of participants was 15.7 years, 60 % were male, with 67 % using multiple daily insulin injections, and 33 % on insulin pumps. Their median HbA1c was 84 mmol/mol, range 52-130. Misreporting behaviour was described for both electronic pump records and written logbooks, as well as verbally. Multiple motivations for misreporting were given, spanning three major themes: Achieving potential benefits; the avoidance of negative consequences; and the avoidance of worry/concern (in self or in others). The main suggestion of participants to reduce misreporting behaviour was to reduce the negative reactions of others to suboptimal blood glucose readings. CONCLUSION: Electronic, written, and verbal SMBG misreporting remains common. This study provides deeper insight into the motivations leading to this behaviour in adolescents, suggesting that further understanding and attention to this aspect of adherence may lead to improvements not only in glycaemic control and safety, but also to the psychological wellbeing of those with T1DM.

First-in-class thyrotropin-releasing hormone (TRH)-based compound binds to a pharmacologically distinct TRH receptor subtype in human brain and is effective in neurodegenerative models.

JAK4D, a first-in-class thyrotropin-releasing hormone (TRH)-based compound, is a prospective therapeutic candidate offering a multifaceted approach to treating neurodegeneration and other CNS conditions. The purpose of these studies was to determine the ability of JAK4D to bind to TRH receptors in human brain and to evaluate its neuropharmacological effects in neurodegenerative animal models. Additionally, JAK4D brain permeation was examined in mouse, and initial toxicology was assessed in vivo and in vitro. We report that JAK4D bound selectively with nanomolar affinity to native TRH receptors in human hippocampal tissue and showed for the first time that these receptors are pharmacologically distinct from TRH receptors in human pituitary, thus revealing a new TRH receptor subtype which represents a promising neurotherapeutic target in human brain. Systemic administration of JAK4D elicited statistically significant and clinically-relevant neuroprotective effects in three established neurodegenerative animal models: JAK4D reduced cognitive deficits when administered post-insult in a kainate (KA)-induced rat model of neurodegeneration; it protected against free radical release and neuronal damage evoked by intrastriatal microdialysis of KA in rat; and it reduced motor decline, weight loss, and lumbar spinal cord neuronal loss in G93A-SOD1 transgenic Amyotrophic Lateral Sclerosis mice. Ability to cross the blood-brain barrier and a clean initial toxicology profile were also shown. In light of these findings, JAK4D is an important tool for investigating the hitherto-unidentified central TRH receptor subtype reported herein and an attractive therapeutic candidate for neurodegenerative disorders.

Challenges for pharmaceutical industry: new partnerships for sustainable human health.

The healthcare burden is increasing in both the developed and the developing world and there is widespread acceptance that the historical pharmaceutical business model is not sustainable. In order to meet the healthcare challenge, companies and academia need to develop new business models to increase the probability of success and decrease the cost of failure. New partnerships have already emerged in the area of neglected diseases and other models for diseases of the developed world are emerging.

A biosensor for inorganic phosphate using a rhodamine-labeled phosphate binding protein.

A novel biosensor for inorganic phosphate (Pi) has been developed based on the phosphate binding protein of Escherichia coli. Two cysteine mutations were introduced and labeled with 6-iodoacetamidotetramethylrhodamine. When physically close to each other and correctly oriented, two rhodamine dyes interact to form a noncovalent dimer. In this state, they have little or no fluorescence, unlike the high fluorescence intensity of monomeric rhodamine. The labeling sites were so placed that the distance and orientation between the rhodamines change as a consequence of the conformational change associated with Pi binding. This movement alters the extent of interaction between the dyes. The best mutant of those tested (A17C, A197C) gives rise on average to approximately 18-fold increase in fluorescence intensity as Pi binds. The kinetics of interaction with Pi were measured at 10 degrees C. Under these conditions, the fluorescence increase associated with Pi binding has a maximum rate of 267 s-1. The Pi dissociation rate is 6.6 s-1, and the dissociation constant is 70 nM. An application of the sensor is demonstrated for measuring ATP hydrolysis in real time as a helicase moves along DNA. Advantages of the new sensor are discussed, both in terms of the use of a rhodamine fluorophore and the potential of this double labeling strategy.

The mechanism of Ras GTPase activation by neurofibromin.

Individual rate constants have been determined for each step of the Ras.GTP hydrolysis mechanism, activated by neurofibromin. Fluorescence intensity and anisotropy stopped-flow measurements used the fluorescent GTP analogue, mantGTP (2'(3')-O-(N-methylanthraniloyl)GTP), to determine rate constants for binding and release of neurofibromin. Quenched flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor, MDCC-PBP was used to measure phosphate release kinetics. Phosphate-water oxygen exchange, using (18)O-substituted GTP and inorganic phosphate (P(i)), was used to determine the extent of reversal of the hydrolysis step and of P(i) binding. The data show that neurofibromin and P(i) dissociate from the NF1.Ras.GDP.P(i) complex with identical kinetics, which are 3-fold slower than the preceding cleavage step. A model is presented in which the P(i) release is associated with the change of Ras from "GTP" to "GDP" conformation. In this model, the conformation change on P(i) release causes the large change in affinity of neurofibromin, which then dissociates rapidly.

ENU mutagenesis reveals a highly mutable locus on mouse Chromosome 4 that affects ear morphogenesis.

Chemical mutagenesis followed by screening for abnormal phenotypes in the mouse holds much promise as a method for revealing gene function. This method is particularly well-suited for discovering genes involved in hearing or balance function, as these defects are relatively easy to screen for in the mouse. We report here the inner ear abnormalities and genetic localization of seven new dominant mutations created by ENU mutagenesis. All seven mutant stocks were identified because of circling and/or head-weaving behavior, which is an indication of balance dysfunction. Investigation of the inner ears of the seven mutant stocks revealed very similar lateral and posterior semicircular canal defects. Studies of the development of the canals in one mutant stock revealed that the affected canals showed reduced outgrowth and delayed canal fusion. Physiological studies performed in one mutant stock showed raised average compound-action-potential thresholds of approximately 10-20 dB sound pressure level (SPL) (depending on frequency), indicating a mild hearing impairment, although scanning electron microscopy performed in several of the mutant stocks revealed no obvious structural defects in the organ of Corti. All seven mutations mapped to the proximal portion of Chromosome (Chr) 4, near the centromere. On the basis of their similar phenotype and map location, we suggest that the seven mutant genes may be allelic and represent a highly mutable locus on Chr 4 that may be particularly susceptible to ENU-induced mutation on the BALB/c genetic background.

A gene-driven ENU-based approach to generating an allelic series in any gene.

N-ethyl-N-nitrosourea (ENU) introduces mutations throughout the mouse genome at relatively high efficiency. Successful high-throughput phenotype screens have been reported and alternative screens using sequence-based approaches have been proposed. For the purpose of generating an allelic series in selected genes by a sequence-based approach, we have constructed an archive of over 4000 DNA samples from individual F1 ENU-mutagenized mice paralleled by frozen sperm samples. Together with our previously reported archive, the total size now exceeds 6000 individuals. A gene-based screen of 27.4 Mbp of DNA, carried out using denaturing high-performance liquid chromatography (DHPLC), found a mutation rate of 1 in 1.01 Mbp of which 1 in 1.82 Mbp were potentially functional. Screening of whole or selected regions of genes on subsets of the archive has allowed us to identify 15 new alleles from 9 genes out of 15 tested. This is a powerful adjunct to conventional mutagenesis strategies and has the advantage of generating a variety of alleles with potentially different phenotypic outcomes that facilitate the investigation of gene function. It is now available to academic collaborators as a community resource.