Gene electrotransfer into murine skeletal muscle: A systematic analysis of parameters for long-term gene expression

Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57BI/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57BI/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 μg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.

Modelling total duration of traffic incidents including incident detection and recovery time

Traffic incidents are key contributors to non-recurrent congestion, potentially generating significant delay. Factors that influence the duration of incidents are important to understand so that effective mitigation strategies can be implemented. To identify and quantify the effects of influential factors, a methodology for studying total incident duration based on historical data from an ‘integrated database’ is proposed. Incident duration models are developed using a selected freeway segment in the Southeast Queensland, Australia network. The models include incident detection and recovery time as components of incident duration. A hazard-based duration modelling approach is applied to model incident duration as a function of a variety of factors that influence traffic incident duration. Parametric accelerated failure time survival models are developed to capture heterogeneity as a function of explanatory variables, with both fixed and random parameters specifications. The analysis reveals that factors affecting incident duration include incident characteristics (severity, type, injury, medical requirements, etc.), infrastructure characteristics (roadway shoulder availability), time of day, and traffic characteristics. The results indicate that event type durations are uniquely different, thus requiring different responses to effectively clear them. Furthermore, the results highlight the presence of unobserved incident duration heterogeneity as captured by the random parameter models, suggesting that additional factors need to be considered in future modelling efforts.

The impact of mobile phone distraction on the braking behaviour of young drivers : a hazard-based duration model

Braking is a crucial driving task with a direct relationship with crash risk, as both excess and inadequate braking can lead to collisions. The objective of this study was to compare the braking profile of young drivers distracted by mobile phone conversations to non-distracted braking. In particular, the braking behaviour of drivers in response to a pedestrian entering a zebra crossing was examined using the CARRS-Q Advanced Driving Simulator. Thirty-two licensed drivers drove the simulator in three phone conditions: baseline (no phone conversation), hands-free, and handheld. In addition to driving the simulator, each participant completed questionnaires related to driver demographics, driving history, usage of mobile phones while driving, and general mobile phone usage history. The drivers were 18–26 years old and split evenly by gender. A linear mixed model analysis of braking profiles along the roadway before the pedestrian crossing revealed comparatively increased decelerations among distracted drivers, particularly during the initial 20 kph of deceleration. Drivers’ initial 20 kph deceleration time was modelled using a parametric accelerated failure time (AFT) hazard-based duration model with a Weibull distribution with clustered heterogeneity to account for the repeated measures experiment design. Factors found to significantly influence the braking task included vehicle dynamics variables like initial speed and maximum deceleration, phone condition, and driver-specific variables such as licence type, crash involvement history, and self-reported experience of using a mobile phone whilst driving. Distracted drivers on average appear to reduce the speed of their vehicle faster and more abruptly than non-distracted drivers, exhibiting excess braking comparatively and revealing perhaps risk compensation. The braking appears to be more aggressive for distracted drivers with provisional licenses compared to drivers with open licenses. Abrupt or excessive braking by distracted drivers might pose significant safety concerns to following vehicles in a traffic stream.

Mismatch negativity in recent-onset and chronic schizophrenia : a current source density analysis

Mismatch negativity (MMN) is a component of the event-related potential elicited by deviant auditory stimuli. It is presumed to index pre-attentive monitoring of changes in the auditory environment. MMN amplitude is smaller in groups of individuals with schizophrenia compared to healthy controls. We compared duration-deviant MMN in 16 recent-onset and 19 chronic schizophrenia patients versus age- and sex-matched controls. Reduced frontal MMN was found in both patient groups, involved reduced hemispheric asymmetry, and was correlated with Global Assessment of Functioning (GAF) and negative symptom ratings. A cortically-constrained LORETA analysis, incorporating anatomical data from each individual's MRI, was performed to generate a current source density model of the MMN response over time. This model suggested MMN generation within a temporal, parietal and frontal network, which was right hemisphere dominant only in controls. An exploratory analysis revealed reduced CSD in patients in superior and middle temporal cortex, inferior and superior parietal cortex, precuneus, anterior cingulate, and superior and middle frontal cortex. A region of interest (ROI) analysis was performed. For the early phase of the MMN, patients had reduced bilateral temporal and parietal response and no lateralisation in frontal ROIs. For late MMN, patients had reduced bilateral parietal response and no lateralisation in temporal ROIs. In patients, correlations revealed a link between GAF and the MMN response in parietal cortex. In controls, the frontal response onset was 17 ms later than the temporal and parietal response. In patients, onset latency of the MMN response was delayed in secondary, but not primary, auditory cortex. However amplitude reductions were observed in both primary and secondary auditory cortex. These latency delays may indicate relatively intact information processing upstream of the primary auditory cortex, but impaired primary auditory cortex or cortico-cortical or thalamo-cortical communication with higher auditory cortices as a core deficit in schizophrenia.

A cost-effectiveness analysis of a telephone-linked care intervention for individuals with Type 2 diabetes

AIM: To assess the cost-effectiveness of an automated telephone-linked care intervention, Australian TLC Diabetes, delivered over 6 months to patients with established Type 2 diabetes mellitus and high glycated haemoglobin level, compared to usual care. METHODS: A Markov model was designed to synthesize data from a randomized controlled trial of TLC Diabetes (n=120) and other published evidence. The 5-year model consisted of three health states related to glycaemic control: 'sub-optimal' HbA1c ≥58mmol/mol (7.5%); 'average' ≥48-57mmol/mol (6.5-7.4%) and 'optimal' <48mmol/mol (6.5%) and a fourth state 'all-cause death'. Key outcomes of the model include discounted health system costs and quality-adjusted life years (QALYS) using SF-6D utility weights. Univariate and probabilistic sensitivity analyses were undertaken. RESULTS: Annual medication costs for the intervention group were lower than usual care [Intervention: £1076 (95%CI: £947, £1206) versus usual care £1271 (95%CI: £1115, £1428) p=0.052]. The estimated mean cost for intervention group participants over five years, including the intervention cost, was £17,152 versus £17,835 for the usual care group. The corresponding mean QALYs were 3.381 (SD 0.40) for the intervention group and 3.377 (SD 0.41) for the usual care group. Results were sensitive to the model duration, utility values and medication costs. CONCLUSION: The Australian TLC Diabetes intervention was a low-cost investment for individuals with established diabetes and may result in medication cost-savings to the health system. Although QALYs were similar between groups, other benefits arising from the intervention should also be considered when determining the overall value of this strategy.

Acoustic identification of 12 species of echolocating bat by discriminant function analysis and artificial neural networks

We recorded echolocation calls from 14 sympatric species of bat in Britain. Once digitised, one temporal and four spectral features were measured from each call. The frequency-time course of each call was approximated by fitting eight mathematical functions, and the goodness of fit, represented by the mean-squared error, was calculated. Measurements were taken using an automated process that extracted a single call from background noise and measured all variables without intervention. Two species of Rhinolophus were easily identified from call duration and spectral measurements. For the remaining 12 species, discriminant function analysis and multilayer back-propagation perceptrons were used to classify calls to species level. Analyses were carried out with and without the inclusion of curve-fitting data to evaluate its usefulness in distinguishing among species. Discriminant function analysis achieved an overall correct classification rate of 79% with curve-fitting data included, while an artificial neural network achieved 87%. The removal of curve-fitting data improved the performance of the discriminant function analysis by 2 %, while the performance of a perceptron decreased by 2 %. However, an increase in correct identification rates when curve-fitting information was included was not found for all species. The use of a hierarchical classification system, whereby calls were first classified to genus level and then to species level, had little effect on correct classification rates by discriminant function analysis but did improve rates achieved by perceptrons. This is the first published study to use artificial neural networks to classify the echolocation calls of bats to species level. Our findings are discussed in terms of recent advances in recording and analysis technologies, and are related to factors causing convergence and divergence of echolocation call design in bats.

Echolocation calls of the long tailed bat: a quantitative analysis of types of calls

The echolocation calls of long-tailed bats (Chalinolobus tuberculatus) were recorded in the Eglinton Valley, Fjordland, New Zealand, and digitized for analysis with the signal-processing software. Univariate and multivariate analyses of measure features facilitated a quantitative classification of the calls. Cluster analysis was used to categorize calls into two groups equating to search and terminal buzz calls described qualitatively for other species. When moving from search to terminal phases, the calls decrease in bandwidth, maximum and minimum frequency of call, and duration. Search calls begin with a steep-downward FM sweep followed by a short, less-modulated component. Buzz calls are FM sweeps. Although not found quantitatively, a broad pre-buzz group of calls also was identified. Ambiguity analysis of calls from the three groups shows that search-phrase calls are well suited to resolving the velocity of targets, and hence, identifying moving targets in a stationary clutter. Pre-buzz and buzz calls are better suited to resolving range, a feature that may aid the bats in capture of evasive prey after it has been identified.

Acoustic identification of twelve species of echolocating bat by discriminant function analysis and artificial neural networks

We recorded echolocation calls from 14 sympatric species of bat in Britain. Once digitised, one temporal and four spectral features were measured from each call. The frequency-time course of each call was approximated by fitting eight mathematical functions, and the goodness of fit, represented by the mean-squared error, was calculated. Measurements were taken using an automated process that extracted a single call from background noise and measured all variables without intervention. Two species of Rhinolophus were easily identified from call duration and spectral measurements. For the remaining 12 species, discriminant function analysis and multilayer back-propagation perceptrons were used to classify calls to species level. Analyses were carried out with and without the inclusion of curve-fitting data to evaluate its usefulness in distinguishing among species. Discriminant function analysis achieved an overall correct classification rate of 79% with curve-fitting data included, while an artificial neural network achieved 87%. The removal of curve-fitting data improved the performance of the discriminant function analysis by 2 %, while the performance of a perceptron decreased by 2 %. However, an increase in correct identification rates when curve-fitting information was included was not found for all species. The use of a hierarchical classification system, whereby calls were first classified to genus level and then to species level, had little effect on correct classification rates by discriminant function analysis but did improve rates achieved by perceptrons. This is the first published study to use artificial neural networks to classify the echolocation calls of bats to species level. Our findings are discussed in terms of recent advances in recording and analysis technologies, and are related to factors causing convergence and divergence of echolocation call design in bats.

Systematic review and meta-analysis of the effects of exercise for those with cancer-related lymphedema

Objective: To evaluate the effects of exercise on cancer-related lymphedema and related symptoms, and to determine the need for those with lymphedema to wear compression during exercise. Data Sources: CINAHL, Cochrane, Ebscohost, MEDLINE, Pubmed, ProQuest Health and Medical Complete, ProQuest Nursing and Allied Health Source, Science Direct and SPORTDiscus databases were searched for trials published prior to 1 January, 2015. Study Selection: Randomised and non-randomised, controlled trials, and single group pre-post studies published in English-language were included. Twenty-one (exercise) and four (compression and exercise) studies met inclusion criteria. Data Extraction: Data was extracted into tabular format using predefined data fields by one reviewer and assessed for accuracy by a second reviewer. Study quality was evaluated using the Effective Public Health Practice Project assessment tool. Data Synthesis: Data was pooled using a random effects model to assess the effects of acute and long-term exercise on lymphedema and lymphedema-associated symptoms, with subgroup analyses for exercise mode and intervention length. There was no effect of exercise (acute or intervention) on lymphedema or associated symptoms with standardised mean differences from all analyses ranging between −0.2 and 0.1 (p-values ≥0.22). Findings from subgroup analyses for exercise mode (aerobic, resistance, mixed, other) and intervention duration (>12 weeks or ≤12 weeks) were consistent with these findings; that is, no effect on lymphedema or associated symptoms. There were too few studies evaluating the effect of compression during regular exercise to conduct a meta-analysis. Conclusions: Individuals with secondary lymphedema can safely participate in progressive, regular exercise without experiencing a worsening of lymphedema or related-symptoms. However, the results also do not suggest any improvements will occur in lymphedema. At present, there is insufficient evidence to support or refute the current clinical recommendation to wear compression garments during regular exercise.

Motorway crash duration and its determinants: Do durations vary across motorways?

Traffic congestion has been a growing issue in many metropolitan areas during recent years, which necessitates the identification of its key contributors and development of sustainable strategies to help decrease its adverse impacts on traffic networks. Road incidents generally and crashes specifically have been acknowledged as the cause of a large proportion of travel delays in urban areas and account for 25% to 60% of traffic congestion on motorways. Identifying the critical determinants of travel delays has been of significant importance to the incident management systems which constantly collect and store the incident duration data. This study investigates the individual and simultaneous differential effects of the relevant determinants on motorway crash duration probabilities. In particular, it applies parametric Accelerated Failure Time (AFT) hazard-based models to develop in-depth insights into how the crash-specific characteristic and the associated temporal and infrastructural determinants impact the duration. AFT models with both fixed and random parameters have been calibrated on one year of traffic crash records from two major Australian motorways in South East Queensland and the differential effects of determinants on crash survival functions have been studied on these two motorways individually. A comprehensive spectrum of commonly used parametric fixed parameter AFT models, including generalized gamma and generalized F families, have been compared to random parameter AFT structures in terms of goodness of fit to the duration data and as a result, the random parameter Weibull AFT model has been selected as the most appropriate model. Significant determinants of motorway crash duration included traffic diversion requirement, crash injury type, number and type of vehicles involved in a crash, day of week and time of day, towing support requirement and damage to the infrastructure. A major finding of this research is that the motorways under study are significantly different in terms of crash durations; such that motorway exhibits durations that are on average 19% shorter compared to the durations on motorway. The differential effects of explanatory variables on crash durations are also different on the two motorways. The detailed presented analysis confirms that, looking at the motorway network as a whole, neglecting the individual differences between roads, can lead to erroneous interpretations of duration and inefficient strategies for mitigating travel delays along a particular motorway.

Statistical analysis of the position of the monsoon trough

Using surface charts at 0330GMT, the movement df the monsoon trough during the months June to September 1990 al two fixed longitudes, namely 79 degrees E and 85 degrees E, is studied. The probability distribution of trough position shows that the median, mean and mode occur at progressively more northern latitudes, especially at 85 degrees E, with a pronounced mode that is close to the northern-most limit reached by the trough. A spectral analysis of the fluctuating latitudinal position of the trough is carried out using FFT and the Maximum Entropy Method (MEM). Both methods show significant peaks around 7.5 and 2.6 days, and a less significant one around 40-50 days. The two peaks at the shorter period are more prominent at the eastern longitude. MEM shows an additional peak around 15 days. A study of the weather systems that occurred during the season shows them to have a duration around 3 days and an interval between systems of around 9 days, suggesting a possible correlation with the dominant short periods observed in the spectrum of trough position.

Analysis of physical activity in emmetropic and myopic university students during semester and holiday periods: A pilot study

Background Previous studies (mostly questionnaire-based in children) suggest that outdoor activity is protective against myopia. There are few studies on young adults investigating both the impact of simply being outdoors versus performing physical activity. The aim was to study the relationship between the refractive error of young adults and their physical activity patterns. Methods Twenty-seven university students, aged 18 to 25 years, wore a pedometer (Omron HJ720ITE) for seven days both during the semester and holiday periods. They simultaneously recorded the type of activity performed, its duration, the number of steps taken (from the pedometer) and their location (indoors/outdoors) in a logbook. Mean spherical refractive error was used to divide participants into three groups (emmetropes: +1.00 to -0.50 D, low myopes: -0.62 to -3.00 D, higher myopes: -3.12 D or greater myopia). Results There were no significant differences between the refractive groups during the semester or holiday periods; the average daily times spent outdoors, the duration of physical activity, the ratio of physical activity performed outdoors to indoors and amount of near work performed were similar. The peak exercise intensity was similar across all groups: approximately 100 steps perminute, a brisk walk. Up to one-third of all physical activity was performed outdoors. There were some significant differences in activities performed during semester and holiday times. For example, lowmyopes spent significantly less time outside (49 ± 47 versus 74 ± 41 minutes, p = 0.005) and performed less physical activity (6,388 ± 1,747 versus 6,779 ± 2,746 steps per day; p = 0.03) during the holidays compared to during semester. Conclusions The fact that all groups had similar low exercise intensity butmany were notmyopic suggests that physical activity levels are not critical. There were differences in the activity patterns of lowmyopes during semester and holiday periods. This study highlights the need for a larger longitudinal-based study with particular emphasis on how discretionary time is spent.

Analysis of an internally mountable accelerometer balance system for use with non-isotropic models in shock tunnels

Knowledge of drag force is an important design parameter in aerodynamics. Measurement of aerodynamic forces at hypersonic speed is a challenge and usually ground test facilities like shock tunnels are used to carry out such tests. Accelerometer based force balances are commonly employed for measuring aerodynamic drag around bodies in hypersonic shock tunnels. In this study, we present an analysis of the effect of model material on the performance of an accelerometer balance used for measurement of drag in impulse facilities. From the experimental studies performed on models constructed out of Bakelite HYLEM and Aluminum, it is clear that the rigid body assumption does not hold good during the short testing duration available in shock tunnels. This is notwithstanding the fact that the rubber bush used for supporting the model allows unconstrained motion of the model during the short testing time available in the shock tunnel. The vibrations induced in the model on impact loading in the shock tunnel are damped out in metallic model, resulting in a smooth acceleration signal, while the signal become noisy and non-linear when we use non-isotropic materials like Bakelite HYLEM. This also implies that careful analysis and proper data reduction methodologies are necessary for measuring aerodynamic drag for non-metallic models in shock tunnels. The results from the drag measurements carried out using a 60 degrees half angle blunt cone is given in the present analysis.

Estimating the duration and cost of weed eradication programmes

Two prerequisites for realistically embarking upon an eradication programme are that cost-benefit analysis favours this strategy over other management options and that sufficient resources are available to carry the programme through to completion. These are not independent criteria, but it is our view that too little attention has been paid to estimating the investment required to complete weed eradication programmes. We deal with this problem by using a two-pronged approach: 1) developing a stochastic dynamic model that provides an estimation of programme duration; and 2) estimating the inputs required to delimit a weed incursion and to prevent weed reproduction over a sufficiently long period to allow extirpation of all infestations. The model is built upon relationships that capture the time-related detection of new infested areas, rates of progression of infestations from the active to the monitoring stage, rates of reversion of infestations from the monitoring to active stage, and the frequency distribution of time since last detection for all infestations. This approach is applied to the branched broomrape (Orobanche ramosa) eradication programme currently underway in South Australia. This programme commenced in 1999 and currently 7450 ha are known to be infested with the weed. To date none of the infestations have been eradicated. Given recent (2008) levels of investment and current eradication methods, model predictions are that it would take, on average, an additional 73 years to eradicate this weed at an average additional cost (NPV) of $AU67.9m. When the model was run for circumstances in 2003 and 2006, the average programme duration and total cost (NPV) were predicted to be 159 and 94 years, and $AU91.3m and $AU72.3m, respectively. The reduction in estimated programme length and cost may represent progress towards the eradication objective, although eradication of this species still remains a long term prospect.

Multivariate analysis of fresh-cut carambola slices stored under different temperatures.

Quality of fresh-cut carambola (Averrhoa carambola L) is related to many chemical and biochemical variables especially those involved with softening and browning, both influenced by storage temperature. To study these effects, a multivariate analysis was used to evaluate slices packaged in vacuum-sealed polyolefin bags, and stored at 2.5 degrees C, 5 degrees C and 10 degrees C, for up to 16 d. The quality of slices at each temperature was correlated with the duration of storage, O(2) and CO(2) concentration in the package, physical chemical constituents, and activity of enzymes involved in softening (PG) and browning (PPO) metabolism. Three quality groups were identified by hierarchical cluster analysis, and the classification of the components within each of these groups was obtained from a principal component analysis (PCA). The characterization of samples by PCA clearly distinguished acceptable and non-acceptable slices. According to PCA, acceptable slices presented higher ascorbic acid content, greater hue angles ((o)h) and final lightness (L-5) in the first principal component (PC1). On the other hand, non-acceptable slices presented higher total pectin content. PPO activity in the PC1. Non-acceptable slices also presented higher soluble pectin content, increased pectin solubilisation and higher CO(2) concentration in the second principal component (PC2) whereas acceptable slices showed lower total sugar content. The hierarchical cluster and PCA analyses were useful for discriminating the quality of slices stored at different temperatures.