Early-stage development of novel cyclodextrin-siRNA nanocomplexes allows for successful postnebulization transfection of bronchial epithelial cells.

BACKGROUND: Successful delivery of small interfering RNA (siRNA) to the lungs remains hampered by poor intracellular delivery, vector-mediated cytotoxicity, and an inability to withstand nebulization. Recently, a novel cyclodextrin (CD), SC12CDClickpropylamine, consisting of distinct lipophilic and cationic subunits, has been shown to transfect a number of cell types. However, the suitability of this vector for pulmonary siRNA delivery has not been assessed to date. To address this, a series of high-content analysis (HCA) and postnebulization assays were devised to determine the potential for CD-siRNA delivery to the lungs. METHODS: SC12CDClickpropylamine-siRNA mass ratios (MRs) were examined for size and zeta potential. In-depth analysis of nanocomplex uptake and toxicity in Calu-3 bronchial epithelial cells was examined using IN Cell(®) HCA assays. Nebulized SC12CDClickpropylamine nanocomplexes were assessed for volumetric median diameter (VMD) and fine particle fraction (FPF) and compared with saline controls. Finally, postnebulization stability was determined by comparing luciferase knockdown elicited by SC12CDClickpropylamine nanocomplexes before and after nebulization. RESULTS: SC12CDClickpropylamine-siRNA complexation formed cationic nanocomplexes of ≤200 nm in size depending on the medium and led to significantly higher levels of siRNA associated with Calu-3 cells compared with RNAiFect-siRNA-treated cells at all MRs (p CONCLUSIONS: SC12CDClickpropylamine nanocomplexes can be effectively nebulized for pulmonary delivery of siRNA using Aeroneb technology to mediate knockdown in airway cells. To the best of our knowledge, this is the first study examining the suitability of SC12CDClickpropylamine-siRNA nanocomplexes for pulmonary delivery. Furthermore, this work provides an integrated nanomedicine-device combination for future in vitro and in vivo preclinical and clinical studies of inhaled siRNA therapeutics

Optimization and Dose Estimation of Aerosol Delivery to Non-Human Primates

Background: In pre-clinical animal studies, the uniformity of dosing across subjects and routes of administration is a crucial requirement. In preparation for a study in which aerosolized live-attenuated measles virus vaccine was administered to cynomolgus monkeys (Macaca fascicularis) by inhalation, we assessed the percentage of a nebulized dose inhaled under varying conditions. Methods: Drug delivery varies with breathing parameters. Therefore we determined macaque breathing patterns (tidal volume, breathing frequency, and inspiratory to expiratory (I:E) ratio) across a range of 3.3-6.5 kg body weight, using a pediatric pneumotachometer interfaced either with an endotracheal tube or a facemask. Subsequently, these breathing patterns were reproduced using a breathing simulator attached to a filter to collect the inhaled dose. Albuterol was nebulized using a vibrating mesh nebulizer and the percentage inhaled dose was determined by extraction of drug from the filter and subsequent quantification. Results: Tidal volumes ranged from 24 to 46 mL, breathing frequencies from 19 to 31 breaths per minute and I: E ratios from 0.7 to 1.6. A small pediatric resuscitation mask was identified as the best fitting interface between animal and pneumotachometer. The average efficiency of inhaled dose delivery was 32.1% (standard deviation 7.5, range 24%-48%), with variation in tidal volumes as the most important determinant. Conclusions: Studies in non-human primates aimed at comparing aerosol delivery with other routes of administration should take both the inter-subject variation and relatively low efficiency of delivery to these low body weight mammals into account

Frequency of microplastics in mesopelagic fishes from the Northwest Atlantic

Microplastics are a ubiquitous pollutant in our seas today and are known to have detrimental effects on a variety of organisms. Over the past decade numerous studies have documented microplastic ingestion by marine species with more recent investigations focussing on the secondary impacts of microplastic ingestion on ecosystem processes. However, few studies so far have examined microplastic ingestion by mesopelagic fish which are one of the most abundant pelagic groups in our oceans and through their vertical migrations are known to contribute significantly to the rapid transport of carbon and nutrients to the deep sea. Therefore, any ingestion of microplastics by mesopelagic fish may adversely affect this cycling and may aid in transport of microplastics from surface waters to the deep-sea benthos. In this study microplastics were extracted from mesopelagic fish under forensic conditions and analysed for polymer type utilising micro-Fourier Transform Infrared Spectroscopy (micro-FTIR) analysis. Fish specimens were collected from depth (300–600 m) in a warm-core eddy located in the Northwest Atlantic, 1,200 km due west of Newfoundland during April and May 2015. In total, 233 fish gut contents from seven different species of mesopelagic fish were examined. An alkaline dissolution of organic materials from extracted stomach contents was performed and the solution filtered over a 0.7 μm borosilicate filter. Filters were examined for microplastics and a subsample originating from 35 fish was further analysed for polymer type through micro-FTIR analysis. Seventy-three percent of all fish contained plastics in their gut contents with Gonostoma denudatum having the highest ingestion rate (100%) followed by Serrivomer beanii (93%) and Lampanyctus macdonaldi (75%). Overall, we found a much higher occurrence of microplastic fragments, mainly polyethylene fibres, in the gut contents of mesopelagic fish than previously reported. Stomach fullness, species and the depth at which fish were caught at, were found to have no effect on the amount of microplastics found in the gut contents. However, these plastics were similar to those sampled from the surface water. Additionally, using forensic techniques we were able to highlight that fibres are a real concern rather than an artefact of airborne contaminatio

Toxicity of lunar dust

The formation, composition and physical properties of lunar dust are incompletely characterised with regard to human health. While the physical and chemical determinants of dust toxicity for materials such as asbestos, quartz, volcanic ashes and urban particulate matter have been the focus of substantial research efforts, lunar dust properties, and therefore lunar dust toxicity may differ substantially. In this contribution, past and ongoing work on dust toxicity is reviewed, and major knowledge gaps that prevent an accurate assessment of lunar dust toxicity are identified. Finally, a range of studies using ground-based, low-gravity, and in situ measurements is recommended to address the identified knowledge gaps. Because none of the curated lunar samples exist in a pristine state that preserves the surface reactive chemical aspects thought to be present on the lunar surface, studies using this material carry with them considerable uncertainty in terms of fidelity. As a consequence, in situ data on lunar dust properties will be required to provide ground truth for ground-based studies quantifying the toxicity of dust exposure and the associated health risks during future manned lunar missions.Comment: 62 pages, 9 figures, 2 tables, accepted for publication in Planetary and Space Scienc

Modified Vaccinia Virus Ankara Preferentially Targets Antigen Presenting Cells In Vitro, Ex Vivo and In Vivo

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies

Erratum to: 36th International Symposium on Intensive Care and Emergency Medicine

[This corrects the article DOI: 10.1186/s13054-016-1208-6.]

Teaching and learning across disciplines : student and staff experiences in a newly modularised system

Within modular degrees it is sometimes possible for students to broaden their education by taking modules from outside their main programme of study. This is one significant aspect of modular degrees which has not been studied. In an effort to better understand this issue, the research reported in this paper explored the experiences (a) of students taking modules from outside their programme of study and (b) of staff teaching modules with significant numbers of students from other programmes. In total, 820 undergraduate students responded to an on-line survey; 12 academic staff members participated in interviews. The survey focused on students’ reasons for choosing the module, their experiences of assessment and their perceptions of workload. Interviews with academic staff focused on the influence of non-programme students on teaching and assessment practices. The discussion addresses the implications of student choice and classroom diversity for teaching and assessment in modular systems.Other funderStrategic Innovation FundEmbargo until May 2012 - AV 27/10/2010 ke - AS 04/11/2010 MD done - OR 10/11/201

Supplementary Material: Evaluation of Cyanea capillata Sting Management Protocols Using Ex Vivo and In Vitro Envenomation Models

Supplementary files for Doyle, T.K.; Headlam, J.L.; Wilcox, C.L.; MacLoughlin, E.; Yanagihara, A.A. Evaluation of Cyanea capillataSting Management Protocols Using Ex Vivo and In Vitro Envenomation Models. Toxins 2017, 9, 215. Video S1: Vinegar Application to Gelatin-Adherent Cnida