Optimization of DNA Extraction from Human Milk

Background: Techniques are emerging for determining the best and most cost-effective way to test for human milk adulteration. Currently, the literature is focused on the use of qPCR testing, a technique used to isolate and amplify pieces of DNA for analysis. However, no recommendation currently exists on the best DNA extraction kit to use to achieve optimal DNA yield or purity from human milk samples for downstream qPCR use. Thus, the objective of this study is to assess and compare two DNA extraction kits for use with human milk samples for future DNA-based analysis in the testing for bovine milk adulteration in human milk. Methods: Forty mothers pumped human milk samples under the observation of a researcher using a brand-new hand pump. Eight unadulterated samples were then randomly chosen for DNA-extraction. The eight samples were thawed, pooled, and DNA was isolated using the Omega Bio-Tek\u27s E.Z.N.A.® Blood DNA Mini Spin Kit and the Norgen Biotek Corporation Plasma/Serum Circulating DNA Purification Mini Kit (Slurry Format) per the protocols included in the kits on arrival. An overnight incubation modification was also added to both kits to try to obtain optimal yield and purity. UV/VIS spectroscopy was used to determine DNA yield and purity using the ThermoFisher Scientific NanoDrop 2000TM 260/280 ratio, and a cost comparison was done between kits. Results: The Norgen kit with no modification provided 143% more DNA than the E.Z.N.A kit with no modifications. Similarly, the average nucleic acid yield was 134% greater when comparing the Norgen and E.Z.N.A kit with an overnight incubation. The Norgen kit provided a 17.0% greater 260/280 ratio and an 11.4% greater 260/280 ratio than the iii E.Z.N.A. kit, with and without modifications, respectively. The Norgen kit costs $2.37 more per extraction than the E.Z.N.A kit. Modifying both DNA extraction kits with an overnight incubation decreased the average nucleic acid yield and purity of the resulting DNA. Conclusion: From these results, the Norgen kit without overnight incubation is a better extraction kit for DNA extraction from raw human milk for both nucleic acid yield and purity. However, the EZNA kit costs less per extraction at$1.45 vs. $3.82. For extraction purposes, purity should be prioritized over nucleic acid yield because contaminants can compromise results and shorten the shelf-life of samples

Reaching older people with PA delivered in football clubs: the reach, adoption and implementation characteristics of the Extra Time Programme.

Background Older adults (OA) represent a core priority group for physical activity and Public Health policy. As a result, significant interest is placed on how to optimise adherence to interventions promoting these approaches. Extra Time (ET) is an example of a national programme of physical activity interventions delivered in professional football clubs for OA aged 55+ years. This paper aims to examine the outcomes from ET, and unpick the processes by which these outcomes were achieved. Methods This paper represents a secondary analysis of data collected during the evaluation of ET. From the 985 OA reached by ET, n=486 adopted the programme and completed post-intervention surveys (typically 12 weeks). We also draw on interview data with 18 ET participants, and 7 staff who delivered the programme. Data were subject to thematic analysis to generate overarching and sub themes. Results Of the 486 participants, the majority 95%, (n= 462) were White British and 59.7% (n=290) were female. Most adopters (65.4%/n=318) had not participated in previous interventions in the host clubs. Social interaction was the most frequently reported benefit of participation (77.2%, n=375). While the reach of the club badge was important in letting people know about the programme, further work enhanced adoption and satisfaction. These factors included (i) listening to participants, (ii) delivering a flexible age-appropriate programme of diverse physical and social activities, (iii) offering activities which satisfy energy drives and needs for learning and (iv) extensive opportunities for social engagement. Conclusions Findings emerging from this study indicate that physical activity and health interventions delivered through professional football clubs can be effective for engaging OA

Food assessment: a discursive analysis of diet talk in interviews with older men who are obese

Objective: Obesity rates are increasing faster in men than in women, with particular concerns raised regarding older men. However, men are less likely than women to engage in weight-loss activities such as dieting, typically constructed as a feminine practice. Previous research has argued that men’s food consumption is notably different and unhealthier than women’s. The novel contribution of this article is an analysis of food assessments in order to explore how older men (mostly) undergoing weight management programmes make sense of changes in their nutritional intake. Design: Semi-structured interviews were conducted with 30 men who were obese, 27 of whom were engaged in weight loss programmes. Discursive psychology was employed to analyse the data. Results: In contrast to other research, participants constructed nutritional advice as enlightening. Participants worked up ‘ownership’ and pleasure assessments to certain food choices which they contrasted with new, less calorific, eating practices. Moreover, new diets were constructed as acceptable. Conclusion: Our study contributes new insights about how nutritional advice impacts upon preconceived (mis)understandings of healthy eating practices. During the interviews, men positioned themselves as educators – health promoters might usefully develop nutritional advice in collaboration with men who have successfully changed their diets for optimum effect

School day intervention opportunities for increasing 7-11 year old children?s moderate to vigorous physical activity

The development and maintenance of healthy physical activity behaviours from an early age is a priority for public health in the UK. Schools provide a number of different opportunities (time inside and outside of the curriculum) and resources in the form of space, equipment and staff for children to learn and develop healthy behaviours, at a time when they are most receptive to behaviour change. The overall aim of the thesis was to identify the different opportunities within the school context whereby children could be physically active and use theoretically driven, whole school interventions to optimise and subsequently increase healthful physical activity. Study 1 demonstrated that primary schools wishing to use pedometers within their curricula can be confident that the EZ-V model is sufficiently accurate to measure physical activity in the form of steps taken (r=0.897). Using the EZ-V pedometer, Study 2 demonstrated that feedback from pedometers along with information on how children can be physically active during the school day, can significantly increase children's mean daily steps-min"' compared to feedback alone or control groups over the course of a school week. Furthermore, boys were significantly more active than girls across each treatment group. Study 3 explored the affect of the primary school travel plan (TP) on the moderate to vigorous physical activity (MVPA) of 7-11 year olds during the winter and summer season. In order to examine the impact of the TP, schools were separated into schools deemed to have an Established TP (implemented for at least 2 years) or `New' schools (i. e., had just drafted their TP and were in the first year of its implementation). Children in the New TP schools accumulated 7.24 (winter) and 24.11 (summer) more minutes of MVPA (5.2% and 15.66% respectively) throughout the day compared with those in the Established TP school children (F(1,35=) 0.955, p=0.207, d=0.33). Overall, children were more active during the summer by 7.81 minutes (F(I, 35) = 0.089, p=0.768, d=0.1). The final study examined the affect of a 12 week, multi-component, whole school intervention which aimed to increase children's school day MVPA. Following baseline physical activity measures via accelerometry, intervention components consisting of a Health Week, Playtime Pals and a Pedometer Challenge were delivered sequentially over the first 6 weeks. Subsequent accelerometer data were collected after each intervention was delivered at 2,4,6 and 12 weeks. Results showed that from baseline to follow-up, children increased their MVPA by 6.57 minutes during the school day, which according to the Q statistic was likely to be beneficial. Results from the Pedometer Challenge found that boy s' mean pedometer steps"day-' were significantly higher than girls' (F(I, 95)= 9.987, p=0.002, d=0.65) and overall, mean pedometer steps-day"' significantly increased from week one to week five (F(,, 93)= 5.845, p=0.018, d=0.24). When the lowest and highest active 50% groups were compared, children in the lowest active 50% group significantly increased their steps from week one to week five (F(l, 47)= 20.847, p=0.000, d=0.93), while the highest active 50% did not (F(1,47)= 0.000, p=0.990, d=0). Furthermore, boys in the highest active 50% group were found to accumulate significantly more steps than the girls, in the highest active 50% group (F(I, 46)= 14.701, p=0.000, d=0.81), while there was no significant difference between the boys' and girls' pedometer steps in the lowest 50% group (F(l, 46)= 0.456, p=0.503, d=0.14). The overall findings of the thesis suggest that schools can successfully optimise the different opportunities during the school day in order to increase children's physical activity, but that larger, controlled and longitudinally designed studies are needed to confirm cause and effect. Most importantly, these changes may have most impact in the least active boys and girls. Interventions such as this should therefore be targeted at the least active children to ensure that they benefit as much as possible from the opportunity to increase their daily physical activity

Physical Activity and Sedentary Behaviour Clustering: Segmentation to Optimise Active Lifestyles.

Background: Increasingly the health impacts of physical inactivity are being distinguished from those of sedentary behaviour. Nevertheless, deleterious health prognoses occur when these behaviours combine, making it a Public Health priority to establish the numbers and salient identifying factors of people who live with this injurious combination. Method: Using an observational between-subjects design, a non-probability sample of 22,836 participants provided data on total daily activity. A 2-step hierarchical cluster analysis identified the optimal number of clusters and the subset of distinguishing variables. Univariate analyses assessed significant cluster differences. Results: High levels of sitting clustered with low physical activity. The ‘Ambulatory & Active’ cluster (n=6,254) sat for 2.5 to 5 h d-1 and were highly active. They were significantly younger, included a greater proportion of males and reported low Indices of Multiple Deprivation compared to other clusters. Conversely, the ‘Sedentary & Low Active’ cluster (n=6,286) achieved ≤60 MET.min.wk-1 of physical activity and sat for ≥8 h d-1. They were the oldest cluster, housed the largest proportion of females and reported moderate Indices of Multiple Deprivation. Conclusions: Public Health systems may benefit from developing policy and interventions that do more to limit sedentary behaviour and encourage light intensity activity in its place

Photoelectrochemistry of Photosystem II in Vitro vs in Vivo.

Factors governing the photoelectrochemical output of photosynthetic microorganisms are poorly understood, and energy loss may occur due to inefficient electron transfer (ET) processes. Here, we systematically compare the photoelectrochemistry of photosystem II (PSII) protein-films to cyanobacteria biofilms to derive: (i) the losses in light-to-charge conversion efficiencies, (ii) gains in photocatalytic longevity, and (iii) insights into the ET mechanism at the biofilm interface. This study was enabled by the use of hierarchically structured electrodes, which could be tailored for high/stable loadings of PSII core complexes and Synechocystis sp. PCC 6803 cells. The mediated photocurrent densities generated by the biofilm were 2 orders of magnitude lower than those of the protein-film. This was partly attributed to a lower photocatalyst loading as the rate of mediated electron extraction from PSII in vitro is only double that of PSII in vivo. On the other hand, the biofilm exhibited much greater longevity (>5 days) than the protein-film (<6 h), with turnover numbers surpassing those of the protein-film after 2 days. The mechanism of biofilm electrogenesis is suggested to involve an intracellular redox mediator, which is released during light irradiation

Hair cells use active zones with different voltage dependence of Ca²⁺ influx to decompose sounds into complementary neural codes.

For sounds of a given frequency, spiral ganglion neurons (SGNs) with different thresholds and dynamic ranges collectively encode the wide range of audible sound pressures. Heterogeneity of synapses between inner hair cells (IHCs) and SGNs is an attractive candidate mechanism for generating complementary neural codes covering the entire dynamic range. Here, we quantified active zone (AZ) properties as a function of AZ position within mouse IHCs by combining patch clamp and imaging of presynaptic Ca2+ influx and by immunohistochemistry. We report substantial AZ heterogeneity whereby the voltage of half-maximal activation of Ca2+ influx ranged over ∼20 mV. Ca2+ influx at AZs facing away from the ganglion activated at weaker depolarizations. Estimates of AZ size and Ca2+ channel number were correlated and larger when AZs faced the ganglion. Disruption of the deafness gene GIPC3 in mice shifted the activation of presynaptic Ca2+ influx to more hyperpolarized potentials and increased the spontaneous SGN discharge. Moreover, Gipc3 disruption enhanced Ca2+ influx and exocytosis in IHCs, reversed the spatial gradient of maximal Ca2+ influx in IHCs, and increased the maximal firing rate of SGNs at sound onset. We propose that IHCs diversify Ca2+ channel properties among AZs and thereby contribute to decomposing auditory information into complementary representations in SGNs

Reaching older people with PA delivered in football clubs: the reach, adoption and implementation characteristics of the Extra Time Programme

Background: Older adults (OA) represent a core priority group for physical activity and Public Health policy. As a result, significant interest is placed on how to optimise adherence to interventions promoting these approaches. Extra Time (ET) is an example of a national programme of physical activity interventions delivered in professional football clubs for OA aged 55+ years. This paper aims to examine the outcomes from ET, and unpick the processes by which these outcomes were achieved. Methods: This paper represents a secondary analysis of data collected during the evaluation of ET. From the 985 OA reached by ET, n=486 adopted the programme and completed post-intervention surveys (typically 12 weeks). We also draw on interview data with 18 ET participants, and 7 staff who delivered the programme. Data were subject to thematic analysis to generate overarching and sub themes. Results: Of the 486 participants, the majority 95%, (n= 462) were White British and 59.7% (n=290) were female. Most adopters (65.4%/n=318) had not participated in previous interventions in the host clubs. Social interaction was the most frequently reported benefit of participation (77.2%, n=375). While the reach of the club badge was important in letting people know about the programme, further work enhanced adoption and satisfaction. These factors included (i) listening to participants, (ii) delivering a flexible age-appropriate programme of diverse physical and social activities, (iii) offering activities which satisfy energy drives and needs for learning and (iv) extensive opportunities for social engagement. Conclusions: Findings emerging from this study indicate that physical activity and health interventions delivered through professional football clubs can be effective for engaging OA