Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea. A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS (TM) clones with insert sizes similar to 20-40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs. Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter beta-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue. This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor > 40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.

A Brassica exon array for whole-transcript gene expression profiling

Affymetrix GeneChip (R) arrays are used widely to study transcriptional changes in response to developmental and environmental stimuli. GeneChip (R) arrays comprise multiple 25-mer oligonucleotide probes per gene and retain certain advantages over direct sequencing. For plants, there are several public GeneChip (R) arrays whose probes are localised primarily in 39 exons. Plant whole-transcript (WT) GeneChip (R) arrays are not yet publicly available, although WT resolution is needed to study complex crop genomes such as Brassica, which are typified by segmental duplications containing paralogous genes and/or allopolyploidy. Available sequence data were sampled from the Brassica A and C genomes, and 142,997 gene models identified. The assembled gene models were then used to establish a comprehensive public WT exon array for transcriptomics studies. The Affymetrix GeneChip (R) Brassica Exon 1.0 ST Array is a 5 mu M feature size array, containing 2.4 million 25-base oligonucleotide probes representing 135,201 gene models, with 15 probes per gene distributed among exons. Discrimination of the gene models was based on an E-value cut-off of 1E(-5), with <= 98 sequence identity. The 135 k Brassica Exon Array was validated by quantifying transcriptome differences between leaf and root tissue from a reference Brassica rapa line (R-o-18), and categorisation by Gene Ontologies (GO) based on gene orthology with Arabidopsis thaliana. Technical validation involved comparison of the exon array with a 60-mer array platform using the same starting RNA samples. The 135 k Brassica Exon Array is a robust platform. All data relating to the array design and probe identities are available in the public domain and are curated within the BrassEnsembl genome viewer at http://www.brassica.info/BrassEnsembl/index.html.

Within-host evolution of Staphylococcus aureus during asymptomatic carriage

Background Staphylococcus aureus is a major cause of healthcare associated mortality, but like many important bacterial pathogens, it is a common constituent of the normal human body flora. Around a third of healthy adults are carriers. Recent evidence suggests that evolution of S. aureus during nasal carriage may be associated with progression to invasive disease. However, a more detailed understanding of within-host evolution under natural conditions is required to appreciate the evolutionary and mechanistic reasons why commensal bacteria such as S. aureus cause disease. Therefore we examined in detail the evolutionary dynamics of normal, asymptomatic carriage. Sequencing a total of 131 genomes across 13 singly colonized hosts using the Illumina platform, we investigated diversity, selection, population dynamics and transmission during the short-term evolution of S. aureus. Principal Findings We characterized the processes by which the raw material for evolution is generated: micro-mutation (point mutation and small insertions/deletions), macro-mutation (large insertions/deletions) and the loss or acquisition of mobile elements (plasmids and bacteriophages). Through an analysis of synonymous, non-synonymous and intergenic mutations we discovered a fitness landscape dominated by purifying selection, with rare examples of adaptive change in genes encoding surface-anchored proteins and an enterotoxin. We found evidence for dramatic, hundred-fold fluctuations in the size of the within-host population over time, which we related to the cycle of colonization and clearance. Using a newly-developed population genetics approach to detect recent transmission among hosts, we revealed evidence for recent transmission between some of our subjects, including a husband and wife both carrying populations of methicillin-resistant S. aureus (MRSA). Significance This investigation begins to paint a picture of the within-host evolution of an important bacterial pathogen during its prevailing natural state, asymptomatic carriage. These results also have wider significance as a benchmark for future systematic studies of evolution during invasive S. aureus disease.

Longitudinal investigation of the role of temperament and stressful life events in childhood anxiety

The current study investigated the longitudinal relationships between BI, life events, and anxiety in a sample of 102 behaviourally inhibited (BI) and 100 uninhibited (BUI) children aged 3 to 4 years. Children’s parents completed questionnaires on BI, stressful life events, and anxiety symptoms, and were administered a diagnostic interview three times in a 5-year period. In line with our hypotheses, negative life events, and negative behaviour- dependent life events (i.e. life events that are related to the children’s own behaviours) in particular, and the impact of negative life events, were predictive of increases in subsequent anxiety symptoms, the likelihood of having an anxiety disorder, and increased number of anxiety diagnoses over the five year follow-up period. Experiencing more positive, behaviour-independent life events decreased the risk of being diagnosed with an anxiety disorder. Furthermore, differences were found in life events between BI and BUI children. That is, BI children experienced fewer positive and specifically positive behaviour-dependent life events, and the impact of these positive life events was also lower in BI children than in BUI children. However, BI did not interact with life events in the prediction of anxiety problems as hypothesized. Therefore, this study seems to indicate that BI and life events act as additive risk factors in the development of anxiety problems.

Informing early intervention: preschool predictors of anxiety disorders in middle childhood

Background: To inform early intervention practice, the present research examines how child anxiety, behavioural inhibition, maternal overinvolvement, maternal negativity, mother-child attachment and maternal anxiety, as assessed at age four, predict anxiety at age nine. Method: 202 children (102 behaviourally inhibited and 100 behaviourally uninhibited) aged 3–4 years were initially recruited and the predictors outlined above were assessed. Diagnostic assessments, using the Anxiety Disorders Interview Schedule, were then conducted five years later. Results: Behavioural inhibition, maternal anxiety, and maternal overinvolvement were significant predictors of clinical anxiety, even after controlling for baseline anxiety (p,.05). No significant effect of negativity or attachment security was found over and above baseline anxiety (p..1). Conclusions: Preschool children who show anxiety, are inhibited, have overinvolved mothers and mothers with anxiety disorders are at increased risk for anxiety in middle childhood. These factors can be used to identify suitable participants for early intervention and can be targeted within intervention programs.

Interpretation bias in preschool children at risk for anxiety: a prospective study

A story-stem paradigm was used to assess interpretation bias in preschool children. Data were available for 131 children. Interpretation bias, behavioural inhibition (BI) and anxiety were assessed when children were aged between 3 years 2 months and 4 years 5 months. Anxiety was subsequently assessed 12-months, 2-years and 5-years later. A significant difference in interpretation bias was found between participants who met criteria for an anxiety diagnosis at baseline, with clinically anxious participants more likely to complete the ambiguous story-stems in a threat-related way. Threat interpretations significantly predicted anxiety symptoms at 12-month follow-up, after controlling for baseline symptoms, but did not predict anxiety symptoms or diagnoses at either 2-year or 5- year follow-up. There was little evidence for a relationship between BI and interpretation bias. Overall, the pattern of results was not consistent with the hypothesis that interpretation bias plays a role in the development of anxiety. Instead, some evidence for a role in the maintenance of anxiety over relatively short periods of time was found. The use of a story-stem methodology to assess interpretation bias in young children is discussed along with the theoretical and clinical implications of the findings.

The role of temperament and family environment in the development of anxiety disorder: two-year follow-up

Objective Behavioural inhibition (BI) in early childhood is associated with increased risk for anxiety. The present research examines BI alongside family environment factors, specifically maternal negativity and overinvolvement, maternal anxiety and mother-child attachment, with a view to providing a broader understanding of the development of child anxiety. Method Participants were 202 children classified at age 4 as either behaviourally inhibited (N=102) or uninhibited (N=100). Family environment, BI and child anxiety were assessed at baseline and child anxiety and BI were assessed again two-years later when participants were aged 6 years. Results After controlling for baseline anxiety, inhibited participants were significantly more likely to meet criteria for a diagnosis of social phobia and generalized anxiety disorder at follow-up. Path analysis suggested that maternal anxiety significantly affected child anxiety over time, even after controlling for the effects of BI and baseline anxiety. No significant paths from parenting or attachment to child anxiety were found. Maternal overinvolvement was significantly associated with BI at follow-up. Conclusions At age 4, BI, maternal anxiety and child anxiety represent risk factors for anxiety at age 6. Furthermore, overinvolved parenting increases risk for BI at age 6, which may then lead to the development of anxiety in later childhood.

Psychopathology in Williams Syndrome: the effect of individual differences across the life span

The present research aimed to comprehensively explore psychopathology in Williams syndrome (WS) across the lifespan and evaluate the relationship between psychopathology and age category (child or adult), gender and cognitive ability. The parents of 50 participants with WS, aged 6-50 years, were interviewed using the Schedule for Affective Disorders and Schizophrenia for School-Age Children (K-SADS-PL). The prevalence of a wide range of Axis I DSM-IV disorders was assessed. In addition to high rates of anxiety and Attention Deficit Hyperactivity Disorder (ADHD) (38% and 20% respectively), 14% of our sample met criteria for a depressive disorder and 42% of participants were not experiencing any significant psychopathological difficulties. There was some evidence for different patterns of psychopathology between children and adults with WS and between males and females. These relationships were largely in keeping with those found in the typically developing population, thus supporting the validity of applying theory and treatment approaches for psychopathology in the typically developing population to WS.

Interpretation of ambiguous situations: evidence for a dissociation between social and physical threat in Williams syndrome

Williams Syndrome (WS) is associated with an unusual profile of anxiety, characterised by increased rates of non-social anxiety but not social anxiety (Dodd & Porter, 2009). The present research examines whether this profile of anxiety is associated with an interpretation bias for ambiguous physical, but not social, situations. Sixteen participants with WS, aged 13-34 years, and two groups of typically developing controls matched to the WS group on chronological age (CA) and mental age (MA), participated. Consistent with the profile of anxiety reported in WS, the WS group were significantly more likely to interpret an ambiguous physical situation as threatening than both control groups. However, no between-group differences were found on the ambiguous social situations.

There’s that scary picture: attention bias to threatening scenes in Williams syndrome

There is increasing evidence that Williams syndrome (WS) is associated with elevated anxiety that is non-social in nature, including generalised anxiety and fears. To date very little research has examined the cognitive processes associated with this anxiety. In the present research, attentional bias for non-social threatening images in WS was examined using a dot-probe paradigm. Participants were 16 individuals with WS aged between 13 and 34 years and two groups of typically developing controls matched to the WS group on chronological age and attentional control ability respectively. The WS group exhibited a significant attention bias towards threatening images. In contrast, no bias was found for group matched on attentional control and a slight bias away from threat was found in the chronological age matched group. The results are contrasted with recent findings suggesting that individuals with WS do not show an attention bias for threatening faces and discussed in relation to neuroimaging research showing elevated amygdala activation in response to threatening non-social scenes in WS.

I see happy people: attention bias towards happy but not angry facial expressions in Williams syndrome

Introduction: Observations of behaviour and research using eye-tracking technology have shown that individuals with Williams syndrome (WS) pay an unusual amount of attention to other people’s faces. The present research examines whether this attention to faces is moderated by the valence of emotional expression. Method: Sixteen participants with WS aged between 13 and 29 years (Mean=19 years 9 months) completed a dot-probe task in which pairs of faces displaying happy, angry and neutral expressions were presented. The performance of the WS group was compared to two groups of typically developing control participants, individually matched to the participants in the WS group on either chronological age or mental age. General mental age was assessed in the WS group using the Woodcock Johnson Test of Cognitive Ability Revised (WJ-COG-R; Woodcock & Johnson, 1989; 1990). Results: Compared to both control groups, the WS group exhibited a greater attention bias for happy faces. In contrast, no between-group differences in bias for angry faces were obtained. Conclusions: The results are discussed in relation to recent neuroimaging findings and the hypersocial behaviour that is characteristic of the WS population.

Beyond behaviour: is social anxiety low in Williams syndrome

Individuals with Williams syndrome (WS) exhibit striking social behaviour that may be indicative of abnormally low social anxiety. The present research aimed to determine whether social anxiety is unusually low in WS and to replicate previous findings of increased generalised anxiety in WS using both parent and self report. Fifteen individuals with WS aged 12-28 years completed the Spence Children’s Anxiety Scale (SCAS) and the Children’s Automatic Thoughts Scale (CATS). Their responses were compared to clinically anxious and community comparison groups matched on mental age. The findings suggest that WS is not associated with unusually low social anxiety but that generalised anxiety symptoms and physical threat thoughts are increased in WS, relative to typically developing children.

Self-ratings and observer-ratings of social skill: a manipulation of state social anxiety in anxious and non-anxious children

The role of state and trait anxiety on observer ratings of social skill and negatively biased self-perception of social skill was examined. Participants were aged between 7 and 13 years (mean=9.65; sd=1.77; N=102), 47 had a current anxiety diagnosis and 55 were non-anxious controls. Participants were randomly allocated to a high or low anxiety condition and asked to complete social tasks. Task instructions were adjusted across conditions to manipulate participants’ state anxiety. Observers rated anxious participants as having poorer social skills than non-anxious controls but there was no evidence that anxious participants exhibited a negative self-perception bias, relative to controls. However, as participants’ ratings of state anxiety increased, their perception of their performance became more negatively biased. The results suggest that anxious children may exhibit real impairments in social skill and that high levels of state anxiety can lead to biased judgements of social skills in anxious and non-anxious children.

Social approach in preschool children with Williams syndrome: the role of the face

Background Indiscriminate social approach behaviour is a salient aspect of the Williams syndrome (WS) behavioural phenotype. The present study examines approach behaviour in preschoolers with WS and evaluates the role of the face in WS social approach behaviour. Method Ten preschoolers with WS (aged 3-6 years) and two groups of typically developing children, matched to the WS group on chronological or mental age, participated in an observed play session. The play session incorporated social and non-social components including two components that assessed approach behaviour towards strangers, one in which the stranger’s face could be seen and one in which the stranger’s face was covered. Results In response to the non-social aspects of the play session, the WS group behaved similarly to both control groups. In contrast, the preschoolers with WS were significantly more willing than either control group to engage with a stranger, even when the stranger’s face could not be seen. Conclusion The findings challenge the hypothesis that an unusual attraction to the face directly motivates social approach behaviour in individuals with WS.

Temperament, family environment and anxiety in preschool children

This research examines the relationship between behavioural inhibition (BI), family environment (overinvolved and negative parenting, parental anxiety and parent-child attachment) and anxiety in a sample of 202 preschool children. Participants were aged between 3 years 2 months and 4 years 5 months, 101 were male. A thorough methodology was used that incorporated data from multiple observations of behaviour, diagnostic interviews and questionnaire measures. The results showed that children categorised as behaviourally inhibited were significantly more likely to meet criteria for a range of anxiety diagnoses. Furthermore, a wide range of family environment factors, including maternal anxiety, parenting and attachment were significantly associated with BI, with inhibited children more likely to experience adverse family environment factors. No interactions between temperament and family environment were found for child anxiety. However, a significant relationship between current maternal anxiety and child anxiety was found consistently even after controlling for BI. Additionally, there was some evidence of a relationship between maternal negativity and child anxiety, after controlling for BI. The results may suggest that temperament and family environment operate as additive, rather than interactive risk factors for child anxiety. This is discussed in the context of theoretical models of child anxiety and directions for future research.