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1.  Misrepresentation of Randomized Controlled Trials in Press Releases and News Coverage: A Cohort Study 
PLoS Medicine  2012;9(9):e1001308.
A study conducted by Amélie Yavchitz and colleagues examines the factors associated with “spin” (specific reporting strategies, intentional or unintentional, that emphasize the beneficial effect of treatments) in press releases of clinical trials.
Background
Previous studies indicate that in published reports, trial results can be distorted by the use of “spin” (specific reporting strategies, intentional or unintentional, emphasizing the beneficial effect of the experimental treatment). We aimed to (1) evaluate the presence of “spin” in press releases and associated media coverage; and (2) evaluate whether findings of randomized controlled trials (RCTs) based on press releases and media coverage are misinterpreted.
Methods and Findings
We systematically searched for all press releases indexed in the EurekAlert! database between December 2009 and March 2010. Of the 498 press releases retrieved and screened, we included press releases for all two-arm, parallel-group RCTs (n = 70). We obtained a copy of the scientific article to which the press release related and we systematically searched for related news items using Lexis Nexis.
“Spin,” defined as specific reporting strategies (intentional or unintentional) emphasizing the beneficial effect of the experimental treatment, was identified in 28 (40%) scientific article abstract conclusions and in 33 (47%) press releases. From bivariate and multivariable analysis assessing the journal type, funding source, sample size, type of treatment (drug or other), results of the primary outcomes (all nonstatistically significant versus other), author of the press release, and the presence of “spin” in the abstract conclusion, the only factor associated, with “spin” in the press release was “spin” in the article abstract conclusions (relative risk [RR] 5.6, [95% CI 2.8–11.1], p<0.001). Findings of RCTs based on press releases were overestimated for 19 (27%) reports. News items were identified for 41 RCTs; 21 (51%) were reported with “spin,” mainly the same type of “spin” as those identified in the press release and article abstract conclusion. Findings of RCTs based on the news item was overestimated for ten (24%) reports.
Conclusion
“Spin” was identified in about half of press releases and media coverage. In multivariable analysis, the main factor associated with “spin” in press releases was the presence of “spin” in the article abstract conclusion.
Editors' Summary
Background
The mass media play an important role in disseminating the results of medical research. Every day, news items in newspapers and magazines and on the television, radio, and internet provide the general public with information about the latest clinical studies. Such news items are written by journalists and are often based on information in “press releases.” These short communications, which are posted on online databases such as EurekAlert! and sent directly to journalists, are prepared by researchers or more often by the drug companies, funding bodies, or institutions supporting the clinical research and are designed to attract favorable media attention to newly published research results. Press releases provide journalists with the information they need to develop and publish a news story, including a link to the peer-reviewed journal (a scholarly periodical containing articles that have been judged by independent experts) in which the research results appear.
Why Was This Study Done?
In an ideal world, journal articles, press releases, and news stories would all accurately reflect the results of health research. Unfortunately, the findings of randomized controlled trials (RCTs—studies that compare the outcomes of patients randomly assigned to receive alternative interventions), which are the best way to evaluate new treatments, are sometimes distorted in peer-reviewed journals by the use of “spin”—reporting that emphasizes the beneficial effects of the experimental (new) treatment. For example, a journal article may interpret nonstatistically significant differences as showing the equivalence of two treatments although such results actually indicate a lack of evidence for the superiority of either treatment. “Spin” can distort the transposition of research into clinical practice and, when reproduced in the mass media, it can give patients unrealistic expectations about new treatments. It is important, therefore, to know where “spin” occurs and to understand the effects of that “spin”. In this study, the researchers evaluate the presence of “spin” in press releases and associated media coverage and analyze whether the interpretation of RCT results based on press releases and associated news items could lead to the misinterpretation of RCT results.
What Did the Researchers Do and Find?
The researchers identified 70 press releases indexed in EurekAlert! over a 4-month period that described two-arm, parallel-group RCTs. They used Lexis Nexis, a database of news reports from around the world, to identify associated news items for 41 of these press releases and then analyzed the press releases, news items, and abstracts of the scientific articles related to each press release for “spin”. Finally, they interpreted the results of the RCTs using each source of information independently. Nearly half the press releases and article abstract conclusions contained “spin” and, importantly, “spin” in the press releases was associated with “spin” in the article abstracts. The researchers overestimated the benefits of the experimental treatment from the press release as compared to the full-text peer-reviewed article for 27% of reports. Factors that were associated with this overestimation of treatment benefits included publication in a specialized journal and having “spin” in the press release. Of the news items related to press releases, half contained “spin”, usually of the same type as identified in the press release and article abstract. Finally, the researchers overestimated the benefit of the experimental treatment from the news item as compared to the full-text peer-reviewed article in 24% of cases.
What Do These Findings Mean?
These findings show that “spin” in press releases and news reports is related to the presence of “spin” in the abstract of peer-reviewed reports of RCTs and suggest that the interpretation of RCT results based solely on press releases or media coverage could distort the interpretation of research findings in a way that favors experimental treatments. This interpretation shift is probably related to the presence of “spin” in peer-reviewed article abstracts, press releases, and news items and may be partly responsible for a mismatch between the perceived and real beneficial effects of new treatments among the general public. Overall, these findings highlight the important role that journal reviewers and editors play in disseminating research findings. These individuals, the researchers conclude, have a responsibility to ensure that the conclusions reported in the abstracts of peer-reviewed articles are appropriate and do not over-interpret the results of clinical research.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001308.
The PLOS Hub for Clinical Trials, which collects PLOS journals relating to clinical trials, includes some other articles on “spin” in clinical trial reports
EurekAlert is an online free database for science press releases
The UK National Health Service Choices website includes Beyond the Headlines, a resource that provides an unbiased and evidence-based analysis of health stories that make the news for both the public and health professionals
The US-based organization HealthNewsReview, a project supported by the Foundation for Informed Medical Decision Making, also provides expert reviews of news stories
doi:10.1371/journal.pmed.1001308
PMCID: PMC3439420  PMID: 22984354
2.  Position of UNC-13 in the active zone regulates synaptic vesicle release probability and release kinetics 
eLife  2013;2:e01180.
The presynaptic active zone proteins UNC-13/Munc13s are essential for synaptic vesicle (SV) exocytosis by directly interacting with SV fusion apparatus. An open question is how their association with active zones, hence their position to Ca2+ entry sites, regulates SV release. The N-termini of major UNC-13/Munc13 isoforms contain a non-calcium binding C2A domain that mediates protein homo- or hetero-meric interactions. Here, we show that the C2A domain of Caenorhabditis elegans UNC-13 regulates release probability of evoked release and its precise active zone localization. Kinetics analysis of SV release supports that the proximity of UNC-13 to Ca2+ entry sites, mediated by the C2A-domain containing N-terminus, is critical for accelerating neurotransmitter release. Additionally, the C2A domain is specifically required for spontaneous release. These data reveal multiple roles of UNC-13 C2A domain, and suggest that spontaneous release and the fast phase of evoked release may involve a common pool of SVs at the active zone.
DOI: http://dx.doi.org/10.7554/eLife.01180.001
eLife digest
Neurons are connected to each other by junctions called synapses. When an electrical signal travelling along a neuron arrives at a synapse, it causes the release of bubble-like structures called synaptic vesicles that contain chemicals called neurotransmitters. When released by the vesicles these neurotransmitters bind to receptors on a second neuron and allow the signal to continue on its way through the nervous system.
The release of synaptic vesicles from the neuron depends largely on the number of calcium ions that enter this neuron via structures called ion channels, and also on the rate at which they enter. Vesicles are released in one of three ways: they can be released quickly (within a few milliseconds) in response to the influx of calcium ions; they can be released slowly (over a period of tens or hundreds of milliseconds) in response to the influx; or they can be released at random times that are not related to the influx.
It is known that the sensitivity of certain calcium sensors near the synapse influences the release of the vesicles. It had been thought that the distance between the “active zone” where the calcium ions enter the neuron and the region where the vesicles reside might also influence rate of release, but the molecular mechanism underlying this hypothesis is poorly understood.
Zhou et al. have now shed new light on this question by performing a series of experiments that involved manipulating a protein called UNC-13 – which is known to be involved in the release of vesicles – in neurons from C. elegans, a nematode worm. First it was shown that the precise position of UNC-13 in the active zone depended on a domain within the protein called the C2A domain. Next it was shown that the distance between the UNC-13 protein and the calcium ion channels strongly influences the quick mode of vesicle release. Finally, Zhou et al. showed that the C2A domain also had a significant influence on the spontaneous release of vesicles, which suggests that a common fleet of vesicles might be used for both the quick and the spontaneous modes of vesicle release. Zhou et al. also generated mutant worms that mimicked a neurological disease, epileptic seizure, and showed that eliminating the C2A domain can relieve some of the symptoms associated with the disease.
Many neurological diseases are caused by signals not being transmitted properly at synapses, so in addition to providing insights into the basic mechanism underlying synaptic action, these results could also assist with the development of new strategies for managing neurological diseases.
DOI: http://dx.doi.org/10.7554/eLife.01180.002
doi:10.7554/eLife.01180
PMCID: PMC3821175  PMID: 24220508
UNC-13; Munc-13; SV release probability; SV release kinetics; C2A domain; miniSOG; Chromophore assisted light inactivation; C. elegans
3.  Design and In Vitro Evaluation of Novel Sustained-Release Double-Layer Tablets of Lornoxicam: Utility of Cyclodextrin and Xanthan Gum Combination 
AAPS PharmSciTech  2009;10(4):1357-1367.
The objective of the present study was to develop new directly compressed, double-layer tablets (DLTs) of lornoxicam, a highly potent nonsteroidal anti-inflammatory drug with short half-life, that are characterized by initial burst drug release in the stomach and comply with the release requirements of sustained-release products. Each of the proposed DLTs is composed of a fast-release layer and a sustained-release layer, anticipating rapid drug release that starts in the stomach to rapidly alleviate the symptoms and continues in the intestine to maintain protracted analgesic effect. An amorphous, freeze-dried inclusion complex of lornoxicam with hydroxypropyl-β-cyclodextrin, present in 1:2 (drug/cyclodextrin) molar ratio, was employed in the fast-release layer to enhance the dissolution of lornoxicam in the stomach and assure rapid onset of its analgesic effect. Xanthan gum (XG), a hydrophilic matrix-forming agent, was integrated in the sustained-release layer to provide appropriate sustainment of drug release. The weight ratios between the sustained-release layer and fast-release layer present in DLTs were adjusted to reach optimal formulations. DLTs composed of sustained-release layer (40% XG) to fast-release layer in 2:1 weight ratio and those composed of sustained-release layer (50% XG) to fast-release layer in 1:1 weight ratio showed the desired release profile. The drug contained in the fast-release layer showed an initial burst drug release of more than 30% of its drug content during the first 30 min of the release study followed by gradual release of the drug for a period of 8 h.
doi:10.1208/s12249-009-9336-9
PMCID: PMC2799602  PMID: 19921543
cyclodextrins; double-layer tablets; lornoxicam; sustained release; xanthan gum
4.  Synchronous and asynchronous modes of synaptic transmission utilize different calcium sources 
eLife  2013;2:e01206.
Asynchronous transmission plays a prominent role at certain synapses but lacks the mechanistic insights of its synchronous counterpart. The current view posits that triggering of asynchronous release during repetitive stimulation involves expansion of the same calcium domains underlying synchronous transmission. In this study, live imaging and paired patch clamp recording at the zebrafish neuromuscular synapse reveal contributions by spatially distinct calcium sources. Synchronous release is tied to calcium entry into synaptic boutons via P/Q type calcium channels, whereas asynchronous release is boosted by a propagating intracellular calcium source initiated at off-synaptic locations in the axon and axonal branch points. This secondary calcium source fully accounts for the persistence following termination of the stimulus and sensitivity to slow calcium buffers reported for asynchronous release. The neuromuscular junction and CNS neurons share these features, raising the possibility that secondary calcium sources are common among synapses with prominent asynchronous release.
DOI: http://dx.doi.org/10.7554/eLife.01206.001
eLife digest
Neurons communicate with one another at junctions called synapses. The arrival of an electrical signal known as an action potential at the first (presynaptic) neuron causes calcium ions to flood into the cell. This in turn causes the neuron to release packages of chemicals called neurotransmitters into the synapse. These activate receptors on the second (postsynaptic) neuron, triggering a new action potential that travels down the axon to the next synapse.
The ions that trigger the release of the neurotransmitters are thought to enter the neuron through special calcium channels on or near the synapse. A sudden discrete influx of calcium ions causes the neuron to release many packages of transmitter simultaneously. This is called synchronous release. By contrast, when successive action potentials occur in the same neuron, the ions entering through the calcium channels accumulate inside the cell. This is thought to account for a sustained release of the neurotransmitter that continues even in the absence of nerve action potentials. This is called asynchronous release.
Wen et al. have now obtained evidence that these two forms of release might be triggered by calcium from different sources. The work was performed using a synapse between nerve and muscle cells in zebrafish: it has been shown that channels called P/Q calcium channels control the release of neurotransmitters at this synapse in zebrafish.
Mutant zebrafish with greatly reduced numbers of P/Q channels showed reduced synchronous release, but normal asynchronous release. Blocking the P/Q channels with a specific toxin in normal zebrafish eliminated synchronous release but left asynchronous release intact. Imaging experiments on these toxin-treated zebrafish revealed that a wave of calcium ions that propagated from a distant source coincided with the onset of asynchronous release. This wave of calcium fully accounted for the delayed onset and the persistence of asynchronous release following termination of the action potentials. This study further demonstrates that asynchronous release can be triggered by calcium ions that do not enter through the P/Q calcium channels.
Waves of calcium have been described in the nervous system before, but their significance has always been unclear. The work of Wen et al. offers the first possible explanation for the role of these waves, and further experiments are now needed to determine whether this process happens at other types of synapses.
DOI: http://dx.doi.org/10.7554/eLife.01206.002
doi:10.7554/eLife.01206
PMCID: PMC3869123  PMID: 24368731
synaptopHluorin; calcium indicators; motor neuron; Zebrafish
5.  Influence of medical journal press releases on the quality of associated newspaper coverage: retrospective cohort study 
Objective To determine whether the quality of press releases issued by medical journals can influence the quality of associated newspaper stories.
Design Retrospective cohort study of medical journal press releases and associated news stories.
Setting We reviewed consecutive issues (going backwards from January 2009) of five major medical journals (Annals of Internal Medicine, BMJ, Journal of the National Cancer Institute, JAMA, and New England Journal of Medicine) to identify the first 100 original research articles with quantifiable outcomes and that had generated any newspaper coverage (unique stories ≥100 words long). We identified 759 associated newspaper stories using Lexis Nexis and Factiva searches, and 68 journal press releases using Eurekalert and journal website searches. Two independent research assistants assessed the quality of journal articles, press releases, and a stratified random sample of associated newspaper stories (n=343) by using a structured coding scheme for the presence of specific quality measures: basic study facts, quantification of the main result, harms, and limitations.
Main outcome Proportion of newspaper stories with specific quality measures (adjusted for whether the quality measure was present in the journal article’s abstract or editor note).
Results We recorded a median of three newspaper stories per journal article (range 1-72). Of 343 stories analysed, 71% reported on articles for which medical journals had issued press releases. 9% of stories quantified the main result with absolute risks when this information was not in the press release, 53% did so when it was in the press release (relative risk 6.0, 95% confidence interval 2.3 to 15.4), and 20% when no press release was issued (2.2, 0.83 to 6.1). 133 (39%) stories reported on research describing beneficial interventions. 24% mentioned harms (or specifically declared no harms) when harms were not mentioned in the press release, 68% when mentioned in the press release (2.8, 1.1 to 7.4), and 36% when no press release was issued (1.5, 0.49 to 4.4). 256 (75%) stories reported on research with important limitations. 16% reported any limitations when limitations were not mentioned in the press release, 48% when mentioned in the press release (3.0, 1.5 to 6.2), and 21% if no press release was issued (1.3, 0.50 to 3.6).
Conclusion High quality press releases issued by medical journals seem to make the quality of associated newspaper stories better, whereas low quality press releases might make them worse.
doi:10.1136/bmj.d8164
PMCID: PMC3267473  PMID: 22286507
6.  Nanoengineered drug-releasing Ti wires as an alternative for local delivery of chemotherapeutics in the brain 
The blood–brain barrier (BBB) blocks the passage of active molecules from the blood which makes drug delivery to the brain a challenging problem. Oral drug delivery using chemically modified drugs to enhance their transport properties or remove the blocking of drug transport across the BBB is explored as a common approach to address these problems, but with limited success. Local delivery of drugs directly to the brain interstitium using implants such as polymeric wafers, gels, and catheters has been recognized as a promising alternative particularly for the treatment of brain cancer (glioma) and neurodegenerative disorders. The aim of this study was to introduce a new solution by engineering a drug-releasing implant for local drug delivery in the brain, based on titanium (Ti) wires with titania nanotube (TNT) arrays on their surfaces. Drug loading and drug release characteristics of this system were explored using two drugs commonly used in oral brain therapy: dopamine (DOPA), a neurotransmitter agent; and doxorubicin (DOXO), an anticancer drug. Results showed that TNT/Ti wires could provide a considerable amount of drugs (>170 μg to 1000 μg) with desirable release kinetics and controllable release time (1 to several weeks) and proved their feasibility for use as drug-releasing implants for local drug delivery in the brain.
Purpose
In this report, a new drug-releasing platform in the form of nanoengineered Ti wires with TNT arrays is proposed as an alternative for local delivery of chemotherapeutics in the brain to bypass the BBB. To prove this concept, drug loading and release characteristics of two drugs important for brain therapy (the neurotransmitter DOPA and the anticancer drug DOXO) were explored.
Methods
Titania nanotube arrays on the surface of Ti wires (TNT/Ti) were fabricated using a simple anodization process, followed by separate loading of two drugs (DOPA and DOXO) inside the nanotube structures. The loading and in vitro release characteristics of prepared TNT/Ti implants were examined using thermogravimetric analysis (TGA) UV-Vis spectroscopy.
Results
Scanning electron microscopy studies confirmed that well-ordered, vertically aligned, densely packed nanotube arrays with an average diameter of 170 nm and length 70 μm were formed on the surface of TNT/Ti wires. TGA results showed a total drug loading of 170 μg and 1200 μg inside the TNTs for DOPA and DOXO respectively. Two-phase drug release behavior was observed including a fast release (burst) for the first 6 hours and a prolonged slow release phase for 8 days, both with acceptable dosage and desirable release kinetics. The physical, structural, loading and release characteristics of prepared TNT/Ti implants showed several advantages in comparison with existing and clinically proved brain implants.
Conclusion
Our results confirmed that TNT/Ti wires can be successfully employed as a suitable platform to release neurotransmitters such as DOPA and anticancer drugs such as DOXO. Hence, they are a feasible alternative as drug-releasing implants for local drug delivery in the brain to combat neurodegenerative disorders or brain tumors.
doi:10.2147/IJN.S29917
PMCID: PMC3356184  PMID: 22619543
titania nanotubes; brain implants; local drug delivery; dopamine; doxorubicin
7.  What is newsworthy? Longitudinal study of the reporting of medical research in two British newspapers 
BMJ : British Medical Journal  2002;325(7355):81-84.
Objective
To assess the characteristics of medical research that is press released by general medical journals and reported in newspapers.
Design
Longitudinal study.
Data sources
All original research articles published in Lancet and BMJ during 1999 and 2000.
Main outcome measures
Inclusion of articles in Lancet or BMJ press releases, and reporting of articles in Times or Sun newspapers.
Results
Of 1193 original research articles, 517 (43%) were highlighted in a press release and 81 (7%) were reported in one or both newspapers. All articles covered in newspapers had been press released. The probability of inclusion in press releases was similar for observational studies and randomised controlled trials, but trials were less likely to be covered in the newspapers (odds ratio 0.15 (95% confidence interval 0.06 to 0.37)). Good news and bad news were equally likely to be press released, but bad news was more likely to be reported in newspapers (1.74 (1.07 to 2.83)). Studies of women's health, reproduction, and cancer were more likely to be press released and covered in newspapers. Studies from industrialised countries other than Britain were less likely to be reported in newspapers (0.51 (0.31 to 0.82)), and no studies from developing countries were covered.
Conclusions
Characteristics of articles were more strongly associated with selection for reporting in newspapers than with selection for inclusion in press releases, although each stage influenced the reporting process. Newspapers underreported randomised trials, emphasised bad news from observational studies, and ignored research from developing countries.
What is already known on this topicNewspapers are an important source of information about the results of medical researchThere are two stages on the path to newspaper coverage—selection by medical journal editors of articles to be press released and the selection of newsworthy articles by journalistsWhat this study addsExamination of press releasing by the Lancet and BMJ and reporting by the Times and Sun showed that selection processes acted at both stagesThe net effect meant that newspapers emphasised results from observational studies, in particular studies of women's health, reproduction, and cancerGood news and bad news were equally likely to be press released, but bad news was more likely to be reported in newspaper articles
PMCID: PMC117129  PMID: 12114239
8.  The association between exaggeration in health related science news and academic press releases: retrospective observational study 
Objective To identify the source (press releases or news) of distortions, exaggerations, or changes to the main conclusions drawn from research that could potentially influence a reader’s health related behaviour.
Design Retrospective quantitative content analysis.
Setting Journal articles, press releases, and related news, with accompanying simulations.
Sample Press releases (n=462) on biomedical and health related science issued by 20 leading UK universities in 2011, alongside their associated peer reviewed research papers and news stories (n=668).
Main outcome measures Advice to readers to change behaviour, causal statements drawn from correlational research, and inference to humans from animal research that went beyond those in the associated peer reviewed papers.
Results 40% (95% confidence interval 33% to 46%) of the press releases contained exaggerated advice, 33% (26% to 40%) contained exaggerated causal claims, and 36% (28% to 46%) contained exaggerated inference to humans from animal research. When press releases contained such exaggeration, 58% (95% confidence interval 48% to 68%), 81% (70% to 93%), and 86% (77% to 95%) of news stories, respectively, contained similar exaggeration, compared with exaggeration rates of 17% (10% to 24%), 18% (9% to 27%), and 10% (0% to 19%) in news when the press releases were not exaggerated. Odds ratios for each category of analysis were 6.5 (95% confidence interval 3.5 to 12), 20 (7.6 to 51), and 56 (15 to 211). At the same time, there was little evidence that exaggeration in press releases increased the uptake of news.
Conclusions Exaggeration in news is strongly associated with exaggeration in press releases. Improving the accuracy of academic press releases could represent a key opportunity for reducing misleading health related news.
doi:10.1136/bmj.g7015
PMCID: PMC4262123  PMID: 25498121
9.  Release of Luminal Exosomes Contributes to TLR4-Mediated Epithelial Antimicrobial Defense 
PLoS Pathogens  2013;9(4):e1003261.
Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.
Author Summary
Exosomes are secreted membranous nanovesicles produced by a variety of cells. Exosomes shuttle various molecules to transfer them to neighboring or distant cells, and have been implicated as mediators in cell-cell communications to modulate physiological and pathological procedures. Here, we report that luminal release of exosomal vesicles is an important component of Toll-like receptor 4 (TLR4)-associated gastrointestinal epithelial defense against infection by Cryptosporidium parvum, an obligate intracellular protozoan that infects gastrointestinal epithelial cells. Activation of TLR4 signaling in host epithelial cells following C. parvum infection promotes luminal release of epithelial exosomes and exosomal shuttling of antimicrobial peptides from the epithelium. By direct binding to the C. parvum surface, exosomal vesicles reveal anti-C. parvum activity. Activation of TLR4 signaling in epithelial cells after LPS stimulation also increases exosomal release and exosome-associated anti-C. parvum activity. Therefore, we speculate that TLR4-mediated exosome release may be relevant to innate mucosal immunity in general, representing a new target for therapeutic intervention for infectious diseases at the mucosal surface.
doi:10.1371/journal.ppat.1003261
PMCID: PMC3617097  PMID: 23592986
10.  Characterizing the Aedes aegypti Population in a Vietnamese Village in Preparation for a Wolbachia-Based Mosquito Control Strategy to Eliminate Dengue 
Background
A life-shortening strain of the obligate intracellular bacteria Wolbachia, called wMelPop, is seen as a promising new tool for the control of Aedes aegypti. However, developing a vector control strategy based on the release of mosquitoes transinfected with wMelPop requires detailed knowledge of the demographics of the target population.
Methodology/Principal Findings
In Tri Nguyen village (611 households) on Hon Mieu Island in central Vietnam, we conducted nine quantitative entomologic surveys over 14 months to determine if Ae. aegypti populations were spatially and temporally homogenous, and to estimate population size. There was no obvious relationship between mosquito (larval, pupal or adult) abundance and temperature and rainfall, and no area of the village supported consistently high numbers of mosquitoes. In almost all surveys, key premises produced high numbers of Ae. aegypti. However, these premises were not consistent between surveys. For an intervention based on a single release of wMelPop-infected Ae. aegypti, release ratios of infected to uninfected adult mosquitoes of all age classes are estimated to be 1.8–6.7∶1 for gravid females (and similarly aged males) or teneral adults, respectively. We calculated that adult female mosquito abundance in Tri Nguyen village could range from 1.1 to 43.3 individuals of all age classes per house. Thus, an intervention could require the release of 2–78 wMelPop-infected gravid females and similarly aged males per house, or 7–290 infected teneral female and male mosquitoes per house.
Conclusions/Significance
Given the variability we encountered, this study highlights the importance of multiple entomologic surveys when evaluating the spatial structure of a vector population or estimating population size. If a single release of wMelPop-infected Ae. aegypti were to occur when wild Ae. aegypti abundance was at its maximum, a preintervention control program would be necessary to ensure that there was no net increase in mosquito numbers. However, because of the short-term temporal heterogeneity, the inconsistent spatial structure and the impact of transient key premises that we observed, the feasibility of multiple releases of smaller numbers of mosquitoes also needs to be considered. In either case, fewer wMelPop-infected mosquitoes would then need to be released, which will likely be more acceptable to householders.
Author Summary
Prevention of dengue disease relies predominantly on controlling the mosquito Aedes aegypti. A new control strategy is being developed based on the use of a life-shortening strain of the intracellular bacteria Wolbachia, which has been microinjected into Ae. aegypti in the laboratory. This strategy aims to eliminate the older and epidemiologically significant individuals from Ae. aegypti populations and thus eliminate dengue transmission. However, designing an intervention strategy involving the release of Wolbachia-infected mosquitoes requires detailed knowledge of the population dynamics of Ae. aegypti at potential release sites. We can then calculate how many mosquitoes need to be released for Wolbachia to spread through the wild population. We examined the abundance and distribution of the Ae. aegypti population over a 14 month period in a Vietnamese village identified by the Vietnam government as a potential pilot release site. We found considerable spatial variability because the houses that produced high numbers of mosquitoes were different in each survey. We estimated that the adult Ae. aegypti female population could be as high as 43 individuals per house. This suggests suppression of the wild Ae. aegypti population prior to a release will be necessary, and multiple releases will be required to counteract unpredictable spatial heterogeneity.
doi:10.1371/journal.pntd.0000552
PMCID: PMC2780318  PMID: 19956588
11.  Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway 
The Journal of Cell Biology  1987;105(1):145-153.
The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.
PMCID: PMC2114904  PMID: 3301864
12.  Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1 
eLife  2013;2:e00411.
Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.
DOI: http://dx.doi.org/10.7554/eLife.00411.001
eLife digest
Ribosomes are complex molecular machines that join amino acids together to form proteins. The order of amino acids in the protein is specified by a strand of messenger RNA (mRNA), and the process of decoding the mRNA into a string of amino acids is called translation. A ribosome consists of two subunits—one large, one small—that come together at a particular site on the mRNA strand called the translation initiation site. The ribosome then moves along the mRNA—joining together amino acids brought to it by transfer RNA (tRNA)—until it reaches a termination site and releases the protein.
The ribosome has three sites; the first amino acid to be delivered by a tRNA molecule to the ribosome occupies the site in the middle—also called the P site—and the second amino acid is delivered to the A site. Once the first two amino acids have been joined together, the ribosome moves along the mRNA so that the first amino acid now occupies the third site, called the E or exit site, and the second amino acid occupies the P site, leaving the A site vacant. The third amino acid is then delivered to the A site, and the whole process repeats itself until the ribosome reaches the termination site. Proteins called release factors are responsible for terminating the translation process and releasing the translated string of amino acids, which folds to form a protein. In bacteria this task can by performed by two releases factors, known as RF1 and RF2. However, the release factor must itself be released to leave the ribosome free to translate another strand of mRNA.
Pallesen et al. have used cryo-electron microscopy (cryo-EM) to study how a third release factor, RF3, helps to release RF1 from the ribosome in bacteria. In cells, RF3 usually forms a complex with a molecule called GDP, and the cryo-EM studies show that this molecule is released shortly after the RF3•GDP complex enters the ribosome. Once inside the ribosome, RF3 comes into contact with RF1 and with a protein called L12 that is part of the ribosome. A molecule called GTP—which is well known as a source of energy within cells—then binds to RF3, and this causes the shape of the ribosome to change. This change of shape results in the release of RF1 and the formation of a new RF3•GDP complex, which then leaves the ribosome.
Further work is needed to fully understand the role of L12 in these events, but a detailed understanding of the mechanism for terminating the translation of mRNA by the ribosome is coming into view.
DOI: http://dx.doi.org/10.7554/eLife.00411.002
doi:10.7554/eLife.00411
PMCID: PMC3677378  PMID: 23755360
Ribosome; Cryo-EM; Structure; RF1; RF3; L7/L12; E. coli
13.  Extracellular ATP Hydrolysis Inhibits Synaptic Transmission by Increasing pH Buffering in the Synaptic Cleft 
PLoS Biology  2014;12(5):e1001864.
A slow mechanism of retinal synaptic inhibition involves hydrolysis of ATP released from pannexin 1 channels (from the tips of horizontal cell dendrites); the resulting protons and phosphates acidify the synaptic cleft, which inhibits neurotransmitter release.
Neuronal computations strongly depend on inhibitory interactions. One such example occurs at the first retinal synapse, where horizontal cells inhibit photoreceptors. This interaction generates the center/surround organization of bipolar cell receptive fields and is crucial for contrast enhancement. Despite its essential role in vision, the underlying synaptic mechanism has puzzled the neuroscience community for decades. Two competing hypotheses are currently considered: an ephaptic and a proton-mediated mechanism. Here we show that horizontal cells feed back to photoreceptors via an unexpected synthesis of the two. The first one is a very fast ephaptic mechanism that has no synaptic delay, making it one of the fastest inhibitory synapses known. The second one is a relatively slow (τ≈200 ms), highly intriguing mechanism. It depends on ATP release via Pannexin 1 channels located on horizontal cell dendrites invaginating the cone synaptic terminal. The ecto-ATPase NTPDase1 hydrolyses extracellular ATP to AMP, phosphate groups, and protons. The phosphate groups and protons form a pH buffer with a pKa of 7.2, which keeps the pH in the synaptic cleft relatively acidic. This inhibits the cone Ca2+ channels and consequently reduces the glutamate release by the cones. When horizontal cells hyperpolarize, the pannexin 1 channels decrease their conductance, the ATP release decreases, and the formation of the pH buffer reduces. The resulting alkalization in the synaptic cleft consequently increases cone glutamate release. Surprisingly, the hydrolysis of ATP instead of ATP itself mediates the synaptic modulation. Our results not only solve longstanding issues regarding horizontal cell to photoreceptor feedback, they also demonstrate a new form of synaptic modulation. Because pannexin 1 channels and ecto-ATPases are strongly expressed in the nervous system and pannexin 1 function is implicated in synaptic plasticity, we anticipate that this novel form of synaptic modulation may be a widespread phenomenon.
Author Summary
At the first retinal synapse, specific cells—horizontal cells (HCs)—inhibit photoreceptors and help to organize the receptive fields of another retinal cell type, bipolar cells. This synaptic interaction is crucial for visual contrast enhancement. Here we show that horizontal cells feed back to photoreceptors via a very fast ephaptic mechanism and a relatively slow mechanism. The slow mechanism requires ATP release via Pannexin 1 (Panx1) channels that are located on HC dendrites near the site where photoreceptors release the neurotransmitter glutamate to HCs and bipolar cells. The released ATP is hydrolyzed to produce AMP, phosphate groups, and protons; these phosphates and protons form a pH buffer, which acidifies the synaptic cleft. This slow acidification inhibits presynaptic calcium channels and consequently reduces the neurotransmitter release of photoreceptors. This demonstrates a new way in which ATP release can be involved in synaptic modulation. Surprisingly, the action of ATP is not purinergic but is mediated via changes in the pH buffer capacity in the synaptic cleft. Given the broad expression of Panx1 channels in the nervous system and the suggestion that Panx1 function underlies stabilization of synaptic plasticity and is needed for learning, we anticipate that this mechanism will be more widespread than just occurring at the first retinal synapse.
doi:10.1371/journal.pbio.1001864
PMCID: PMC4028192  PMID: 24844296
14.  The Smart Aerial Release Machine, a Universal System for Applying the Sterile Insect Technique 
PLoS ONE  2014;9(7):e103077.
Background
Beyond insecticides, alternative methods to control insect pests for agriculture and vectors of diseases are needed. Management strategies involving the mass-release of living control agents have been developed, including genetic control with sterile insects and biological control with parasitoids, for which aerial release of insects is often required. Aerial release in genetic control programmes often involves the use of chilled sterile insects, which can improve dispersal, survival and competitiveness of sterile males. Currently available means of aerially releasing chilled fruit flies are however insufficiently precise to ensure homogeneous distribution at low release rates and no device is available for tsetse.
Methodology/Principal Findings
Here we present the smart aerial release machine, a new design by the Mubarqui Company, based on the use of vibrating conveyors. The machine is controlled through Bluetooth by a tablet with Android Operating System including a completely automatic guidance and navigation system (MaxNav software). The tablet is also connected to an online relational database facilitating the preparation of flight schedules and automatic storage of flight reports. The new machine was compared with a conveyor release machine in Mexico using two fruit flies species (Anastrepha ludens and Ceratitis capitata) and we obtained better dispersal homogeneity (% of positive traps, p<0.001) for both species and better recapture rates for Anastrepha ludens (p<0.001), especially at low release densities (<1500 per ha). We also demonstrated that the machine can replace paper boxes for aerial release of tsetse in Senegal.
Conclusions/Significance
This technology limits damages to insects and allows a large range of release rates from 10 flies/km2 for tsetse flies up to 600 000 flies/km2 for fruit flies. The potential of this machine to release other species like mosquitoes is discussed. Plans and operating of the machine are provided to allow its use worldwide.
doi:10.1371/journal.pone.0103077
PMCID: PMC4103892  PMID: 25036274
15.  The Effect of Hatchery Release Strategy on Marine Migratory Behaviour and Apparent Survival of Seymour River Steelhead Smolts (Oncorhynchus mykiss) 
PLoS ONE  2011;6(3):e14779.
Early marine migratory behaviour and apparent survival of hatchery-reared Seymour River steelhead (Oncorhynchus mykiss) smolts was examined over a four year period (2006–2009) to assess the impact of various management strategies on improving early marine survival. Acoustically tagged smolts were released to measure their survival using estuary and coastal marine receivers forming components of the Pacific Ocean Shelf Tracking (POST) array. Early marine survival was statistically indistinguishable between releases of summer run and winter run steelhead races, night and day releases, and groups released 10 days apart. In 2009, the survival of summer run steelhead released into the river was again trialed against groups released directly into the ocean at a distance from the river mouth. Apparent survival was improved significantly for the ocean released groups. The health and physiological status of the various release groups were monitored in years 2007–2009, and results indicate that the fish were in good health, with no clinical signs of disease at the time of release. The possibility of a disease event contributing to early marine mortality was further examined in 2009 by vaccinating half of the released fish against common fish diseases (vibriosis, furunculosis). The results suggest that marine survival may be enhanced using this approach, although not to the extent observed when the smolts were transported away from the river mouth before release. In summary, direct experimental testing of different release strategies using the POST array to measure ocean survival accelerated the scientific process by allowing rapid collection of data which enabled the rejection of several existing theories and allowed tentative identification of several new alternative approaches that might improve early marine survival of Seymour River steelhead.
doi:10.1371/journal.pone.0014779
PMCID: PMC3066170  PMID: 21468320
16.  Regulating Drug Release Behavior and Kinetics from Matrix Tablets Based on Fine Particle-Sized Ethyl Cellulose Ether Derivatives: An In Vitro and In Vivo Evaluation 
The Scientific World Journal  2012;2012:842348.
The design and fabrication of sustained/controlled release dosage forms, employing new excipients capable of extending/controlling the release of drugs from the dosage forms over prolonged periods, has worked well in achieving optimally enhanced therapeutic levels of the drugs. In this sense, the objective of this study was to investigate the suitability of selected cellulose ether derivatives for use in direct compression (DC) and as efficient drug release controlling agents. Controlled release matrix tablets of ciprofloxacin were prepared at different drug-to-polymer (D : P) ratios by direct compression using a fine particle sized ethylcellulose ether derivative (ETHOCEL Standard Premium 7FP) as rate controlling polymer. The tablets obtained were evaluated for various physico-chemical characteristics and in-vitro drug release studies were conducted in phosphate buffer (pH 7.4) using PharmaTest dissolution apparatus at constant temperature of 37°C ± 0.1. Similarity factor f2 was employed to the release profiles of test formulations and were compared with marketed ciprofloxacin conventional tablets. Drug release mechanism and the kinetics involved were investigated by fitting the release profile data to various kinetic models. It was found that with increasing the proportion of ethylcellulose ether derivative in the matrix, the drug release was significantly extended up to 24 hours. The tablets exhibited zero order or nearly zero order drug transport mechanism. In vivo drug release performance of the developed controlled release tablets and reference conventional tablets containing ciprofloxacin were determined in rabbit serum according to randomized two-way crossover study design using High Performance Liquid Chromatography. Several bioavailability parameters of both the test tablets and conventional tablets including Cmax⁡, Tmax⁡ and AUC0-t were compared which showed an optimized Cmax⁡ and Tmax⁡ (P < 0.05). A good correlation was obtained between in vitro drug release and in vivo drug absorption with correlation value (R2 = 0.934). Relative bioavailability was found to be 93%. Reproducibility of manufacturing process and accelerated stability of the developed tablets were performed in stability chamber at 40 ± 2°C and 75 ± 5% relative humidity for a period of 6 months and were found to be stable throughout the stability period.
doi:10.1100/2012/842348
PMCID: PMC3350987  PMID: 22649325
17.  Accelerating protein release from microparticles for regenerative medicine applications 
There is a need to control the spatio-temporal release kinetics of growth factors in order to mitigate current usage of high doses. A novel delivery system, capable of providing both structural support and controlled release kinetics, has been developed from PLGA microparticles. The inclusion of a hydrophilic PLGA–PEG–PLGA triblock copolymer altered release kinetics such that they were decoupled from polymer degradation. A quasi zero order release profile over four weeks was produced using 10% w/w PLGA–PEG–PLGA with 50:50 PLGA whereas complete and sustained release was achieved over ten days using 30% w/w PLGA–PEG–PLGA with 85:15 PLGA and over four days using 30% w/w PLGA–PEG–PLGA with 50:50 PLGA. These three formulations are promising candidates for delivery of growth factors such as BMP-2, PDGF and VEGF. Release profiles were also modified by mixing microparticles of two different formulations providing another route, not previously reported, for controlling release kinetics. This system provides customisable, localised and controlled delivery with adjustable release profiles, which will improve the efficacy and safety of recombinant growth factor delivery.
Graphical abstract
Highlights
► A new delivery system providing controlled release kinetics has been developed. ► Inclusion of hydrophilic PLGA–PEG–PLGA decoupled release kinetics from degradation. ► Using 10% triblock copolymer produced quasi zero order release over four weeks. ► Mixing microparticle formulations provided another route for controlling release. ► This system provides customisable, localised and controlled delivery of growth factors.
doi:10.1016/j.msec.2013.02.020
PMCID: PMC3654200  PMID: 23623071
Poly (d,l-lactic -co-glycolic acid) (PLGA); Microparticles; Microspheres; Controlled release; Double emulsion solvent evaporation; Growth factor delivery
18.  Release of DNA from Polyelectrolyte Multilayers Fabricated Using ‘Charge-Shifting’ Cationic Polymers: Tunable Temporal Control and Sequential, Multi-Agent Release 
We report an approach to the design of multilayered polyelectrolyte thin films (or ‘polyelectrolyte multilayers’, PEMs) that can be used to provide tunable control over the release of plasmid DNA (or multiple different DNA constructs) from film-coated surfaces. Our approach is based upon methods for the layer-by-layer assembly of DNA-containing thin films, and exploits the properties of a new class of cationic ‘charge-shifting’ polymers (or amine-functionalized polymers that undergo gradual changes in net charge upon side-chain ester hydrolysis) to provide control over the rates at which these films erode and release DNA. We synthesized two ‘charge-shifting’ polymers (polymers 1 and 2) containing different side chain structures by ring-opening reactions of poly(2-alkenyl azlactone)s with two different tertiary amine functionalized alcohols (2-dimethylaminoethanol and 3-dimethyl-1-propanol, respectively). Subsequent characterization revealed large changes in the rates of side chain ester hydrolysis for these two polymers; whereas the half-life for the hydrolysis of the esters in polymer 1 was ~200 days, the half-life for polymer 2 was ~6 days. We demonstrate that these large differences in side chain hydrolysis make possible the design of PEMs that erode and promote the surface-mediated release of DNA either rapidly (e.g., over ~3 days for films fabricated using polymer 2) or slowly (e.g., over ~1 month for films fabricated using polymer 1). We demonstrate further that it is possible to design films with release profiles that are intermediate to these two extremes by fabricating films using solutions containing different mixtures of these two polymers. This approach can thus expand the usefulness of these two polymers and achieve a broader range of DNA release profiles without the need to synthesize polymers with new structures or properties. Finally, we demonstrate that polymers 1 and 2 can be used to fabricate multilayered films with hierarchical structures that promote the sequential release of two different DNA constructs with separate and distinct release profiles (e.g., the release of a first construct over a period of ~3 days, followed by the sustained release of a second for a period of ~70 days). With further development, this approach could contribute to the design of functional thin films and surface coatings that provide sophisticated control over the timing and the order of the release of two or more DNA constructs (or other agents) of interest in a range of biomedical contexts.
doi:10.1016/j.jconrel.2010.07.112
PMCID: PMC3005143  PMID: 20678530
Layer-by-Layer; Polyelectrolyte; Thin Film; DNA Delivery; Surface-Mediated
19.  Characterization of drug-release kinetics in trabecular bone from titania nanotube implants 
Purpose
The aim of this study was to investigate the application of the three-dimensional bone bioreactor for studying drug-release kinetics and distribution of drugs in the ex vivo cancellous bone environment, and to demonstrate the application of nanoengineered titanium (Ti) wires generated with titania nanotube (TNT) arrays as drug-releasing implants for local drug delivery
Methods
Nanoengineered Ti wires covered with a layer of TNT arrays implanted in bone were used as a drug-releasing implant. Viable bovine trabecular bone was used as the ex vivo bone substrate embedded with the implants and placed in the bone reactor. A hydrophilic fluorescent dye (rhodamine B) was used as the model drug, loaded inside the TNT–Ti implants, to monitor drug release and transport in trabecular bone. The distribution of released model drug in the bone was monitored throughout the bone structure, and concentration profiles at different vertical (0–5 mm) and horizontal (0–10 mm) distances from the implant surface were obtained at a range of release times from 1 hour to 5 days.
Results
Scanning electron microscopy confirmed that well-ordered, vertically aligned nanotube arrays were formed on the surface of prepared TNT–Ti wires. Thermogravimetric analysis proved loading of the model drug and fluorescence spectroscopy was used to show drug-release characteristics in-vitro. The drug release from implants inserted into bone ex vivo showed a consistent gradual release of model drug from the TNT–Ti implants, with a characteristic three-dimensional distribution into the surrounding bone, over a period of 5 days. The parameters including the flow rate of bone culture medium, differences in trabecular microarchitecture between bone samples, and mechanical loading were found to have the most significant influence on drug distribution in the bone.
Conclusion
These results demonstrate the utility of the Zetos™ system for ex vivo drug-release studies in bone, which can be applied to optimize the delivery of specific therapies and to assist in the design of new drug delivery systems. This method has the potential to provide new knowledge to understand drug distribution in the bone environment and to considerably improve existing technologies for local administration in bone, including solving some critical problems in bone therapy and orthopedic implants.
doi:10.2147/IJN.S33655
PMCID: PMC3446838  PMID: 23028217
local drug delivery; Zetos bone bioreactor; drug-releasing implant; drug diffusion
20.  Re-examining how complexin inhibits neurotransmitter release 
eLife  2014;3:e02391.
Complexins play activating and inhibitory functions in neurotransmitter release. The complexin accessory helix inhibits release and was proposed to insert into SNARE complexes to prevent their full assembly. This model was supported by ‘superclamp’ and ‘poor-clamp’ mutations that enhanced or decreased the complexin-I inhibitory activity in cell–cell fusion assays, and by the crystal structure of a superclamp mutant bound to a synaptobrevin-truncated SNARE complex. NMR studies now show that the complexin-I accessory helix does not insert into synaptobrevin-truncated SNARE complexes in solution, and electrophysiological data reveal that superclamp mutants have slightly stimulatory or no effects on neurotransmitter release, whereas a poor-clamp mutant inhibits release. Importantly, increasing or decreasing the negative charge of the complexin-I accessory helix inhibits or stimulates release, respectively. These results suggest a new model whereby the complexin accessory helix inhibits release through electrostatic (and perhaps steric) repulsion enabled by its location between the vesicle and plasma membranes.
DOI: http://dx.doi.org/10.7554/eLife.02391.001
eLife digest
The instructions sent to, from and within the brain are rapidly transmitted along neurons in the form of electrical signals. These signals cannot pass across the small gaps—called synapses—that separate neighboring neurons. Instead, neurons release chemicals called neurotransmitters into the synapses, and these relay the signal to the next neuron.
The neurotransmitters are stored inside neurons in small bubbles called vesicles. To release these neurotransmitters into the synapse, the membrane that encloses the vesicle fuses with the membrane that surrounds the neuron. To fuse the membranes, proteins embedded in the vesicle membrane interact with similar proteins in the neuron membrane to form a structure called a SNARE complex. Additional proteins control membrane fusion to ensure that the signal is passed to the other neuron at the right time and with the appropriate efficiency.
Among these proteins are the complexins, which are often found attached to SNARE complexes. Although different parts of complexins can both help and hinder membrane fusion, a part known as an accessory helix is thought to have only one role—to stop the membranes from fusing together. Several models have been suggested for how the accessory helix interferes with fusion. However, after performing a range of analyses by diverse biophysical techniques, Trimbuch, Xu et al. suggest these models are unlikely to describe the process accurately.
Instead, Trimbuch, Xu et al. propose a new model based on the electrostatic properties of two molecules that are both negatively charged. An accessory helix taken from a fruit fly complexin was more negatively charged than a mammalian version, and experiments showed it was also better at preventing the release of neurotransmitters. It is thought that the negative charges on the helix hold the membranes apart because the helix is located between the membranes, which are also negatively charged. Consistent with this model, Trimbuch, Xu et al. showed that the membranes fused more easily when some of the negative charges on the accessory helix were replaced with positive charges. The next challenges are to test the model further with additional studies, and to explain how other proteins work with complexins to control neurotransmitter release.
DOI: http://dx.doi.org/10.7554/eLife.02391.002
doi:10.7554/eLife.02391
PMCID: PMC4040926  PMID: 24842998
membrane fusion; synaptic vesicle exocytosis; protein interactions; human; mouse
21.  Press Releases Issued by Supplements Industry Organisations and Non-Industry Organisations in Response to Publication of Clinical Research Findings: A Case-Control Study 
PLoS ONE  2014;9(7):e101533.
Background
Dietary supplement use is increasing despite lack of evidence of benefits, or evidence of harm. Press releases issued by the supplements industry might contribute to this situation by using ‘spin’ (strategies to hype or denigrate findings) to distort the results of clinical studies. We assessed press releases issued in response to publication of clinical studies on dietary supplements.
Methods and Findings
We analyzed 47 supplements industry press releases and 91 non-industry press releases and news stories, generated in response to 46 clinical studies of dietary supplements published between 1/1/2005 and 5/31/2013. The primary outcome was ‘spin’ content and direction. We also assessed disposition towards use of dietary supplements, reporting of study information, and dissemination of industry press releases. More supplements industry press releases (100%) contained ‘spin’ than non-industry media documents (55%, P<0.001). Hyping ‘spin’ scores were higher in industry than non-industry media documents for studies reporting benefit of supplements (median ‘spin’ score 3.3, 95% CI 1.0–5.5 vs 0.5, 0–1.0; P<0.001). Denigratory ‘spin’ scores were higher in industry than non-industry media documents for studies reporting no effect (6.0, 5.0–7.0 vs 0, 0–0; P<0.001) or harm (6.0, 5.5–7.5 vs 0, 0–0.5; P<0.001) from a supplement. Industry press releases advocated supplement use in response to >90% of studies that reported no benefit, or harm, of the supplement. Industry press releases less frequently reported study outcomes, sample size, and estimates of effect size than non-industry media documents (all P<0.001), particularly for studies that reported no benefit of supplements. Industry press releases were referenced by 148 news stories on the websites of 6 organizations that inform manufacturers, retailers and consumers of supplements.
Conclusions
Dietary supplements industry press releases issued in response to clinical research findings are characterized by ‘spin’ that hypes results that are favourable to supplement use and denigrates results that are not.
doi:10.1371/journal.pone.0101533
PMCID: PMC4081644  PMID: 24992571
22.  Designing a Community Engagement Framework for a New Dengue Control Method: A Case Study from Central Vietnam 
Background
The Wolbachia strategy aims to manipulate mosquito populations to make them incapable of transmitting dengue viruses between people. To test its efficacy, this strategy requires field trials. Public consultation and engagement are recognized as critical to the future success of these programs, but questions remain regarding how to proceed. This paper reports on a case study where social research was used to design a community engagement framework for a new dengue control method, at a potential release site in central Vietnam.
Methodology/Principal Findings
The approach described here, draws on an anthropological methodology and uses both qualitative and quantitative methods to design an engagement framework tailored to the concerns, expectations, and socio-political setting of a potential trial release site for Wolbachia-infected Aedes aegypti mosquitoes. The process, research activities, key findings and how these were responded to are described. Safety of the method to humans and the environment was the most common and significant concern, followed by efficacy and impact on local lives. Residents expected to be fully informed and engaged about the science, the project, its safety, the release and who would be responsible should something go wrong. They desired a level of engagement that included regular updates and authorization from government and at least one member of every household at the release site.
Conclusions/Significance
Results demonstrate that social research can provide important and reliable insights into public concerns and expectations at a potential release site, as well as guidance on how these might be addressed. Findings support the argument that using research to develop more targeted, engagement frameworks can lead to more sensitive, thorough, culturally comprehensible and therefore ethical consultation processes. This approach has now been used successfully to seek public input and eventually support for releases Wolbachia-infected mosquitoes, in two different international settings - Australia and Vietnam.
Author Summary
In recent years, a number of new strategies using novel technologies for the control of dengue fever control have emerged. These strategies are notably different from their predecessors and not without controversy. Many also require open release field trials to test their efficacy. Public consultation and engagement are recognized as critical to the future success of these programs, but questions remain regarding how to proceed. In this paper we describe an approach to public engagement that uses social research to design an engagement framework and communication materials tailored to the concerns, expectations, and socio-political setting of potential trial release sites. This approach was developed and implemented in Australia (2008–2010) where the first publicly supported field trials occurred January 2011. We report here on the implementation of this approach in Vietnam (2009–2010) where the second release will occur in 2014. This paper describes the process, research activities, outcomes and key findings from the Vietnamese field site. It highlights key public concerns and expectations about engagement and authorization and shows how these were used to develop a more targeted, culturally appropriate and comprehensible engagement framework and communication materials. The paper demonstrates the viability of this approach to community engagement for new dengue control strategies, in a ‘developing’ country context.
doi:10.1371/journal.pntd.0002794
PMCID: PMC4031131  PMID: 24853391
23.  Solventless photocurable film coating: Evaluation of drug release, mechanical strength, and photostability 
AAPS PharmSciTech  2007;8(3):E42-E51.
A new solventless photocurable film-coating system was investigated in which nonpareil beads were coated in a minicoating pan with liquid prepolymer (L) and powdered solid pore-forming agents (S) and cured by UV light. Release from the coating could by altered by changing the material, the number of layers, and the coating thickness. Immediate release of a blue dye contained in the nonpareils was obtained with sodium starch glycolate as a pore former that swelled the coating and yielded large pores; through these pores the dye quickly released while leaving behind the scaffold provided by the photocured prepolymer. Simple pore formers (lactose and sodium chloride) dissolved away without swelling and provided a more sustained release. The nature of the scaffold and pore structure of the coating were determined by simultaneously monitoring the release of sodium chloride from the coating and blue dye from the beads. At least 50% of the sodium chloride that was incorporated into the coating released before the dye released through the coating, except at an S/L ratio (ratio of the amount of solid pore-forming agent to the volume of liquid prepolymer) of 2.4, where 40% of the sodium chloride was released before the release of dye. The coupling between dye release and pore formation was found to be dependent on the S/L ratio of the coating. Simulation based on percolation theory showed that the coupling of pore formation and dye release was higher when the variance in tortuosity was lower. The coating was photostable and could withstand normal handling stress.
doi:10.1208/pt0803057
PMCID: PMC2750553  PMID: 17915807
Coating; photocurable; solventless; photoinitiator; process and formulation parameters; functional release; photostability
24.  Preparation and In-vivo Pharmacokinetic Study of a Novel Extended Release Compression Coated Tablets of Fenoterol Hydrobromide 
AAPS PharmSciTech  2008;9(3):1016-1024.
The aim of this study was to formulate extended release compression coated core tablets of fenoterol hydrobromide, a selective β2 adrenergic receptor agonist, in an attempt to prevent nocturnal asthma. Two hydrophilic polymers viz Kollidon® SR, Polyox® WSR 303 and a hydrophobic one (Precirol® ATO5) were employed. Compression coated tablets were formulated by preparing a core tablet containing 7.5 mg drug and various amounts of polymer and Emcompress® then compressed coated with the same polymeric materials. For comparison purpose different matrix tablets were also prepared employing the same polymers. In-vitro release studies were carried out at different pH (1.2 and 6.8). Pharmacokinetics of extended release tablets as well as commercially available immediate release tablets (Berotec®) were studied after oral administration to beagle dogs using a new developed LC-MS/MS method with a lower limit of quantification of 1 ng/ml. Fenoterol release from compression coated tablets was significantly lower than matrix tablets. The mechanism of release was changed with the nature and content of polymer. The release pattern of drug from F16 containing 40 mg Kollidon® SR divided in the core tablet (15 mg) and the rest in the compressed coat (25 mg) showed a typical zero order release kinetic that could extend drug release >10 h and reasonable time for 75% to be released (t75) (8.92 h). When compared to immediate release Berotec® tablet the MRT was significantly extended from 7.03 ± 0.76 to 10.93 ± 1.25 h (P < 0.001) and HVDt 50%Cmax was also significantly extended from 2.71 ± 0.68 to 6.81 ± 0.67 h with expected prevention of nocturnal asthma.
doi:10.1208/s12249-008-9135-8
PMCID: PMC2977029  PMID: 18770048
compression coat; extended release; fenoterol hydrobromide; LC/MS/MS; pharmacokinetics; tablets
25.  Nanosized sustained-release pyridostigmine bromide microcapsules: process optimization and evaluation of characteristics 
Background
Pyridostigmine bromide (3-[[(dimethylamino)-carbonyl]oxy]-1-methylpyridinium bromide), a reversible inhibitor of cholinesterase, is given orally in tablet form, and a treatment schedule of multiple daily doses is recommended for adult patients. Nanotechnology was used in this study to develop an alternative sustained-release delivery system for pyridostigmine, a synthetic drug with high solubility and poor oral bioavailability, hence a Class III drug according to the Biopharmaceutics Classification System. Novel nanosized pyridostigmine-poly(lactic acid) microcapsules (PPNMCs) were expected to have a longer duration of action than free pyridostigmine and previously reported sustained-release formulations of pyridostigmine.
Methods
The PPNMCs were prepared using a double emulsion-solvent evaporation method to achieve sustained-release characteristics for pyridostigmine. The preparation process for the PPNMCs was optimized by single-factor experiments. The size distribution, zeta potential, and sustained-release behavior were evaluated in different types of release medium.
Results
The optimal volume ratio of inner phase to external phase, poly(lactic acid) concentration, polyvinyl alcohol concentration, and amount of pyridostigmine were 1:10, 6%, 3% and 40 mg, respectively. The negatively charged PPNMCs had an average particle size of 937.9 nm. Compared with free pyridostigmine, PPNMCs showed an initial burst release and a subsequent very slow release in vitro. The release profiles for the PPNMCs in four different types of dissolution medium were fitted to the Ritger-Peppas and Weibull models. The similarity between pairs of dissolution profiles for the PPNMCs in different types of medium was statistically significant, and the difference between the release curves for PPNMCs and free pyridostigmine was also statistically significant.
Conclusion
PPNMCs prepared by the optimized protocol described here were in the nanometer range and had good uniformity, with significantly slower pyridostigmine release than from free pyridostigmine. This novel sustained-release delivery nanosystem for pyridostigmine might alleviate the need to identify new acetylcholinesterase inhibitors.
doi:10.2147/IJN.S40860
PMCID: PMC3582480  PMID: 23459707
nanosized microcapsules; process optimization; characteristics; sustained-release; pyridostigmine bromide

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