Development of a method to quantify the DNA content in DNA- cationic peptide nanoparticles

Gene therapy has the potential to provide safe and targeted therapies for a variety of diseases. A range of intracellular gene delivery vehicles have been proposed for this purpose. Non-viral vectors are a particularly attractive option and among them cationic peptides have emerged as promising candidates. For the pharmaceutical formulation and application to clinical studies it is necessary to quantify the amount of pDNA condensed with the delivery system. There is a severe deficiency in this area, thus far no methods have been reported specifically for pDNA condensed with cationic peptide to form nanoparticles. The current study seeks to address this and describes the evaluation of a range of disruption agents to extract DNA from nanoparticles formed by condensation with cationic fusogenic peptides RALA and KALA. Only proteinase K exhibited efficient and reproducible results and compatibility with the PicoGreen reagent based quantification assay. Thus we report for the first time a simple and reliable method that can quantify the pDNA content in pDNA cationic peptide nanoparticles.

A randomised controlled trial of increasing fruit and vegetable intake and how this influences the carotenoid concentration and activities of PON-1 and LCAT in HDL from subjects with type 2 diabetes

Background

High density lipoproteins (HDL) have many cardioprotective roles; however, in subjects with type 2 diabetes (T2D) these cardioprotective properties are diminished. Conversely, increased fruit and vegetable (F&V) intake may reduce cardiovascular disease risk, although direct trial evidence of a mechanism by which this occurs in subjects with T2D is lacking. Therefore, the aim of this study was to examine if increased F&V consumption influenced the carotenoid content and enzymes associated with the antioxidant properties of HDL in subjects with T2D.

Eighty obese subjects with T2D were randomised to a 1- or ≥6-portion/day F&V diet for 8-weeks. Fasting serum was collected pre- and post-intervention. HDL was subfractionated into HDL₂ and HDL₃ by rapid ultracentrifugation. Carotenoids were measured in serum, HDL₂ and HDL₃ by high performance liquid chromatography. The activity of paraoxonase-1 (PON-1) was measured in serum, HDL₂ and HDL₃ by a spectrophotometric assay, while the activity of lecithin cholesterol acyltransferase (LCAT) was measured in serum, HDL₂ and HDL₃ by a fluorometric assay.

In the ≥6- vs. 1-portion post-intervention comparisons, carotenoids increased in serum, HDL₂ and particularly HDL₃, (α-carotene, p = 0.008; β-cryptoxanthin, p = 0.042; lutein, p = 0.012; lycopene, p = 0.016), as did the activities of PON-1 and LCAT in HDL₃ (p = 0.006 and 0.044, respectively).

To our knowledge, this is the first study in subjects with T2D to demonstrate that increased F&V intake augmented the carotenoid content and influenced enzymes associated with the antioxidant properties of HDL. We suggest that these changes would enhance the cardioprotective properties of this lipoprotein.

Effect of carbohydrate type and concentration on polyhydroxy alcohol and trehalose content of conidia of three entomopathogenic fungi

The entomopathogenic fungi Beauveria bassiana, Metarhizium anisopliae and Paecilomyces farinosus were cultured on solid agar media containing different carbohydrate components (glycerol, glucose, trehalose or starch) at concentrations of ≤ 142.7 g added carbon 1-1 for 30 d at 25°C. The water activity (a(w)) of the media ranged from 0.925 to 0.998. Growth of M. anisopliae and P. farinosus was stimulated between 0.975 and 0.995 a(w) on glucose media and that of P. farinosus at 0. 975 a(w) on glycerol media. At < 0.970 a(w), growth of each fungal species was significantly reduced (P < 0.05). Polyhydroxy alcohols (polyols) and trehalose were extracted from conidia produced on different media and quantified using HPLC. Total polyol content of conidia produced on glucose media varied between 5.2 and 52.2 mg g-1 for B. bassiana, 77.3 and 90.3 mg g-1 for M. anisopliae, and 26.7 and 76.1 mg g-1 for P. farinosus. The amounts of specific polyols in conidia varied significantly from media of different glucose concentrations. Mannitol was the predominant polyol in conidia of all three species, with conidia of M. anisopliae, for example, containing as much as 75.2 mg mannitol g-1 when cultured on glucose media. The amount of the lower molecular mass polyols glycerol and erythritol was greater in conidia produced on glucose media with > 50.0 g added carbon 1-1 than that in conidia produced at lower glucose concentrations. Conidia contained between 10.8 and 20.8 mg glycerol plus erythritol g-1 on glucose media with 142.7 g added carbon 1-1, depending on species. Conversely, conidia of B. bassiana and P. farinosus contained maximum amounts of trehalose ( ≤ 23.5 mg g-1) when produced on glucose media with < 50.0 g added carbon l-1, and trehalose content was considerably less at higher glucose concentrations. There were accumulations of glycerol and erythritol in conidia of all three species when grown on glycerol media with > 25.0 g added carbon 1-1; conidia of B. bassiana contained up to 154.0 mg glycerol plus erythritol g-1. hen B. bassiana and P. farinosus were grown on trehalose media, conidia contained up to 222.1 mg trehalose g-1. By contrast, conidia of M. anisopliae contained < 17.0 mg trehalose g-1 under all conditions tested. The water availability of solutions of different polyols is discussed in relation to their potential to act in osmotic adjustment during germination. The ability to manipulate polyol and trehalose content of fungal propagules may be critical in enhancing the storage life and efficacy of biological control agents.

Implications of FPS1 deletion and membrane ergosterol content for glycerol efflux from Saccharomyces cerevisiae

The deletion of the gene encoding the glycerol facilitator Fps1p was associated with an altered plasma membrane lipid composition in Saccharomyces cerevisiae. The S. cerevisiae fps1delta strain respectively contained 18 and 26% less ergosterol than the wild-type strain, at the whole-cell level and at the plasma membrane level. Other mutants with deficiencies in glycerol metabolism were studied to investigate any possible link between membrane ergosterol content and intracellular glycerol accumulation. In these mutants a modification in intracellular glycerol concentration, or in intra- to extracellular glycerol ratio was accompanied by a reduction in plasma membrane ergosterol content. However, there was no direct correlation between ergosterol content and intracellular glycerol concentration. Lipid composition influences the membrane permeability for solutes during adaptation of yeast cells to osmotic stress. In this study, ergosterol supplementation was shown to partially suppress the hypo-osmotic sensitivity phenotype of the fps1delta strain, leading to more efficient glycerol efflux, and improved survival. The erg-1 disruption mutant, which is unable to synthesise ergosterol, survived and recovered from the hypo-osmotic shock more successfully when the concentration of exogenously supplied ergosterol was increased. The results obtained suggest that a higher ergosterol content facilitates the flux of glycerol across the plasma membrane of S. cerevisiae cells.

High content analysis: A sensitive tool to detect and quantify the cytotoxic, synergistic and antagonistic effects of chemical contaminants in foods

Aflatoxin B1 (AFB1), ochratoxin A (OTA) and fumonisin B1 (FB1) are important mycotoxins in terms of
human exposure via food, their toxicity and regulatory limits that exist worldwide. Mixtures of toxins can frequently be present in foods, however due to the complications of determining their combined toxicity,
legal limits of exposure are determined for single compounds, based on long standing toxicological
techniques. High content analysis (HCA) may be a useful tool to determine total toxicity of complex
mixtures of mycotoxins. Endpoints including cell number (CN), nuclear intensity (NI), nuclear area (NA),
plasma membrane permeability (PMP), mitochondrial membrane potential (MMP) and mitochondrial
mass (MM) were compared to the conventional 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium
bromide (MTT) and neutral red (NR) endpoints in MDBK cells. Individual concentrations of each
mycotoxin (OTA 3mg/ml, FB1 8mg/ml and AFB11.28mg/ml) revealed no cytotoxicity with MTTor NR but
HCA showed significant cytotoxic effects up to 41.6% (p0.001) and 10.1% (p0.05) for OTA and AFB1,
respectively. The tertiary mixture (OTA 3mg/ml, FB1 8mg/ml and AFB1 1.28mg/ml) detected up to 37.3%
and 49.8% more cytotoxicity using HCA over MTT and NR, respectively. Whilst binary combinations of
OTA (3mg/ml) and FB1 (8mg/ml) revealed synergistic interactions using HCA (MMP, MM, NI endpoints)
not detected using MTT or NR. HCA is a highly novel and sensitive tool that could substantially help
determine future regulatory limits, for single and combined toxins present in food, ensuring legislation is based on true risks to human health exposure.

Knowledge translation in men’s health research: development and delivery of content for use online

Background: Men can be hard to reach with face-to-face health-related information, while increasingly, research shows that they are seeking health information from online sources. Recognizing this trend, there is merit in developing innovative online knowledge translation (KT) strategies capable of translating research on men’s health into engaging health promotion materials. While the concept of KT has become a new mantra for researchers wishing to bridge the gap between research evidence and improved health outcomes, little is written about the process, necessary skills, and best practices by which researchers can develop online knowledge translation.
Objective: Our aim was to illustrate some of the processes and challenges involved in, and potential value of, developing research knowledge online to promote men’s health.

Methods: We present experiences of KT across two case studies of men’s health. First, we describe a study that uses interactive Web apps to translate knowledge relating to Canadian men’s depression. Through a range of mechanisms, study findings were repackaged with the explicit aim of raising awareness and reducing the stigma associated with men’s depression and/or help-seeking. Second, we describe an educational resource for teenage men about unintended pregnancy, developed for delivery in the formal Relationship and Sexuality Education school curricula of Ireland, Northern Ireland (United Kingdom), and South Australia. The intervention is based around a Web-based interactive film drama entitled “If I Were Jack”.

Results: For each case study, we describe the KT process and strategies that aided development of credible and well-received online content focused on men’s health promotion. In both case studies, the original research generated the inspiration for the interactive online content and the core development strategy was working with a multidisciplinary team to develop this material through arts-based approaches. In both cases also, there is an acknowledgment of the need for gender and culturally sensitive information. Both aimed to engage men by disrupting stereotypes about men, while simultaneously addressing men through authentic voices and faces. Finally, in both case studies we draw attention to the need to think beyond placement of content online to delivery to target audiences from the outset.

Conclusions: The case studies highlight some of the new skills required by academics in the emerging paradigm of translational research and contribute to the nascent literature on KT. Our approach to online KT was to go beyond dissemination and diffusion to actively repackage research knowledge through arts-based approaches (videos and film scripts) as health promotion tools, with optimal appeal, to target male audiences. Our findings highlight the importance of developing a multidisciplinary team to inform the design of content, the importance of adaptation to context, both in terms of the national implementation context and consideration of gender-specific needs, and an integrated implementation and evaluation framework in all KT work.

Online health information - what the newspapers tell their readers: a systematic content analysis

Background: This study investigated the nature of newspaper reporting about online health information in the UK and US. Internet users frequently search for health information online, although the accuracy of the information retrieved varies greatly and can be misleading. Newspapers have the potential to influence public health behaviours, but information has been lacking in relation to how newspapers portray online health information to their readers.

Methods: The newspaper database Nexis (R) UK was searched for articles published from 2003 - 2012 relating to online health information. Systematic content analysis of articles published in the highest circulation newspapers in the UK and US was performed. A second researcher coded a 10% sample to establish inter-rater reliability of coding.

Results: In total, 161 newspaper articles were included in the analysis. Publication was most frequent in 2003, 2008 and 2009, which coincided with global threats to public health. UK broadsheet newspapers were significantly more likely to cover online health information than UK tabloid newspapers (p = 0.04) and only one article was identified in US tabloid newspapers. Articles most frequently appeared in health sections. Among the 79 articles that linked online health information to specific diseases or health topics, diabetes was the most frequently mentioned disease, cancer the commonest group of diseases and sexual health the most frequent health topic. Articles portrayed benefits of obtaining online health information more frequently than risks. Quotations from health professionals portrayed mixed opinions regarding public access to online health information. 108 (67.1%) articles directed readers to specific health-related web sites. 135 (83.9%) articles were rated as having balanced judgement and 76 (47.2%) were judged as having excellent quality reporting. No difference was found in the quality of reporting between UK and US articles.

Conclusions: Newspaper coverage of online health information was low during the 10-year period 2003 to 2012. Journalists tended to emphasise the benefits and understate the risks of online health information and the quality of reporting varied considerably. Newspapers directed readers to sources of online health information during global epidemics although, as most articles appeared in the health sections of broadsheet newspapers, coverage was limited to a relatively small readership.

Defining the content and delivery of an intervention to Change AdhereNce to treatment in BonchiEctasis (CAN-BE): a qualitative approach incorporating the Theoretical Domains Framework, behavioural change techniques and stakeholder expert panels

Background
Low patient adherence to treatment is associated with poorer health outcomes in bronchiectasis. We sought to use the Theoretical Domains Framework (TDF) (a framework derived from 33 psychological theories) and behavioural change techniques (BCTs) to define the content of an intervention to change patients’ adherence in bronchiectasis (Stage 1 and 2) and stakeholder expert panels to define its delivery (Stage 3).

Methods
We conducted semi-structured interviews with patients with bronchiectasis about barriers and motivators to adherence to treatment and focus groups or interviews with bronchiectasis healthcare professionals (HCPs) about their ability to change patients’ adherence to treatment. We coded these data to the 12 domain TDF to identify relevant domains for patients and HCPs (Stage 1). Three researchers independently mapped relevant domains for patients and HCPs to a list of 35 BCTs to identify two lists (patient and HCP) of potential BCTs for inclusion (Stage 2). We presented these lists to three expert panels (two with patients and one with HCPs/academics from across the UK). We asked panels who the intervention should target, who should deliver it, at what intensity, in what format and setting, and using which outcome measures (Stage 3).

Results
Eight TDF domains were perceived to influence patients’ and HCPs’ behaviours: Knowledge, Skills, Beliefs about capability, Beliefs about consequences, Motivation, Social influences, Behavioural regulation and Nature of behaviours (Stage 1). Twelve BCTs common to patients and HCPs were included in the intervention: Monitoring, Self-monitoring, Feedback, Action planning, Problem solving, Persuasive communication, Goal/target specified:behaviour/outcome, Information regarding behaviour/outcome, Role play, Social support and Cognitive restructuring (Stage 2). Participants thought that an individualised combination of these BCTs should be delivered to all patients, by a member of staff, over several one-to-one and/or group visits in secondary care. Efficacy should be measured using pulmonary exacerbations, hospital admissions and quality of life (Stage 3).

Conclusions
Twelve BCTs form the intervention content. An individualised selection from these 12 BCTs will be delivered to all patients over several face-to-face visits in secondary care. Future research should focus on developing physical materials to aid delivery of the intervention prior to feasibility and pilot testing. If effective, this intervention may improve adherence and health outcomes for those with bronchiectasis in the future.

The Role of Water Content and Paste Proportion on Physico-mechanical Properties of Alkali Activated Fly Ash–Ggbs Concrete

The growth of the construction industry worldwide poses a serious concern on the sustainability of the building material production chain, mainly due to the carbon emissions related to the production of Portland cement. On the other hand, valuable materials from waste streams, particularly from the metallurgical industry, are not used at their full potential. Alkali activated concrete (AAC) has emerged in the last years as a promising alternative to traditional Portland cement based concrete for some applications. However, despite showing remarkable strength and durability potential, its utilisation is not widespread, mainly due to the lack of broadly accepted standards for the selection of suitable mix recipes fulfilling design requirements, in particular workability, setting time and strength. In this paper, a contribution towards the design development of AAC synthetized from pulverised fuel ash (60%) and ground granulated blast furnace slag (40%) activated with a solution of sodium hydroxide and sodium silicate is proposed. Results from a first batch of mixes indicated that water content influences the setting time and that paste content is a key parameter for controlling strength development and workability. The investigation indicated that, for the given raw materials and activator compositions, a minimum water to solid (w/s) ratio of 0.37 was needed for an initial setting time of about 1 hour. Further work with paste content in the range of 30% to 33% determined the relationship between workability and strength development and w/s ratio and paste content. Strengths in the range of 50 - 60 MPa were achieved.

Acute Cocoa Supplementation Increases Postprandial HDL Cholesterol and Insulin in Obese Adults with Type 2 Diabetes after Consumption of a High-Fat Breakfast

BACKGROUND: Dietary cocoa is an important source of flavonoids and is associated with favorable cardiovascular disease effects, such as improvements in vascular function and lipid profiles, in nondiabetic adults. Type 2 diabetes (T2D) is associated with adverse effects on postprandial serum glucose, lipids, inflammation, and vascular function.

OBJECTIVE: We examined the hypothesis that cocoa reduces metabolic stress in obese T2D adults after a high-fat fast-food-style meal.

METHODS: Adults with T2D [n = 18; age (means ± SEs): 56 ± 3 y; BMI (in kg/m(2)): 35.3 ± 2.0; 14 women; 4 men) were randomly assigned to receive cocoa beverage (960 mg total polyphenols; 480 mg flavanols) or flavanol-free placebo (110 mg total polyphenols; <0.1 mg flavanols) with a high-fat fast-food-style breakfast [766 kcal, 50 g fat (59% energy)] in a crossover trial. After an overnight fast (10-12 h), participants consumed the breakfast with cocoa or placebo, and blood sample collection [glucose, insulin, lipids, and high-sensitivity C-reactive protein (hsCRP)] and vascular measurements were conducted at 0.5, 1, 2, 4, and 6 h postprandially on each study day. Insulin resistance was evaluated by homeostasis model assessment.

RESULTS: Over the 6-h study, and specifically at 1 and 4 h, cocoa increased HDL cholesterol vs. placebo (overall Δ: 1.5 ± 0.8 mg/dL; P ≤ 0.01) but had no effect on total and LDL cholesterol, triglycerides, glucose, and hsCRP. Cocoa increased serum insulin concentrations overall (Δ: 5.2 ± 3.2 mU/L; P < 0.05) and specifically at 4 h but had no overall effects on insulin resistance (except at 4 h, P < 0.05), systolic or diastolic blood pressure, or small artery elasticity. However, large artery elasticity was overall lower after cocoa vs. placebo (Δ: -1.6 ± 0.7 mL/mm Hg; P < 0.05), with the difference significant only at 2 h.

CONCLUSION: Acute cocoa supplementation showed no clear overall benefit in T2D patients after a high-fat fast-food-style meal challenge. Although HDL cholesterol and insulin remained higher throughout the 6-h postprandial period, an overall decrease in large artery elasticity was found after cocoa consumption. This trial was registered at clinicaltrials.gov as NCT01886989.

Content Validity in Landscape Valuation: A transdisciplinary approach to valuing the landscape of the peninsula of Sorrento, Italy.

This paper addresses the representation of landscape complexity in stated preferences research. It integrates landscape ecology and landscape economics and conducts the landscape analysis in a three-dimensional space to provide ecologically meaningful quantitative landscape indicators that are used as variables for the monetary valuation of landscape in a stated preferences study. Expected heterogeneity in taste intensity across respondents is addressed with a mixed logit model in Willingness to Pay space. The results suggest that the integration of landscape ecology metrics in a stated preferences model provides useful insights for valuing landscape and landscape changes