Application of dried blood spots to determine vitamin D status in a large nutritional study with unsupervised sampling: the Food4Me project

An efficient and robust method to measure vitamin D (25-hydroxy vitamin D3 (25(OH)D3) and 25-hydroxy vitamin D2 in dried blood spots (DBS) has been developed and applied in the pan-European multi-centre, internet-based, personalised nutrition intervention study Food4Me. The method includes calibration with blood containing endogenous 25(OH)D3, spotted as DBS and corrected for haematocrit content. The methodology was validated following international standards. The performance characteristics did not reach those of the current gold standard liquid chromatography-MS/MS in plasma for all parameters, but were found to be very suitable for status-level determination under field conditions. DBS sample quality was very high, and 3778 measurements of 25(OH)D3 were obtained from 1465 participants. The study centre and the season within the study centre were very good predictors of 25(OH)D3 levels (P<0·001 for each case). Seasonal effects were modelled by fitting a sine function with a minimum 25(OH)D3 level on 20 January and a maximum on 21 July. The seasonal amplitude varied from centre to centre. The largest difference between winter and summer levels was found in Germany and the smallest in Poland. The model was cross-validated to determine the consistency of the predictions and the performance of the DBS method. The Pearson's correlation between the measured values and the predicted values was r 0·65, and the sd of their differences was 21·2 nmol/l. This includes the analytical variation and the biological variation within subjects. Overall, DBS obtained by unsupervised sampling of the participants at home was a viable methodology for obtaining vitamin D status information in a large nutritional study.

The relationship between challenging parenting behaviour and childhood anxiety disorders

Background: This research investigates the relationship between challenging parenting behaviour and childhood anxiety disorders proposed by Bögels and Phares (2008). Challenging parenting behaviour involves the playful encouragement of children to go beyond their own limits, and may decrease children’s risk for anxiety (Bögels & Phares, 2008). Method: Parents (n = 164 mothers, 144 fathers) of 164 children aged between 3.4 and 4.8 years participated in the current study. A multi-method, multi-informant assessment of anxiety was used, incorporating data from diagnostic interviews as well as questionnaire measures. Parents completed self-report measures of their parenting behaviour (n = 147 mothers, 138 fathers) and anxiety (n = 154 mothers, 143 fathers). Mothers reported on their child’s anxiety via questionnaire as well as diagnostic interview (n = 156 and 164 respectively). Of these children, 74 met criteria for an anxiety disorder and 90 did not. Results: Fathers engaged in challenging parenting behaviour more often than mothers. Both mothers’ and fathers’ challenging parenting behaviour was associated with lower report of child anxiety symptoms. However, only mothers’ challenging parenting behaviour was found to predict child clinical anxiety diagnosis. Limitations: Shared method variance from mothers confined the interpretation of these results. Moreover, due to study design, it is not possible to delineate cause and effect. Conclusions: The finding with respect to maternal challenging parenting behaviour was not anticipated, prompting replication of these results. Future research should investigate the role of challenging parenting behaviour by both caregivers as this may have implications for parenting interventions for anxious children.

Dystrophin-related protein, utrophin, in normal and dystrophic human fetal skeletal muscle.

Dystrophin is the product of the Duchenne muscular dystrophy (DMD) gene. Dystrophin-related protein (utrophin), an autosomal homologue of dystrophin, was studied in skeletal muscle from normal fetuses aged 9-26 weeks and one stillbirth of 41 weeks' gestation, and compared with low- and high-risk DMD fetuses aged 9-20 weeks. Utrophin was present at the sarcolemma from before 9 weeks' gestation, although there was variability in intensity both within and between myotubes. Sarcolemmal immunolabelling became more uniform, and levels of utrophin increased to a maximum at approximately 17-18 weeks. Levels then declined, until by 26 weeks sarcolemmal labelling was negligible and levels were similar to adult control muscle. By 41 weeks there was virtually no sarcolemmal labelling, although immunolabelling of capillaries was bright. Similar results were obtained with normal and DMD fetal muscle. Utrophin is therefore expressed in the presence and absence of dystrophin and down-regulated before birth in normal fetal muscle fibres. Samples were not available to determine whether or when, utrophin levels decline in DMD fetal muscle. On Western blots, utrophin was shown to have a smaller relative molecular mass than adult dystrophin, but similar to the fetal isoform. Blood vessels were brightly immunolabelled at all ages, although utrophin immunolabelling of peripheral nerves increased with gestational age.

Fat mass-and obesity-associated genotype, dietary intakes and anthropometric measures in European adults: the Food4Me study

The interplay between the fat mass- and obesity-associated (FTO) gene variants and diet has been implicated in the development of obesity. The aim of the present analysis was to investigate associations between FTO genotype, dietary intakes and anthropometrics among European adults. Participants in the Food4Me randomised controlled trial were genotyped for FTO genotype (rs9939609) and their dietary intakes, and diet quality scores (Healthy Eating Index and PREDIMED-based Mediterranean diet score) were estimated from FFQ. Relationships between FTO genotype, diet and anthropometrics (weight, waist circumference (WC) and BMI) were evaluated at baseline. European adults with the FTO risk genotype had greater WC (AAv. TT: +1·4 cm; P=0·003) and BMI (+0·9 kg/m2; P=0·001) than individuals with no risk alleles. Subjects with the lowest fried food consumption and two copies of the FTO risk variant had on average 1·4 kg/m2 greater BMI (Ptrend=0·028) and 3·1 cm greater WC (Ptrend=0·045) compared with individuals with no copies of the risk allele and with the lowest fried food consumption. However, there was no evidence of interactions between FTO genotype and dietary intakes on BMI and WC, and thus further research is required to confirm or refute these findings.

Genome-wide association study of response to cognitive behavioural therapy in children with anxiety disorders

Background Anxiety disorders are common, and cognitive–behavioural therapy (CBT) is a first-line treatment. Candidate gene studies have suggested a genetic basis to treatment response, but findings have been inconsistent. Aims To perform the first genome-wide association study (GWAS) of psychological treatment response in children with anxiety disorders (n = 980). Method Presence and severity of anxiety was assessed using semi-structured interview at baseline, on completion of treatment (post-treatment), and 3 to 12 months after treatment completion (follow-up). DNA was genotyped using the Illumina Human Core Exome-12v1.0 array. Linear mixed models were used to test associations between genetic variants and response (change in symptom severity) immediately post-treatment and at 6-month follow-up. Results No variants passed a genome-wide significance threshold (P = 5×10−8) in either analysis. Four variants met criteria for suggestive significance (P<5×10−6) in association with response post-treatment, and three variants in the 6-month follow-up analysis. Conclusions This is the first genome-wide therapygenetic study. It suggests no common variants of very high effect underlie response to CBT. Future investigations should maximise power to detect single-variant and polygenic effects by using larger, more homogeneous cohorts.

A low mortality, high morbidity reduced intensity status epilepticus (RISE) model of epilepsy and epileptogenesis in the rat

Animal models of acquired epilepsies aim to provide researchers with tools for use in understanding the processes underlying the acquisition, development and establishment of the disorder. Typically, following a systemic or local insult, vulnerable brain regions undergo a process leading to the development, over time, of spontaneous recurrent seizures. Many such models make use of a period of intense seizure activity or status epilepticus, and this may be associated with high mortality and/or global damage to large areas of the brain. These undesirable elements have driven improvements in the design of chronic epilepsy models, for example the lithium-pilocarpine epileptogenesis model. Here, we present an optimised model of chronic epilepsy that reduces mortality to 1% whilst retaining features of high epileptogenicity and development of spontaneous seizures. Using local field potential recordings from hippocampus in vitro as a probe, we show that the model does not result in significant loss of neuronal network function in area CA3 and, instead, subtle alterations in network dynamics appear during a process of epileptogenesis, which eventually leads to a chronic seizure state. The model’s features of very low mortality and high morbidity in the absence of global neuronal damage offer the chance to explore the processes underlying epileptogenesis in detail, in a population of animals not defined by their resistance to seizures, whilst acknowledging and being driven by the 3Rs (Replacement, Refinement and Reduction of animal use in scientific procedures) principles.

Investigation and optimization of parameters affecting the multiply charged ion yield in AP-MALDI MS

Liquid matrix-assisted laser desorption/ionization (MALDI) allows the generation of predominantly multiply charged ions in atmospheric pressure (AP) MALDI ion sources for mass spectrometry (MS) analysis. The charge state distribution of the generated ions and the efficiency of the ion source in generating such ions crucially depend on the desolvation regime of the MALDI plume after desorption in the AP-tovacuum inlet. Both high temperature and a flow regime with increased residence time of the desorbed plume in the desolvation region promote the generation of multiply charged ions. Without such measures the application of an electric ion extraction field significantly increases the ion signal intensity of singly charged species while the detection of multiply charged species is less dependent on the extraction field. In general, optimization of high temperature application facilitates the predominant formation and detection of multiply charged compared to singly charged ion species. In this study an experimental setup and optimization strategy is described for liquid AP-MALDI MS which improves the ionization effi- ciency of selected ion species up to 14 times. In combination with ion mobility separation, the method allows the detection of multiply charged peptide and protein ions for analyte solution concentrations as low as 2 fmol/lL (0.5 lL, i.e. 1 fmol, deposited on the target) with very low sample consumption in the low nL-range.

Reproducibility of the online Food4Me food-frequency questionnaire for estimating dietary intakes across Europe

Background: Accurate dietary assessment is key to understanding nutrition-related outcomes and is essential for estimating dietary change in nutrition-based interventions. Objective: The objective of this study was to assess the pan-European reproducibility of the Food4Me food-frequency questionnaire (FFQ) in assessing the habitual diet of adults. Methods: Participantsfromthe Food4Me study, a 6-mo,Internet-based, randomizedcontrolled trial of personalized nutrition conducted in the United Kingdom, Ireland, Spain, Netherlands, Germany, Greece, and Poland were included. Screening and baseline data (both collected before commencement of the intervention) were used in the present analyses, and participants were includedonly iftheycompleted FFQs at screeningand at baselinewithin a 1-mo timeframebeforethe commencement oftheintervention. Sociodemographic (e.g., sex andcountry) andlifestyle[e.g.,bodymass index(BMI,inkg/m2)and physical activity] characteristics were collected. Linear regression, correlation coefficients, concordance (percentage) in quartile classification, and Bland-Altman plots for daily intakes were used to assess reproducibility. Results: In total, 567 participants (59% female), with a mean 6 SD age of 38.7 6 13.4 y and BMI of 25.4 6 4.8, completed bothFFQswithin 1 mo(mean 6 SD: 19.26 6.2d).Exact plus adjacent classification oftotal energy intakeinparticipants was highest in Ireland (94%) and lowest in Poland (81%). Spearman correlation coefficients (r) in total energy intake between FFQs ranged from 0.50 for obese participants to 0.68 and 0.60 in normal-weight and overweight participants, respectively. Bland-Altman plots showed a mean difference between FFQs of 210 kcal/d, with the agreement deteriorating as energy intakes increased. There was little variation in reproducibility of total energy intakes between sex and age groups. Conclusions: The online Food4Me FFQ was shown to be reproducible across 7 European countries when administered within a 1-mo period to a large number of participants. The results support the utility of the online Food4Me FFQ as a reproducible tool across multiple European populations. This trial was registered at clinicaltrials.gov as NCT01530139.

Genetic and phenotypic characterization of complex hereditary spastic paraplegia

The hereditary spastic paraplegias are a heterogeneous group of degenerative disorders that are clinically classified as either pure with predominant lower limb spasticity, or complex where spastic paraplegia is complicated with additional neurological features, and are inherited in autosomal dominant, autosomal recessive or X-linked patterns. Genetic defects have been identified in over 40 different genes, with more than 70 loci in total. Complex recessive spastic paraplegias have in the past been frequently associated with mutations in SPG11 (spatacsin), ZFYVE26/SPG15, SPG7 (paraplegin) and a handful of other rare genes, but many cases remain genetically undefined. The overlap with other neurodegenerative disorders has been implied in a small number of reports, but not in larger disease series. This deficiency has been largely due to the lack of suitable high throughput techniques to investigate the genetic basis of disease, but the recent availability of next generation sequencing can facilitate the identification of disease- causing mutations even in extremely heterogeneous disorders. We investigated a series of 97 index cases with complex spastic paraplegia referred to a tertiary referral neurology centre in London for diagnosis or management. The mean age of onset was 16 years (range 3 to 39). The SPG11 gene was first analysed, revealing homozygous or compound heterozygous mutations in 30/97 (30.9%) of probands, the largest SPG11 series reported to date, and by far the most common cause of complex spastic paraplegia in the UK, with severe and progressive clinical features and other neurological manifestations, linked with magnetic resonance imaging defects. Given the high frequency of SPG11 mutations, we studied the autophagic response to starvation in eight affected SPG11 cases and control fibroblast cell lines, but in our restricted study we did not observe correlations between disease status and autophagic or lysosomal markers. In the remaining cases, next generation sequencing was carried out revealing variants in a number of other known complex spastic paraplegia genes, including five in SPG7 (5/97), four in FA2H (also known as SPG35) (4/97) and two in ZFYVE26/SPG15. Variants were identified in genes usually associated with pure spastic paraplegia and also in the Parkinson’s disease-associated gene ATP13A2, neuronal ceroid lipofuscinosis gene TPP1 and the hereditary motor and sensory neuropathy DNMT1 gene, highlighting the genetic heterogeneity of spastic paraplegia. No plausible genetic cause was identified in 51% of probands, likely indicating the existence of as yet unidentified genes.