Quantification of functionalised gold nanoparticle-targeted knockdown of gene expression in HeLa cells

Introduction: Gene therapy continues to grow as an important area of research, primarily because of its potential in the treatment of disease. One significant area where there is a need for better understanding is in improving the efficiency of oligonucleotide delivery to the cell and indeed, following delivery, the characterization of the effects on the cell. Methods: In this report, we compare different transfection reagents as delivery vehicles for gold nanoparticles functionalized with DNA oligonucleotides, and quantify their relative transfection efficiencies. The inhibitory properties of small interfering RNA (siRNA), single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) sequences targeted to human metallothionein hMT-IIa are also quantified in HeLa cells. Techniques used in this study include fluorescence and confocal microscopy, qPCR and Western analysis. Findings: We show that the use of transfection reagents does significantly increase nanoparticle transfection efficiencies. Furthermore, siRNA, ssRNA and ssDNA sequences all have comparable inhibitory properties to ssDNA sequences immobilized onto gold nanoparticles. We also show that functionalized gold nanoparticles can co-localize with autophagosomes and illustrate other factors that can affect data collection and interpretation when performing studies with functionalized nanoparticles. Conclusions: The desired outcome for biological knockdown studies is the efficient reduction of a specific target; which we demonstrate by using ssDNA inhibitory sequences targeted to human metallothionein IIa gene transcripts that result in the knockdown of both the mRNA transcript and the target protein

A qualitative study of the impact of severe asthma and its treatment showing that treatment burden is neglected in existing asthma assessment scales

Background People with severe asthma experience significant respiratory symptoms and suffer adverse effects of oral corticosteroids (OCS), including disturbed mood and physical symptoms. OCS impacts on health-related quality of life (HRQoL) have not been quantified. Asthma HRQoL scales are valid as outcome measures for patients requiring OCS only if they assess the deficits imposed by OCS. Aims The aim of this study was to compare the burden of disease and treatment in patients with severe asthma with items in eight asthma-specific HRQoL scales. Methods Twenty-three patients with severe asthma recruited from a severe asthma clinic were interviewed about the impact of their respiratory symptoms and the burden of their treatment. The domains from a thematic analysis of these interviews were compared with the items of eight asthma-specific HRQoL scales. Results In addition to the burden caused by symptoms, ten domains of OCS impact on HRQoL were identified: depression, irritability, sleep, hunger, weight, skin, gastric, pain, disease anxiety, and medication anxiety. Some patients experienced substantial HRQoL deficits attributed to OCS. Although all HRQoL scales include some OCS-relevant items, all eight scales fail to adequately assess the several types of burden experienced by some patients while on OCS. Conclusion The burden of OCS in severe asthma is neglected in policy and practice because it is not assessed in outcome studies. Existing asthma HRQoL scales provide an overly positive estimation of HRQoL in patients with frequent exposure to OCS and underestimate the benefit of interventions that reduce OCS exposure. Changes to existing measurement procedures are needed

Layered convection as the origin of Saturn's luminosity anomaly

As they keep cooling and contracting, Solar System giant planets radiate more energy than they receive from the Sun. Applying the first and second principles of thermodynamics, one can determine their cooling rate, luminosity, and temperature at a given age. Measurements of Saturn's infrared intrinsic luminosity, however, reveal that this planet is significantly brighter than predicted for its age. This excess luminosity is usually attributed to the immiscibility of helium in the hydrogen-rich envelope, leading to "rains" of helium-rich droplets. Existing evolution calculations, however, suggest that the energy released by this sedimentation process may not be sufficient to resolve the puzzle. Here, we demonstrate using planetary evolution models that the presence of layered convection in Saturn's interior, generated, like in some parts of Earth oceans, by the presence of a compositional gradient, significantly reduces its cooling. It can explain the planet's present luminosity for a wide range of configurations without invoking any additional source of energy. This suggests a revision of the conventional homogeneous adiabatic interior paradigm for giant planets, and questions our ability to assess their heavy element content. This reinforces the possibility for layered convection to help explaining the anomalously large observed radii of extrasolar giant planets.Comment: Published in Nature Geoscience. Online publication date: April 21st, 2013. Accepted version before journal editing and with Supplementary Informatio

El Niño Modulations Over The Past Seven Centuries (Letter to the editor)

postprin

Recommendations for a core outcome set for measuring standing balance in adult populations: a consensus-based approach

Standing balance is imperative for mobility and avoiding falls. Use of an excessive number of standing balance measures has limited the synthesis of balance intervention data and hampered consistent clinical practice.To develop recommendations for a core outcome set (COS) of standing balance measures for research and practice among adults.A combination of scoping reviews, literature appraisal, anonymous voting and face-to-face meetings with fourteen invited experts from a range of disciplines with international recognition in balance measurement and falls prevention. Consensus was sought over three rounds using pre-established criteria.The scoping review identified 56 existing standing balance measures validated in adult populations with evidence of use in the past five years, and these were considered for inclusion in the COS.Fifteen measures were excluded after the first round of scoring and a further 36 after round two. Five measures were considered in round three. Two measures reached consensus for recommendation, and the expert panel recommended that at a minimum, either the Berg Balance Scale or Mini Balance Evaluation Systems Test be used when measuring standing balance in adult populations.Inclusion of two measures in the COS may increase the feasibility of potential uptake, but poses challenges for data synthesis. Adoption of the standing balance COS does not constitute a comprehensive balance assessment for any population, and users should include additional validated measures as appropriate.The absence of a gold standard for measuring standing balance has contributed to the proliferation of outcome measures. These recommendations represent an important first step towards greater standardization in the assessment and measurement of this critical skill and will inform clinical research and practice internationally

Implementing a Non-Specialist Delivered Psychological Intervention for Young Adolescents in a Protracted Refugee Setting: a Qualitative Process Evaluation in Lebanon

There has been an increase in the evaluation and implementation of non-specialist delivered psychological interventions to address unmet mental health needs in humanitarian emergencies. While randomized controlled trials (RCTs) provide important evidence about intervention impact, complementary qualitative process evaluations are essential to understand key implementation processes and inform future scaling up of the intervention. This study was conducted as part of an RCT of the Early Adolescents Skills for Emotions (EASE) psychological intervention for young adolescents with elevated psychological distress (predominantly with a Syrian refugee background) in Lebanon. Our aims were firstly to conduct a qualitative process evaluation to understand stakeholder experiences and perceived impact of the intervention and identify barriers and facilitators for implementation, and secondly to explore considerations for scaling up. Eleven key informant interviews and seven focus groups were conducted with 39 respondents including adolescent and caregiver participants, trainers, providers, outreach workers, and local stakeholders. Data were analyzed using inductive and deductive thematic analysis. Respondents perceived the intervention to be highly needed and reported improvements in adolescent mental health and wellbeing. Key implementation factors that have potential to influence engagement, adherence, and perceived impact included the socio-economic situation of families, mental health stigma, coordination within and between sectors (particularly for scaling up), embedding the intervention within existing service pathways, having clear quality and accountability processes including training and supervision for non-specialists, and sustainable funding. Our findings provide important context for understanding effectiveness outcomes of the RCT and highlights factors that need to be considered when implementing a mental health intervention on a larger scale in a complex crisis

Exploring pig trade patterns to inform the design of risk-based disease surveillance and control strategies

An understanding of the patterns of animal contact networks provides essential information for the design of risk-based animal disease surveillance and control strategies. This study characterises pig movements throughout England and Wales between 2009 and 2013 with a view to characterising spatial and temporal patterns, network topology and trade communities. Data were extracted from the Animal and Plant Health Agency (APHA)’s RADAR (Rapid Analysis and Detection of Animal-related Risks) database, and analysed using descriptive and network approaches. A total of 61,937,855 pigs were moved through 872,493 movements of batches in England and Wales during the 5-year study period. Results show that the network exhibited scale-free and small-world topologies, indicating the potential for diseases to quickly spread within the pig industry. The findings also provide suggestions for how risk-based surveillance strategies could be optimised in the country by taking account of highly connected holdings, geographical regions and time periods with the greatest number of movements and pigs moved, as these are likely to be at higher risk for disease introduction. This study is also the first attempt to identify trade communities in the country, information which could be used to facilitate the pig trade and maintain disease-free status across the country in the event of an outbreak

Incidence and risk factors for poor ankle functional recovery, and the development and progression of posttraumatic ankle osteoarthritis after significant ankle ligament injury (SALI) : the SALI cohort study protocol

Background: Ankle sprains are one of the most common musculoskeletal injuries, accounting for up to 5% of all Emergency Department visits in the United Kingdom. Ankle injury may be associated with future ankle osteoarthritis. Up to 70% of ankle osteoarthritis cases may be associated with previous ankle injury. There is limited research regarding the association between ankle sprain and ankle osteoarthritis development. The current study aims to phenotype those who suffer significant ankle ligament injuries, identify potential risk factors for ankle injuries and subsequent poor recovery, examine why individuals may develop osteoarthritis, and what factors influence this chance. Methods: In this multicentre cohort study participants were recruited from nine Emergency Departments and two Urgent Care Centres in the United Kingdom. Participants (aged 18–70 years old) were defined as those who had suffered an isolated acute ankle sprain, which was Ottawa Ankle Rules positive, but negative for a significant ankle fracture on x-ray. Age and sex matched controls were also recruited. The controls were individuals who had not suffered a significant ankle injury, including ankle pain, function affected for more than 7 days, or the ankle caused them to report to an Emergency Department. Data is collected through a series of seven questionnaires (at baseline, 3 months, 1 year, 3 years, 5 years, 10 years, and 15 years later). The questionnaires include four sections (demographic questions; index injury, and injury history questions; functional assessment questions; and quality of life questions) and are designed to collect detailed information about the individual, their injury, potential risk factors for ankle sprains and ankle osteoarthritis, plus their medical history and any medication consumed. Discussion: The Significant Ankle Ligament Injury (SALI) study aims to add to the limited knowledge regarding which factors can predict ankle sprains, complaints, and osteoarthritis. This is important because despite ankle sprains being regarded as a benign injury that resolves quickly, residual symptoms are not uncommon months and years after the injury

Energy expenditure during egg laying is equal for early and late breeding free-living female great tits

In many bird populations, variation in the timing of reproduction exists but it is not obvious how this variation is maintained as timing has substantial fitness consequences. Daily energy expenditure (DEE) during the egg laying period increases with decreasing temperatures and thus perhaps only females that can produce eggs at low energetic cost will lay early in the season, at low temperatures. We tested whether late laying females have a higher daily energy expenditure during egg laying than early laying females in 43 great tits (Parus major), by comparing on the same day the DEE of early females late in their laying sequence with DEE of late females early in their egg laying sequence. We also validated the assumption that there are no within female differences in DEE within the egg laying sequence. We found a negative effect of temperature and a positive effect of female body mass on DEE but no evidence for differences in DEE between early and late laying females. However, costs incurred during egg laying may have carry-over effects later in the breeding cycle and if such carry-over effects differ for early and late laying females this could contribute to the maintenance of phenotypic variation in laying dates

Stage-Specific Inhibition of MHC Class I Presentation by the Epstein-Barr Virus BNLF2a Protein during Virus Lytic Cycle

gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8+ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8+ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (ΔBNLF2a) and compared the ability of ΔBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8+ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by ΔBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8+ T cells remained equally poor when presented by both wild-type and ΔBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet ΔBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle