Evaluation of massively parallel sequencing for forensic DNA methylation profiling.

Epigenetics is an emerging area of interest in forensic science. DNA methylation, a type of epigenetic modification, can be applied to chronological age estimation, identical twin differentiation and body fluid identification. However, there is not yet an agreed, established methodology for targeted detection and analysis of DNA methylation markers in forensic research. Recently, a massively parallel sequencing-based approach has been suggested. The use of massively parallel sequencing is well established in clinical epigenetics and is emerging as a new technology in the forensic field. This review investigates the potential benefits, limitations, and considerations of this technique for the analysis of DNA methylation in a forensic context. The importance of a robust protocol, regardless of the methodology used, which minimizes potential sources of bias is highlighted.

Children's neural response to contrast-negated faces is species specific.

Face recognition abilities develop dramatically during the first year of life, but comparatively little is known about the nature of face-specific perceptual development during early childhood. Face-specific effects of image appearance on recognition, including face inversion and contrast negation, are a useful means of understanding the functional properties of face perception developmentally. Here, we examined the generality of the impact of contrast negation on face perception during early childhood using event-related potentials (ERPs). Specifically, we recorded continuous electroencephalography (EEG) while adult participants and children between 4 and 6 years of age viewed human and non-human primate faces presented in either positive or negative contrast. We examined both the P100 and N170 components to determine whether or not sensitivity to contrast polarity was evident in face-sensitive components during early childhood and also whether or not that sensitivity was specific to species category. We found evidence of a species-specific effect of contrast negation at the N170, suggesting that by early childhood some aspects of face-specific processing have been restricted to a relatively narrow class of face stimuli. However, this effect is of the opposite sign relative to adults, suggesting that there is continued maturation of face-specific processing during childhood.

A randomised controlled trial of a community-based healthy lifestyle program for overweight and obese adolescents: the Loozit study protocol.

BACKGROUND: There is a need to develop sustainable and clinically effective weight management interventions that are suitable for delivery in community settings where the vast majority of overweight and obese adolescents should be treated. This study aims to evaluate the effect of additional therapeutic contact as an adjunct to the Loozit group program -- a community-based, lifestyle intervention for overweight and lower grade obesity in adolescents. The additional therapeutic contact is provided via telephone coaching and either mobile phone Short Message Service or electronic mail, or both. METHODS AND DESIGN: The study design is a two-arm randomised controlled trial that aims to recruit 168 overweight and obese 13-16 year olds (Body Mass Index z-score 1.0 to 2.5) in Sydney, Australia. Adolescents with secondary causes of obesity or significant medical illness are excluded. Participants are recruited via schools, media coverage, health professionals and several community organisations. Study arm one receives the Loozit group weight management program (G). Study arm two receives the same Loozit group weight management program plus additional therapeutic contact (G+ATC). The 'G' intervention consists of two phases. Phase 1 involves seven weekly group sessions held separately for adolescents and their parents. This is followed by phase 2 that involves a further seven group sessions held regularly, for adolescents only, until two years follow-up. Additional therapeutic contact is provided to adolescents in the 'G+ATC' study arm approximately once per fortnight during phase 2 only. Outcome measurements are assessed at 2, 12 and 24 months post-baseline and include: BMI z-score, waist z-score, metabolic profile indicators, physical activity, sedentary behaviour, eating patterns, and psychosocial well-being. DISCUSSION: The Loozit study is the first randomised controlled trial of a community-based adolescent weight management intervention to incorporate additional therapeutic contact via a combination of telephone coaching, mobile phone Short Message Service, and electronic mail. If shown to be successful, the Loozit group weight management program with additional therapeutic contact has the potential to be readily translatable to a range of health care settings. TRIAL REGISTRATION: The protocol for this study is registered with the Australian Clinical Trials Registry (ACTRNO12606000175572).

STRmix™ collaborative exercise on DNA mixture interpretation.

An intra and inter-laboratory study using the probabilistic genotyping (PG) software STRmix™ is reported. Two complex mixtures from the PROVEDIt set, analysed on an Applied Biosystems™ 3500 Series Genetic Analyzer, were selected. 174 participants responded. For Sample 1 (low template, in the order of 200 rfu for major contributors) five participants described the comparison as inconclusive with respect to the POI or excluded him. Where LRs were assigned, the point estimates ranging from 2 × 104 to 8 × 106. For Sample 2 (in the order of 2000 rfu for major contributors), LRs ranged from 2 × 1028 to 2 × 1029. Where LRs were calculated, the differences between participants can be attributed to (from largest to smallest impact): This study demonstrates a high level of repeatability and reproducibility among the participants. For those results that differed from the mode, the differences in LR were almost always minor or conservative.

Characterization of catecholamine release from deer adrenal medullary chromaffin cells.

Isolated adrenal medullary chromaffin cells maintained in culture have been widely used to study neurosecretory events. Many of these studies have been conducted using cells obtained from the bovine adrenal. In this study we have cultured chromaffin cells from an alternative large animal model, the deer, and have conducted the first characterization of secretion from this preparation. Cervine chromaffin cells, preloaded with [3H]noradrenalin, displayed a strong secretory response to the cholinergic agonist carbachol, with a maximal secretion of approximately 28% cell content over 15 min. This response was reproduced by nicotinic but not muscarinic agonists and was similarly inhibited by nicotinic but not muscarinic antagonists. Nicotine-evoked secretion measured over a 15 min time period was inhibited approximately 50% by the L-type Ca2+-channel antagonist nifedipine and approximately 20% by N-type (omega-conotoxin GVIA) or N, P/Q-type (omega-conotoxin MVIIC) antagonists. In contrast the response was unaffected by omega-agatoxin IVA, a P/Q-type antagonist. In addition to nicotinic receptor stimulation, activation of PACAP or histamine H1 receptors resulted in a concentration-dependent increase in secretion. PACAP was approximately two-fold more effective than histamine although both were weaker secretagogues than nicotine. In contrast, cervine chromaffin cells did not respond to angiotensin II or bradykinin, two agents known to stimulate secretion from bovine chromaffin cells. These data provide an initial characterization of the secretory response from cervine adrenal medullary chromaffin cells indicating that there are marked similarities but also potentially significant differences between them and their far more extensively described bovine counterparts.

The variability in likelihood ratios due to different mechanisms.

Recently there has been a drive towards standardisation of forensic DNA interpretation methods resulting in the uptake of probabilistic interpretation software. Some of these software solutions utilise Markov chain Monte Carlo techniques (MCMC). They will not produce an identical answer after repeat interpretations of the same evidence profile because of the Monte Carlo aspect. This is a new source of variability within the forensic DNA analysis process. In this paper we explore the size of the MCMC variability within the interpretation software STRmix™ compared to other sources of variability in forensic DNA profiling including PCR, capillary electrophoresis load and injection, and the makeup of allele frequency databases. The MCMC variability within STRmix™ was shown to be the smallest source of variability in this process.

Characterising the STR locus D6S1043 and examination of its effect on stutter rates.

The forensic analysis of DNA is most often undertaken by the amplification of short tandem repeats (STR) using the polymerase chain reaction (PCR). DNA amplification can result in production of the target allele amplicon and a by-product called stutter. Stutter is the result of the miscopy of the target allele and is typically one repeat smaller. Stutter is traditionally described as a ratio of stutter and allele height; stutter ratio (SR). The challenge to DNA profile interpretation is most serious whenever stutter products are of a similar height to the minor allelic peaks in a mixed DNA profile. An accurate assignment of peaks and the prediction of their height is important when objectively interpreting forensic DNA profiles. The longest uninterrupted stretch (LUS) of tandem repeats within the allele has previously been shown to be a good predictor of stutter ratio. LUS is determined by sequencing a range of observed alleles at a locus. The locus D6S1043 is a relatively new locus to appear in commercial forensic DNA testing kits. To date however, there has been no comprehensive report of sequencing of this locus. In this work, we sequence a sample of D6S1043 alleles to determine LUS values and investigate allele repeat number and LUS as explanatory variables for SR.

Species-specific effects of pigmentation negation on the neural response to faces.

Face processing is limited in scope as a function of experience - discrimination ability and face-specific behavioral effects are reduced in out-group faces. Nonetheless, other-species faces phylogenetically close to our own may be processed by similar mechanisms as human faces. Presently, we asked whether or not the well-known effect of contrast-negation on face recognition (Galper, 1970) was exclusive to human faces or generalized to monkey faces. Negation disrupts face pigmentation substantially, allowing us to examine species-specific use of surface cues as a function of expertise. We tested adult observers behaviorally and electrophysiologically: participants completed a 4AFC discrimination task subject to manipulations of face species and independent negation of image luminance and image chroma, and the same stimuli were used to collect event-related potentials in a go/no-go task. We predicted that expertise for human faces would lead to larger deleterious effects of negation for human faces in both tasks, reflected in longer RTs for correct responses in the discrimination task and species-specific modulation of the N170 and P200 by contrast-negation. Our results however, indicate that behaviorally, luminance and chroma negation affect discrimination performance in a species-independent manner, while similar effects of contrast-negation effects are evident in each species at different components of the ERP response.

The effect of cleaning agents on the ability to obtain DNA profiles using the Identifiler™ and PowerPlex® Y multiplex kits.

A year after the introduction of Identifiler™ into the forensic DNA laboratories of the Institute of Environmental Science and Research Limited (ESR), increasing occurrences of dropout of the three loci, D7S820, D18S51, and FGA, were observed in samples where the DNA was not degraded and sufficient DNA was present that full DNA profiles were to be expected. The dropout was either partial or complete at these loci. Full profiles could sometimes be obtained by reamplification of samples using the same input amount of DNA. After a thorough investigation of the methods and procedures used in the laboratory, the cause of this inhibition was identified as the cleaning agent TriGene™ ADVANCE. This was determined after the deliberate addition of varying amounts of different cleaning reagents into the DNA amplification reactions. At concentrations of 0.004% TriGene™ ADVANCE caused inhibition resulting in tri-loci dropout. At concentrations of 0.04% and higher, complete inhibition was observed. An effect was also seen on the amplification of samples using the Y STR profiling system PowerPlex(®) Y. This work highlights the importance of checking all reagents and chemicals prior to use, even those with no apparent direct influence on the DNA profiling process.