Can dogs reduce stress levels in school children? effects of dog-assisted interventions on salivary cortisol in children with and without special educational needs using randomized controlled trials.

Prolonged or excessive stress negatively affects learning, behavior and health across the lifespan. To alleviate adverse effects of stress in school children, stressors should be reduced, and support and effective interventions provided. Animal-assisted interventions (AAI) have shown beneficial effects on health and wellbeing, however, robust knowledge on stress mediation in children is lacking. Despite this, AAIs are increasingly employed in settings world-wide, including schools, to reduce stress and support learning and wellbeing. This study is the first randomized controlled trial to investigate dog-assisted interventions as a mediator of stress in school children with and without special educational needs (SEN) over the school term. Interventions were carried out individually and in small groups twice a week for 20 minutes over the course of 4 weeks. We compared physiological changes in salivary cortisol in a dog intervention group with a relaxation intervention group and a no treatment control group. We compared cortisol level means before and after the 4 weeks of interventions in all children as well as acute cortisol in mainstream school children. Dog interventions lead to significantly lower stress in children with and without special educational needs compared to their peers in relaxation or no treatment control groups. In neurotypical children, those in the dog interventions showed no baseline stress level increases over the school term. In addition, acute cortisol levels evidenced significant stress reduction following the interventions. In contrast, the no treatment control group showed significant rises in baseline cortisol levels from beginning to end of school term. Increases also occurred in the relaxation intervention group. Children with SEN showed significantly decreased cortisol levels after dog group interventions. No changes occurred in the relaxation or no treatment control groups. These findings provide crucial evidence that dog interventions can successfully attenuate stress levels in school children with important implications for AAI implementation, learning and wellbeing.

CACNA1C methylation: association with cortisol, perceived stress, rs1006737 and childhood trauma in males.

Aim: We investigated morning cortisol, stress, rs1006737 and childhood trauma relationship with CACNA1C methylation. Materials & methods: Morning cortisol release, childhood trauma and perceived stress were collected and genotyping for rs1006737 conducted in 103 adult males. Genomic DNA extracted from saliva was bisulphite converted and using pyrosequencing methylation determined at 11 CpG sites within intron 3 of CACNA1C. Results: A significant negative correlation between waking cortisol and overall mean methylation was found and a positive correlation between CpG5 methylation and perceived stress. Conclusion:CACNA1C methylation levels may be related to cortisol release and stress perception. Future work should evaluate the influence of altered CACNA1C methylation on stress reactivity to investigate this as a potential mechanism for mental health vulnerability.

Effects of exercise, cognitive, and dual-task interventions on cognition in type 2 diabetes mellitus: A systematic review and meta-analysis.

INTRODUCTION: Previous evidence has shown significant effects of exercise, cognitive and dual-task training for improving cognition in healthy cohorts. The effects of these types of interventions in type 2 diabetes mellitus is unclear. The aim of this research was to systematically review evidence, and estimate the effect, of exercise, cognitive, and dual-task interventions on cognition in type 2 diabetes mellitus. METHOD: Electronic databases including PubMed, EMBASE, CINAHL, PsycINFO, SPORTDiscus, and MEDLINE were searched for ongoing and completed interventional trials investigating the effect of either an exercise, cognitive or dual-task intervention on cognition in type 2 diabetes mellitus. RESULTS: Nine trials met the inclusion criteria-one dual-task, two cognitive, and six exercise. Meta-analyses of exercise trials showed no significant effects of exercise on measures of executive function (Stroop task, SMD = -0.31, 95% CI -0.71-0.09, P = 0.13, trail making test part A SMD = 0.28, 95% CI -0.20-0.77 P = 0.25, trail making test part B SMD = -0.15, 95% CI -0.64-0.34 P = 0.54, digit symbol SMD = 0.09, 95% CI -0.39-0.57 P = 0.72), and memory (immediate memory SMD = 0.20, 95% CI -0.28-0.69, P = 0.41 and delayed memory SMD = -0.06, 95% CI -0.55-0.42, P = 0.80). A meta-analysis could not be conducted using cognitive or dual-task data, but individual trials did report a favourable effect of interventions on cognition. Risk of bias was considered moderate to high for the majority of included trials. CONCLUSIONS: Meta-analyses of exercise trials identified a small effect size (0.31), which whilst not significant warrants further investigation. Larger and more robust trials are needed that report evidence using appropriate reporting guidelines (e.g. CONSORT) to increase confidence in the validity of results. TRIAL REGISTRATION: Protocol was registered (CRD42017058526) on the International Prospective Register of Systematic Reviews (http://www.crd.york.ac.uk/PROSPERO).

Dopamine and Working Memory: Genetic Variation, Stress and Implications for Mental Health.

At the molecular level, the neurotransmitter dopamine (DA) is a key regulatory component of executive function in the prefrontal cortex (PFC) and dysfunction in dopaminergic (DAergic) circuitry has been shown to result in impaired working memory (WM). Research has identified multiple common genetic variants suggested to impact on the DA system functionally and also behaviourally to alter WM task performance. In addition, environmental stressors impact on DAergic tone, and this may be one mechanism by which stressors confer vulnerability to the development of neuropsychiatric conditions. This chapter aims to evaluate the impact of key DAergic gene variants suggested to impact on both synaptic DA levels (COMT, DAT1, DBH, MAOA) and DA receptor function (ANKK1, DRD2, DRD4) in terms of their influence on visuospatial WM. The role of stressors and interaction with the genetic background is discussed in addition to discussion around some of the implications for precision psychiatry. This and future work in this area aim to disentangle the neural mechanisms underlying susceptibility to stress and their impact and relationship with cognitive processes known to influence mental health vulnerability.

The effect of ANKK1 Taq1A and DRD2 C957T polymorphisms on executive function: A systematic review and meta-analysis.

Research in healthy adults suggests that C957T polymorphism of the dopamine D2 receptor encoding DRD2 and the Taq1A polymorphism of the neighbouring gene ankyrin repeat and kinase domain containing 1 (ANKK1) alter dopaminergic signalling and may influence prefrontally-mediated executive functions. A systematic review and meta-analysis was carried out on the evidence for the association of DRD2 C957T and ANKK1 Taq1A polymorphisms in performance on tasks relating to the three core domains of executive function: working memory, response inhibition and cognitive flexibility in healthy adults. CINAHL, MEDLINE, PsycARTICLES and PsychINFO databases were searched for predefined key search terms associated with the two polymorphisms and executive function. Studies were included if they investigated a healthy adult population with the mean age of 18-65 years, no psychiatric or neurological disorder and only the healthy adult arm were included in studies with any case-control design. Data from 17 independent studies were included in meta-analysis, separated by the Taq1A and C957T polymorphisms and by executive function tests: working memory (Taq1A, 6 samples, n = 1270; C957 T, 6 samples, n = 977), cognitive flexibility (C957 T, 3 samples, n = 620), and response inhibition (C957 T, 3 samples, n = 598). The meta-analyses did not establish significant associations between these gene polymorphisms of interest and any of the executive function domains. Theoretical implications and methodological considerations of these findings are discussed.

Overnight retention of emotional memories is influenced by BDNF Val66Met but not 5-HTTLPR.

Emotional memory may be modulated by BDNF Val66Met and 5-HTTLPR polymorphisms. However, the influence of these genetic variants on the overnight retention of emotional memories has not been investigated in humans. Thirty-six healthy female students were selected to participate in this study based on 5-HTTLPR genotype status (L'/L', L'/S', S'/S'). Participants were also genotyped for BDNF Val66Met (Val/Val, Met carriers). We measured recognition performance for positive, neutral and negative images before and after overnight sleep. We found a significant interaction between BDNF Val66Met genotype group and image valence on post-sleep recognition performance. This interaction was driven by greater memory for negative and positive images, relative to neutral images, in Met carriers. We also found that longer Rapid Eye Movement (REM) sleep duration predicted greater post-sleep recognition performance for negative images in Met carriers, but not in Val homozygotes. We observed no influence of 5-HTTLPR polymorphisms on post- sleep recognition performance for positive, neutral or negative images. Our findings support a modulatory role for BDNF Val66Met in overnight emotional memory retention in females. We discuss the implications of this finding for understanding the influence of BDNF Val66Met on depression vulnerability.

Eye Movements in the "Morris Maze" Spatial Working Memory Task Reveal Deficits in Strategic Planning.

Analysis of eye movements can provide insights into processes underlying performance of cognitive tasks. We recorded eye movements in healthy participants and people with idiopathic Parkinson disease during a token foraging task based on the spatial working memory component of the widely used Cambridge Neuropsychological Test Automated Battery. Participants selected boxes (using a mouse click) to reveal hidden tokens. Tokens were never hidden under a box where one had been found before, such that memory had to be used to guide box selections. A key measure of performance in the task is between search errors (BSEs) in which a box where a token has been found is selected again. Eye movements were found to be most commonly directed toward the next box to be clicked on, but fixations also occurred at rates higher than expected by chance on boxes farther ahead or back along the search path. Looking ahead and looking back in this way was found to correlate negatively with BSEs and was significantly reduced in patients with Parkinson disease. Refixating boxes where tokens had already been found correlated with BSEs and the severity of Parkinson disease symptoms. It is concluded that eye movements can provide an index of cognitive planning in the task. Refixations on locations where a token has been found may also provide a sensitive indicator of visuospatial memory integrity. Eye movement measures derived from the spatial working memory task may prove useful in the assessment of executive functions as well as neurological and psychiatric diseases in the future.

Measuring Cortisol in the Classroom with School-Aged Children-A Systematic Review and Recommendations.

The collection of salivary cortisol has been chosen as one of the least intrusive, easiest to collect, analyze, and store methods of obtaining information on physiological changes. It is, however, not clear what the best practice is when collecting salivary cortisol from children within the school setting. The aim of this systematic review is to evaluate the feasibility of cortisol collection in schools for future research and to make recommendations for best practice. The review included 25 peer-reviewed articles from seven databases. The hypotheses of the included studies vary, but they all use cortisol as a diurnal, baseline, or acute measure, or to measure the effect of an intervention. Two methods of salivary cortisol collection were preferred by most of the research, i.e., passive drool or cotton Salivettes. The review has concluded that cortisol is a physiological marker that can be successfully measured in school-based research. However, there are discrepancies across studies when evaluating the collection guidelines, protocols, and instructions to participants as well as transparency of the success rate of obtaining all samples. Recommendations are made for future research to address and avoid such discrepancies and improve cross-study comparisons by implementing standard protocol guidelines.

The influence of REM sleep and SWS on emotional memory consolidation in participants reporting depressive symptoms.

Negative emotional memory bias is thought to play a causal role in the onset and maintenance of major depressive disorder. Rapid Eye Movement (REM) sleep has been shown to selectively consolidate negative emotional memories in healthy participants, and is greater in quantity and density in depressed patients. Slow-Wave Sleep (SWS) is typically associated with the consolidation of non-emotional memories. However, the effects of REM sleep and SWS on emotional memory consolidation have not been investigated in participants reporting depressive symptoms. In this study, we recruited two groups of healthy participants; one reporting mild-to-moderate depressive symptoms, and another reporting minimal depressive symptoms (assessed using the Beck Depression Inventory; BDI-II). Using a within-subjects split-night design, we measured consolidation of positive, neutral and negative images across a 3 h retention interval rich in either REM sleep or SWS. We found a significant sleep condition x image valence interaction in participants reporting depressive symptoms [F (2, 20) = 4.73, p = .021], but not participants reporting minimal depressive symptoms [F (2, 22) = .17, p = .845]. Participants reporting depressive symptoms consolidated significantly more neutral memories during SWS, and marginally more negative memories during REM sleep, than those reporting minimal depressive symptoms [t (21) = 2.44, p = .023; t (21) = 1.96, p = .064, respectively]. Our preliminary results demonstrate that REM sleep and SWS have differential effects on the consolidation of emotional and neutral images in participants reporting depressive symptoms. Further studies including larger sample sizes are required to investigate whether REM sleep alterations promote the development of negative memory bias in major depressive disorder.

Elevated cortisol awakening response associated with early life stress and impaired executive function in healthy adult males.

Experiencing early life stress (ELS) and subsequent dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis may play a role in the aetiology of mental health disorders. However, the exact mechanisms linking HPA-axis dysregulation with the development of psychopathology have not been fully delineated. Progress in this area is hampered by the complex and often conflicting associations found between markers of HPA-axis function and risk factors for mental health disorders such as impaired executive function (EF) and ELS. This study investigated the association of the cortisol awakening response (CAR) with ELS and EF in a healthy adult male population (n=109, aged 21-63). As previous inconsistencies in CAR and ELS association studies may be the result of not considering ELS-related factors such as cumulative exposure, type of stressor and developmental timing of ELS, these were also investigated. The main findings were that the CAR was significantly elevated in individuals reporting ELS compared to those reporting no ELS (p=0.007) and that an elevated CAR predicted poorer problem solving/planning (p=0.046). Cumulative exposure, type of stressor and developmental timing of ELS were also found to impact significantly on the CAR. These results suggest that ELS is associated with chronic changes in HPA-axis function and that these changes may be associated with impairments in problem solving/planning. Future work should investigate further the neurobiological mechanisms linking ELS, the CAR and EF and their role in conferring risk for the development of mental health disorders.

The effect of COMT Val158Met and DRD2 C957T polymorphisms on executive function and the impact of early life stress.

INTRODUCTION: Previous research has indicated that variation in genes encoding catechol-O-methyltransferase (COMT) and dopamine receptor D2 (DRD2) may influence cognitive function and that this may confer vulnerability to the development of mental health disorders such as schizophrenia. However, increasing evidence suggests environmental factors such as early life stress may interact with genetic variants in affecting these cognitive outcomes. This study investigated the effect of COMT Val158Met and DRD2 C957T polymorphisms on executive function and the impact of early life stress in healthy adults. METHODS: One hundred and twenty-two healthy adult males (mean age 35.2 years, range 21-63) were enrolled in the study. Cognitive function was assessed using Cambridge Neuropsychological Test Automated Battery and early life stress was assessed using the Childhood Traumatic Events Scale (Pennebaker & Susman, 1988). RESULTS: DRD2 C957T was significantly associated with executive function, with CC homozygotes having significantly reduced performance in spatial working memory and spatial planning. A significant genotype-trauma interaction was found in Rapid Visual Information Processing test, a measure of sustained attention, with CC carriers who had experienced early life stress exhibiting impaired performance compared to the CC carriers without early life stressful experiences. There were no significant findings for COMT Val158Met. CONCLUSIONS: This study supports previous findings that DRD2 C957T significantly affects performance on executive function related tasks in healthy individuals and shows for the first time that some of these effects may be mediated through the impact of childhood traumatic events. Future work should aim to clarify further the effect of stress on neuronal systems that are known to be vulnerable in mental health disorders and more specifically what the impact of this might be on cognitive function.

The 'affect tagging and consolidation' (ATaC) model of depression vulnerability.

Since the 1960's polysomnographic sleep research has demonstrated that depressive episodes are associated with REM sleep alterations. Some of these alterations, such as increased REM sleep density, have also been observed in first-degree relatives of patients and remitted patients, suggesting that they may be vulnerability markers of major depressive disorder (MDD), rather than mere epiphenomena of the disorder. Neuroimaging studies have revealed that depression is also associated with heightened amygdala reactivity to negative emotional stimuli, which may also be a vulnerability marker for MDD. Several models have been developed to explain the respective roles of REM sleep alterations and negatively-biased amygdala activity in the pathology of MDD, however the possible interaction between these two potential risk-factors remains uncharted. This paper reviews the roles of the amygdala and REM sleep in the encoding and consolidation of negative emotional memories, respectively. We present our 'affect tagging and consolidation' (ATaC) model, which argues that increased REM sleep density and negatively-biased amygdala activity are two separate, genetically influenced risk-factors for depression which interact to promote the development of negative memory bias - a well-known cognitive vulnerability marker for depression. Predictions of the ATaC model may motivate research aimed at improving our understanding of sleep dependent memory consolidation in depression aetiology.

The neuroproteomics of schizophrenia.

Proteomics is the study of global gene expression of an organ, body system, fluid, or cellular compartment at the protein level. Proteomic findings are reflective of complex gene × environment interactions, and the importance of this is increasingly appreciated in schizophrenia research. In this review, we outline the main proteomic methods available to researchers in this area and summarize, for the first time, the findings of the main quantitative neuroproteomic investigations of schizophrenia brain. Our review of these data revealed 16 gray matter proteins, and eight white matter proteins that were differentially expressed in the same direction in two or more investigations. Pathway analysis identified cellular assembly and organization as particularly disrupted in both gray and white matter, whereas the glycolysis-gluconeogenesis pathway was the major signaling pathway significantly altered in both. Reassuringly, these findings show remarkable convergence with functional pathways and positional candidate genes implicated from genomic studies. The specificity of schizophrenia proteomic findings are also addressed in the context of neuroproteomic investigations of neurodegenerative disorders and bipolar disorder. Finally, we discuss the major challenges in the field of neuroproteomics, such as the need for high throughput validation methods and optimal sample preparation. Future directions in the neuroproteomics of schizophrenia, including the use of blood-based biomarker work, the need to focus on subproteomes, and the increasing use of mass spectrometry-based methods are all discussed. This area of research is still in its infancy and offers huge potential to our understanding of schizophrenia on a cellular level.

Differential effects of wild-type and A53T mutant isoform of alpha-synuclein on the mitochondrial proteome of differentiated SH-SY5Y cells.

Increased levels of wild-type (WT) alpha-synuclein (alpha-syn) and mutant A53T alpha-syn are associated with Parkinson's disease (PD), a disease linked to abnormal mitochondrial function. This study compared mitochondria prepared from differentiated SH-SY5Y cells overexpressing WT or A53T alpha-syn with control cells, using 2-D difference in-gel electrophoresis. Statistical analysis was carried out primarily using ANOVA (p < 0.01; Host:WT:A53T) and subsequently using independent t tests (host vs WT, host vs A53T). Of the protein spots found to be differentially expressed (n = 71; p < 0.01, >1.8/<-1.8 fold change), 63 proteins were identified by LC-MS/MS, with the majority (77%) significantly altered in WT samples only. Twenty-three proteins known to be integral components of the mitochondria were abnormally expressed including those with roles in ATP synthesis, oxidoreduction, motor activity, carbohydrate metabolism, protein transcription, and protein folding. Thirteen forms of cytoskeletal proteins were also found to be overexpressed in the mitochondrial preparations from WT alpha-syn cells, suggesting an increased interaction of mitochondria with the cytoskeletal network. Altered levels of four mitochondrial proteins (HSPA9 (mortalin), NDUFS1, DLAT, ATP5A1) were confirmed using Western blot analysis. Furthermore, a significant reduction in OXPHOS 1 activity was observed in the WT alpha-syn cells, suggesting that there are functional consequences of the observed altered protein expression changes in the mitochondria.

alpha-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells.

Parkinson's disease (PD) is characterized in part by the presence of alpha-synuclein (alpha-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the alpha-synuclein gene (SNCA) are associated with familial PD. Since Ca2+ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca2+ homeostasis in cells stably transfected with either wild-type alpha-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca2+ influx evoked by exposure of cells to 50 mM K+ was enhanced in cells expressing all three forms of alpha-syn, an effect which was due specifically to increased Ca2+ entry via L-type Ca2+ channels. Mobilization of Ca2+ by muscarine was not strikingly modified by any of the alpha-syn forms, but they all reduced capacitative Ca2+ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca2+](i) in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca2+ content was unaffected by any form of alpha-synuclein. However, only WT alpha-syn transfected cells displayed significantly impaired viability. Our findings suggest that alpha-syn regulates Ca2+ entry pathways and, consequently, that abnormal alpha-syn levels may promote neuronal damage through dysregulation of Ca2+ homeostasis.

Proteomic analysis of increased Parkin expression and its interactants provides evidence for a role in modulation of mitochondrial function.

Parkin is an ubiquitin-protein ligase (E3), mutations of which cause juvenile onset - autosomal recessive Parkinson's disease, and result in reduced enzymic activity. In contrast, increased levels are protective against mitochondrial dysfunction and neurodegeneration, the mechanism of which is largely unknown. In this study, 2-DE and MS proteomic techniques were utilised to investigate the effects of increased Parkin levels on protein expression in whole cell lysates using in an inducible Parkin expression system in HEK293 cells, and also to isolate potential interactants of Parkin using tandem affinity purification and MS. Nine proteins were significantly differentially expressed (+/-2-fold change; p<0.05) using 2-DE analysis. MS revealed the identity of these proteins to be ACAT2, HNRNPK, HSPD1, PGK1, PRDX6, VCL, VIM, TPI1, and IMPDH2. The first seven of these were reduced in expression. Western blot analysis confirmed the reduction in one of these proteins (HNRNPK), and that its levels were dependent on 26S proteasomal activity. Tandem affinity purification/MS revealed 14 potential interactants of Parkin; CKB, DBT, HSPD1, HSPA9, LRPPRC, NDUFS2, PRDX6, SLC25A5, TPI1, UCHL1, UQCRC1, VCL, YWHAZ, YWHAE. Nine of these are directly involved in mitochondrial energy metabolism and glycolysis; four were also identified in the 2-DE study (HSP60, PRDX6, TPI1, and VCL). This study provides further evidence for a role for Parkin in regulating mitochondrial activity within cells.

Proteomic analysis reveals protein changes within layer 2 of the insular cortex in schizophrenia.

Abnormalities in the size and activity of the insular cortex (IC), a brain region involved in auditory hallucinations and language, have been previously found in brain imaging studies in schizophrenia. In addition, cortical layer 2 has been shown to be abnormal in many brain regions in schizophrenia. In this study, 2-D DIGE was used to quantitatively analyse protein expression in schizophrenia and control cases (n = 15/group) in microdissected layer 2 IC tissue. Proteomic analyses revealed 57 significantly differentially expressed (p<0.05) protein spots in schizophrenia. Validation of differential expression of two of the proteins differentially expressed was subsequently confirmed using Western blotting. This work provides evidence of abnormal protein expression in layer 2 of the IC in schizophrenia, supporting previous work of reduced neuronal size in this cortical layer in the IC. Over half of proteins abnormally expressed in this study have not been reported previously in proteomic studies investigating schizophrenia or neurodegenerative disorders. Proteins found to be abnormally expressed appear to collectively impact on neuronal plasticity through roles in neurite outgrowth, cellular morphogenesis and synaptic function.

BSPR/EBI 2007 meeting report--Integrative Proteomics: From Molecules to Systems. July 25-27, 2007 Wellcome Trust Conference Centre, Hinxton, UK.

This report reviews the joint British Society for Proteome Research (BSPR) and European Bioinformatics Institute (EBI) 2007 meeting, 'Integrative Proteomics: From Molecules to Systems' which took place at the Wellcome Trust Conference Centre, Hinxton, UK, from 25th to 27th July. The aim of this year's meeting was to explore how the integration of 'omic' technologies can lead to a comprehensive understanding of cellular organization, differentiation and signalling. Studies investigating protein-protein interactions and trafficking illustrated how the combination of proteomics and bioinformatics is allowing systems biology to develop as a discipline in its own right.

From genome to proteome: back to the future. Report on the 7th Siena meeting, September 3-7, 2006.

This report reviews the 7th Siena Meeting 'From Genome to Proteome: Back to the Future' which took place in Italy from 3-7 September, 2006. There was a significant rise in the number of delegates attending compared with previous Siena meetings. A diversity of speakers and presentations addressed the theme of the meeting in moving proteomics forward to integrate with biology as a whole entity rather than in isolated fractions. In addition, technological advancements in sample preparation and separation as well as identification were discussed.

Examination of 2-DE in the Human Proteome Organisation Brain Proteome Project pilot studies with the new RAIN gel matching technique.

The Human Proteome Organisation (HUPO) Brain Proteome Project (BPP) pilot studies have generated over 200 2-D gels from eight participating laboratories. This data includes 67 single-channel and 60 DIGE gels comparing 30 whole frozen C57/BL6 female mouse brains, ten each at embryonic day 16, postnatal day 7 (juvenile) and postnatal day 54-56 (adult); and ten single-channel and three DIGE gels comparing human epilepsy surgery of the temporal front lobe with a corresponding post-mortem specimen. The samples were generated centrally and distributed to the participating laboratories, but otherwise no restrictions were placed on sample preparation, running and staining protocols, nor on the 2-D gel analysis packages used. Spots were characterised by MS and the annotated gel images published on a ProteinScape web server. In order to examine the resultant differential expression and protein identifications, we have reprocessed a large subset of the gels using the newly developed RAIN (Robust Automated Image Normalisation) 2-D gel matching algorithm. Traditional approaches use symbolic representation of spots at the very early stages of the analysis, which introduces persistent errors due to inaccuracies in spot modelling and matching. With RAIN, image intensity distributions, rather than selected features, are used, where smooth geometric deformation and expression bias are modelled using multi-resolution image registration and bias-field correction. The method includes a new approach of volume-invariant warping which ensures the volume of protein expression under transformation is preserved. An image-based statistical expression analysis phase is then proposed, where small insignificant expression changes over one gel pair can be revealed when reinforced by the same consistent changes in others. Results of the proposed method as applied to the HUPO BPP data show significant intra-laboratory improvements in matching accuracy over a previous state-of-the-art technique, Multi-resolution Image Registration (MIR), and the commercial Progenesis PG240 package.