Functional studies with membrane-bound and detergent-solubilized alpha2-adrenergic receptors expressed in Sf9 cells

A chip-based biosensor technology using surface plasmon resonance (SPR) was developed for studying the interaction of ligands and G protein-coupled receptors (GPCRs). GPCRs, the fourth largest superfamily in the human genome, are the largest class of targets for drug discovery. We have expressed the three subtypes of alpha(2)-adrenergic receptor (alpha(2)-AR), a prototypical GPCR as functional fusion proteins in baculovirus-infected insect cells. The localization of the expressed receptor was observed in intracellular organelles, as detected by eGFP fluorescence. In addition, the deletion mutants of alpha(2B)-AR, with a deletion in the 3rd intracellular loop, exhibited unaltered K(d) values and enhanced stability, thus making them more promising candidates for crystallization. SPR demonstrated that small molecule ligands can bind the detergent-solubilized receptor, thus proving that alpha(2)-AR is active even in a lipid-free environment. The K(d) values obtained from the biosensor analysis and traditional ligand binding studies correlate well with each other. This is the first demonstration of the binding of a small molecule to the detergent-solubilized state of alpha(2)-ARs and interaction of low-molecular mass-ligands in real time in a label-free environment. This technology will also allow the development of high throughput platform for screening a large number of compounds for generation of leads.     

An emperor penguin population estimate: the first global, synoptic survey of a species from space

Our aim was to estimate the population of emperor penguins (Aptenodytes fosteri) using a single synoptic survey. We examined the whole continental coastline of Antarctica using a combination of medium resolution and Very High Resolution (VHR) satellite imagery to identify emperor penguin colony locations. Where colonies were identified, VHR imagery was obtained in the 2009 breeding season. The remotely-sensed images were then analysed using a supervised classification method to separate penguins from snow, shadow and guano. Actual counts of penguins from eleven ground truthing sites were used to convert these classified areas into numbers of penguins using a robust regression algorithm.We found four new colonies and confirmed the location of three previously suspected sites giving a total number of emperor penguin breeding colonies of 46. We estimated the breeding population of emperor penguins at each colony during 2009 and provide a population estimate of ~238,000 breeding pairs (compared with the last previously published count of 135,000-175,000 pairs). Based on published values of the relationship between breeders and non-breeders, this translates to a total population of ~595,000 adult birds.There is a growing consensus in the literature that global and regional emperor penguin populations will be affected by changing climate, a driver thought to be critical to their future survival. However, a complete understanding is severely limited by the lack of detailed knowledge about much of their ecology, and importantly a poor understanding of their total breeding population. To address the second of these issues, our work now provides a comprehensive estimate of the total breeding population that can be used in future population models and will provide a baseline for long-term research.     

Visualisation and Quantitative Analysis of the Rodent Malaria Liver Stage by Real Time Imaging

The quantitative analysis of Plasmodium development in the liver in laboratory animals in cultured cells is hampered by low parasite infection rates and the complicated methods required to monitor intracellular development. As a consequence, this important phase of the parasite''s life cycle has been poorly studied compared to blood stages, for example in screening anti-malarial drugs. Here we report the use of a transgenic P. berghei parasite, PbGFP-Luc_con, expressing the bioluminescent reporter protein luciferase to visualize and quantify parasite development in liver cells both in culture and in live mice using real-time luminescence imaging. The reporter-parasite based quantification in cultured hepatocytes by real-time imaging or using a microplate reader correlates very well with established quantitative RT-PCR methods. For the first time the liver stage of Plasmodium is visualized in whole bodies of live mice and we were able to discriminate as few as 1–5 infected hepatocytes per liver in mice using 2D-imaging and to identify individual infected hepatocytes by 3D-imaging. The analysis of liver infections by whole body imaging shows a good correlation with quantitative RT-PCR analysis of extracted livers. The luminescence-based analysis of the effects of various drugs on in vitro hepatocyte infection shows that this method can effectively be used for in vitro screening of compounds targeting Plasmodium liver stages. Furthermore, by analysing the effect of primaquine and tafenoquine in vivo we demonstrate the applicability of real time imaging to assess parasite drug sensitivity in the liver. The simplicity and speed of quantitative analysis of liver-stage development by real-time imaging compared to the PCR methodologies, as well as the possibility to analyse liver development in live mice without surgery, opens up new possibilities for research on Plasmodium liver infections and for validating the effect of drugs and vaccines on the liver stage of Plasmodium.     

Structure-activity relationships for analogs of the tuberculosis drug bedaquiline with the naphthalene unit replaced by bicyclic heterocycles

Replacing the naphthalene C-unit of the anti-tuberculosis drug bedaquiline with a range of bicyclic heterocycles of widely differing lipophilicity gave analogs with a 4.5-fold range in clogP values. The biological results for these compounds indicate on average a lower clogP limit of about 5.0 in this series for retention of potent inhibitory activity (MIC₉₀s) against M.tb in culture. Some of the compounds also showed a significant reduction in inhibition of hERG channel potassium current compared with bedaquiline, but there was no common structural feature that distinguished these.     

Impact of Pregnancy-Associated Malaria on Infant Malaria Infection in Southern Benin

Background

Infants of mothers with placental Plasmodium falciparum infections at delivery are themselves more susceptible to malaria attacks or to infection in early life.

Methodology/ Principal Findings

To assess the impact of either the timing or the number of pregnancy-associated malaria (PAM) infections on the incidence of parasitemia or malaria attacks in infancy, we followed 218 mothers through pregnancy (monthly visits) up to delivery and their infants from birth to 12 months of age (fortnightly visits), collecting detailed clinical and parasitological data. After adjustment on location, mother’s age, birth season, bed net use, and placental malaria, infants born to a mother with PAM during the third trimester of pregnancy had a significantly increased risk of infection (OR [95% CI]: 4.2 [1.6; 10.5], p = 0.003) or of malaria attack (4.6 [1.7; 12.5], p = 0.003). PAM during the first and second trimesters had no such impact. Similarly significant results were found for the effect of the overall number of PAM episodes on the time to first parasitemia and first malaria attack (HR [95% CI]: 2.95 [1.58; 5.50], p = 0.001 and 3.19 [1.59; 6.38], p = 0.001) respectively.

Conclusions/ Significance

This study highlights the importance of protecting newborns by preventing repeated episodes of PAM in their mothers.     

Searching for the Oldest Baobab of Madagascar: Radiocarbon Investigation of Large Adansonia rubrostipa Trees

We extended our research on the architecture, growth and age of trees belonging to the genus Adansonia, by starting to investigate large individuals of the most widespread Malagasy species. Our research also intends to identify the oldest baobabs of Madagascar. Here we present results of the radiocarbon investigation of the two most representative Adansonia rubrostipa (fony baobab) specimens, which are located in south-western Madagascar, in the Tsimanampetsotse National Park. We found that the fony baobab called “Grandmother” consists of 3 perfectly fused stems of different ages. The radiocarbon date of the oldest sample was found to be 1136 ± 16 BP. We estimated that the oldest part of this tree, which is mainly hollow, has an age close to 1,600 yr. This value is comparable to the age of the oldest Adansonia digitata (African baobab) specimens. By its age, the Grandmother is a major candidate for the oldest baobab of Madagascar. The second investigated specimen, called the “polygamous baobab”, consists of 6 partially fused stems of different ages. According to dating results, this fony baobab is 1,000 yr old. This research is the first investigation of the structure and age of Malagasy baobabs.     

Patient-Reported Outcome (PRO) Assessment in Clinical Trials: A Systematic Review of Guidance for Trial Protocol Writers

Background

Evidence suggests there are inconsistencies in patient-reported outcome (PRO) assessment and reporting in clinical trials, which may limit the use of these data to inform patient care. For trials with a PRO endpoint, routine inclusion of key PRO information in the protocol may help improve trial conduct and the reporting and appraisal of PRO results; however, it is currently unclear exactly what PRO-specific information should be included. The aim of this review was to summarize the current PRO-specific guidance for clinical trial protocol developers.

Methods and Findings

We searched the MEDLINE, EMBASE, CINHAL and Cochrane Library databases (inception to February 2013) for PRO-specific guidance regarding trial protocol development. Further guidance documents were identified via Google, Google scholar, requests to members of the UK Clinical Research Collaboration registered clinical trials units and international experts. Two independent investigators undertook title/abstract screening, full text review and data extraction, with a third involved in the event of disagreement. 21,175 citations were screened and 54 met the inclusion criteria. Guidance documents were difficult to access: electronic database searches identified just 8 documents, with the remaining 46 sourced elsewhere (5 from citation tracking, 27 from hand searching, 7 from the grey literature review and 7 from experts). 162 unique PRO-specific protocol recommendations were extracted from included documents. A further 10 PRO recommendations were identified relating to supporting trial documentation. Only 5/162 (3%) recommendations appeared in ≥50% of guidance documents reviewed, indicating a lack of consistency.

Conclusions

PRO-specific protocol guidelines were difficult to access, lacked consistency and may be challenging to implement in practice. There is a need to develop easily accessible consensus-driven PRO protocol guidance. Guidance should be aimed at ensuring key PRO information is routinely included in appropriate trial protocols, in order to facilitate rigorous collection/reporting of PRO data, to effectively inform patient care.