Metabolites are small molecules involved in cellular metabolism where they act as reaction substrates or products. The term ‘metabolomics’ refers to the comprehensive study of these molecules. The concentrations of metabolites in biological tissues are under genetic control, but this is limited by environmental factors such as diet. In adult mono- and dizygotic twin pairs, we estimated the contribution of genetic and shared environmental influences on metabolite levels by structural equation modeling and tested whether the familial resemblance for metabolite levels is mainly explained by genetic or by environmental factors that are shared by family members. Metabolites were measured across three platforms: two based on proton nuclear magnetic resonance techniques and one employing mass spectrometry. These three platforms comprised 237 single metabolic traits of several chemical classes. For the three platforms, metabolites were assessed in 1407, 1037 and 1116 twin pairs, respectively. We carried out power calculations to establish what percentage of shared environmental variance could be detected given these sample sizes. Our study did not find evidence for a systematic contribution of shared environment, defined as the influence of growing up together in the same household, on metabolites assessed in adulthood. Significant heritability was observed for nearly all 237 metabolites; significant contribution of the shared environment was limited to 6 metabolites. The top quartile of the heritability distribution was populated by 5 of the 11 investigated chemical classes. In this quartile, metabolites of the class lipoprotein were significantly overrepresented, whereas metabolites of classes glycerophospholipids and glycerolipids were significantly underrepresented.

In lexical development, the specificity of phonological representations is important. The ability to build phonologically specific lexical representations predicts the number of words a child knows (vocabulary breadth), but it is not clear if it also fosters how well words are known (vocabulary depth). Sixty-six children were studied in kindergarten (age 5;7) and first grade (age 6;8). The predictive value of the ability to learn phonologically similar new words, phoneme discrimination ability, and phonological awareness on vocabulary breadth and depth were assessed using hierarchical regression. Word learning explained unique variance in kindergarten and first-grade vocabulary depth, over the other phonological factors. It did not explain unique variance in vocabulary breadth. Furthermore, even after controlling for kindergarten vocabulary breadth, kindergarten word learning still explained unique variance in first-grade vocabulary depth. Skill in learning phonologically similar words appears to predict knowledge children have about what words mean.

Children's abilities to process the phonological structure of words are important predictors of their literacy development. In the current study, we examined the interrelatedness between implicit (i.e., speech decoding) and explicit (i.e., phonological awareness) phonological abilities, and especially the role therein of lexical specificity (i.e., the ability to learn to recognize spoken words based on only minimal acoustic-phonetic differences). We tested 75 Dutch monolingual and 64 Turkish–Dutch bilingual kindergartners. SEM analyses showed that speech decoding predicted lexical specificity, which in turn predicted rhyme awareness in the first language learners but phoneme awareness in the second language learners. Moreover, in the latter group there was an impact of the second language: Dutch speech decoding and lexical specificity predicted Turkish phonological awareness, which in turn predicted Dutch phonological awareness. We conclude that language-specific phonological characteristics underlie different patterns of transfer from implicit to explicit phonological abilities in first and second language learners.

European legislation states that after stunning regular checks should be performed to guarantee animals are unconscious between the end of the stunning process and death. When animals are killed without prior stunning these checks should be performed before the animal is released from restraint. The validity of certain indicators used to assess unconsciousness under different stunning and slaughter conditions is under debate. The aim of this study was to validate the absence of threat-, withdrawal-, corneal- and eyelid reflex as indicators to assess unconsciousness in calves subjected to different stunning and slaughter methods. Calves (201±22 kg) were randomly assigned to one of the following four treatments: (1) Captive bolt stunning followed by neck cut in an inverted position (n=25); (2) Non-stunned slaughter in an upright position (n=7); (3) Non-stunned slaughter in an inverted position (180° rotation) (n=25); (4) Non-stunned slaughter in an upright position followed by captive bolt stunning 40 s after the neck cut (n=25). Each calf was equipped with non-invasive electroencephalogram (EEG) electrodes before the slaughter procedure. All reflexes were verified once before the slaughter procedure. At the beginning of the procedure (T=0 s) calves were stunned (treatment 1) or neck cut in an upright position (treatment 2, 4) or inverted position (treatment 3). Calves of treatment 4 were captive bolt stunned 34±8 s after the neck cut. Reflexes were assessed every 20 s from T=15 s for all treatments until all reflex tests resulted in a negative response three times in a row and a flat line EEG was observed. In addition, reflexes were assessed 5 s after captive bolt stunning in calves of treatments 1 and 4. Visual assessment of changes in the amplitude and frequency of EEG traces was used to determine loss of consciousness. Timing of loss of consciousness was related to timing of loss of reflexes. After captive bolt stunning, absence of threat-, withdrawal-, corneal- and eyelid reflex indicated unconsciousness as determined by EEG recordings. After non-stunned slaughter, both threat- and withdrawal reflex were on average lost before calves were unconscious based on EEG recordings. The eyelid- and corneal reflex were on average lost after calves had lost consciousness based on EEG recordings and appeared to be distinctly conservative indicators of unconsciousness in non-stunned slaughtered calves since they were observed until 76±50 and 85±45 s (mean±SD), respectively, after EEG-based loss of consciousness.

Assessing unconsciousness is important to safeguard animal welfare shortly after stunning at the slaughter plant. Indicators that can be visually evaluated are most often used when assessing unconsciousness, as they can be easily applied in slaughter plants. These indicators include reflexes originating from the brain stem (e.g. eye reflexes) or from the spinal cord (e.g. pedal reflex) and behavioural indicators such as loss of posture, vocalisations and rhythmic breathing. When physically stunning an animal, for example, captive bolt, most important indicators looked at are posture, righting reflex, rhythmic breathing and the corneal or palpebral reflex that should all be absent if the animal is unconscious. Spinal reflexes are difficult as a measure of unconsciousness with this type of stunning, as they may occur more vigorous. For stunning methods that do not physically destroy the brain, for example, electrical and gas stunning, most important indicators looked at are posture, righting reflex, natural blinking response, rhythmic breathing, vocalisations and focused eye movement that should all be absent if the animal is unconscious. Brain stem reflexes such as the cornea reflex are difficult as measures of unconsciousness in electrically stunned animals, as they may reflect residual brain stem activity and not necessarily consciousness. Under commercial conditions, none of the indicators mentioned above should be used as a single indicator to determine unconsciousness after stunning. Multiple indicators should be used to determine unconsciousness and sufficient time should be left for the animal to die following exsanguination before starting invasive dressing procedures such as scalding or skinning. The recording and subsequent assessment of brain activity, as presented in an electroencephalogram (EEG), is considered the most objective way to assess unconsciousness compared with reflexes and behavioural indicators, but is only applied in experimental set-ups. Studies performed in an experimental set-up have often looked at either the EEG or reflexes and behavioural indicators and there is a scarcity of studies that correlate these different readout parameters. It is recommended to study these correlations in more detail to investigate the validity of reflexes and behavioural indicators and to accurately determine the point in time at which the animal loses consciousness.

Prader–Willi syndrome (PWS) is a genetically determined disorder associated with the loss of the paternal contribution to the proximal part of the long arm of chromosome 15. Its pathophysiology is dominated by hypothalamic dysfunctions. The psychopathological phenotype comprises affective and psychotic symptoms as well as an increase of pre-existent obsessive-compulsive behaviors. The present study comprises 19 PWS patients who were referred for neuropsychiatric evaluation because of psychotic deterioration. Patients were assessed by using the elements of semistructured symptom checklists. In the majority a genetic analysis was performed to detect the underlying chromosomal defect. In 16 of the 19 patients a diagnosis of cycloid psychosis could be established. The other three showed a bipolar affective disorder. Of the psychotic patients, 11 were diagnosed as UPD and one as del 15q11–13. The remaining four patients were diagnosed clinically. For various reasons the genetic etiology could not be established. In PWS patients with a psychotic disorder (cycloid psychosis) a disproportional number of UPD is found.

In the present study, the development of semantic categorization and its relationship with reading was investigated across Dutch primary grade students. Three Exemplar-level tasks (Experiment 1) and two Superordinate-level tasks (Experiment 2) with different types of distracters (phonological, semantic and perceptual) were administered to assess semantic categorization skills. Reading was measured with a standardized word-reading test. Results of both experiments demonstrated that children in the higher grades had shorter reaction times and fewer errors than children in the lower grades. Reading skill, however, was not related to semantic categorization performance. Moreover, neither grade level nor reading skill was related to the effect of distracter type on error percentages. Based on the results of this study, we suggest a substantial development of semantic categorization skills over time, and reject the notion that Dutch poor readers have less advanced semantic categorization skills than typical readers.

The plasma lipid-lowering effect of PUFA, one of their main beneficial effects, is considered to be related to the regulation of lipid biosynthesis through transcription factors including sterol regulatory element binding proteins (SREBP). In the present study, we compared the effect of different PUFA on SREBP activity in HepG2 cells, using a sterol regulatory element–luciferase reporter construct as a probe. Supplementation with different fatty acids reduced SREBP activity in the order 20 : 5n-3 = 18 : 2n-6 = 20 : 4n-6≫18 : 3n-3 = 22 : 6n-3 = 22 : 5n-6≫18 : 1n-9. The suppression of SREBP activity greatly depended on the degree of incorporation of the supplemented PUFA into cellular lipids, and correlated positively with the unsaturation index (r 0·831; P < 0·01) of total cell lipids. Supplemented PUFA were also metabolised to longer and more unsaturated species. These processing activities were higher for n-3 than n-6 PUFA (P < 0·01). We studied the effect of PUFA on the intracellular distribution of non-esterified cholesterol, using filipin staining and fluorescence microscopy with or without the cholesterol traffic blocker U18666A. The data show that the incorporation of PUFA increases non-esterified cholesterol flow from the plasma membrane to intracellular membranes. We conclude that suppression of SREBP activity by PUFA depends on the degree of incorporation into cellular lipids, and is associated with increased flow of non-esterified cholesterol between the plasma membrane and intracellular membranes.

In Sub-Saharan Africa, cowpea is well known for its ability to survive under conditions of water stress and it plays an important role in regions where drought is the factor most limiting to crop yield. In the present study, the drought tolerance levels of 20 African cowpea accessions from three countries were evaluated. A number of the genotypes showed drought tolerance, the merits of stomatal behaviour and cell membrane stability to assess drought tolerance was demonstrated. Damage to the cell membranes caused by drought was less in tolerant accessions. Stomatal opening was also better regulated; the opening was smaller under drought conditions, thus reducing transpiration (T). The wide range of drought tolerance observed among the accessions suggests the possibility of breeding drought-tolerant cultivars in cowpea. Drought-tolerant accessions included Bafoussam 1, M.66, Bafoussam 3, Hluhluwa, Bafoussam 4, Balen, Makueni, Bafoussam 2 and Okhalweni and these could be recommended to breeders as valuable material for drought tolerance improvement in cowpea.

A 63nm Twin Flash memory cell with a size of 0.0225μm2 per 2 (or 4) bits is presented. To achieve small cell areas, a buried bit line and an aggressive gate length of 100 nm are the key features of this cell together with a minimum thermal budget processing. A novel epitaxial CoSi2 process allows the salicidation of local buried bitlines with only a few tens of nanometer width.