Not all voxels are created equal: Reducing estimation bias in regional NODDI metrics using tissue-weighted means.

Neurite orientation dispersion and density imaging (NODDI) estimates microstructural properties of brain tissue relating to the organisation and processing capacity of neurites, which are essential elements for neuronal communication. Descriptive statistics of NODDI tissue metrics are commonly analyzed in regions-of-interest (ROI) to identify brain-phenotype associations. Here, the conventional method to calculate the ROI mean weights all voxels equally. However, this produces biased estimates in the presence of CSF partial volume. This study introduces the tissue-weighted mean, which calculates the mean NODDI metric across the tissue within an ROI, utilising the tissue fraction estimate from NODDI to reduce estimation bias. We demonstrate the proposed mean in a study of white matter abnormalities in young onset Alzheimer's disease (YOAD). Results show the conventional mean induces significant bias that correlates with CSF partial volume, primarily affecting periventricular regions and more so in YOAD subjects than in healthy controls. Due to the differential extent of bias between healthy controls and YOAD subjects, the conventional mean under- or over-estimated the effect size for group differences in many ROIs. This demonstrates the importance of using the correct estimation procedure when inferring group differences in studies where the extent of CSF partial volume differs between groups. These findings are robust across different acquisition and processing conditions. Bias persists in ROIs at higher image resolution, as demonstrated using data obtained from the third phase of the Alzheimer's disease neuroimaging initiative (ADNI); and when performing ROI analysis in template space. This suggests that conventional ROI means of NODDI metrics are biased estimates under most contemporary experimental conditions, the correction of which requires the proposed tissue-weighted mean. The tissue-weighted mean produces accurate estimates of ROI means and group differences when ROIs contain voxels with CSF partial volume. In addition to NODDI, the technique can be applied to other multi-compartment models that account for CSF partial volume, such as the free water elimination method. We expect the technique to help generate new insights into normal and abnormal variation in tissue microstructure of regions typically confounded by CSF partial volume, such as those in individuals with larger ventricles due to atrophy associated with neurodegenerative disease.

Outcomes of a Community-Based Paediatric Weight Management Programme in an Irish Midlands Setting.

Ongoing investigation is needed into feasible approaches which reduce excess weight in childhood. This study aimed to assess the effectiveness of an adapted version of the Scottish Childhood Overweight Treatment Trial (SCOTT) in an Irish primary care setting. Families were offered monthly dietitian-led sessions for six months. These sessions targeted dietary habits, family meals, screen time and exercise. Of the 95 children (mean age 7.6 years) referred, 90.5% (n86) were obese and 9.5% (n9) were overweight. Fifty-one (53.7%) families opted into the programme from referral, and 18 completed the programme (64.7% attrition). Statistically significant reductions in body mass index (BMI) were observed between sessions one and six (25.7±4.2kg/m2 and 25.3±4.8kg/m2, respectively, p<0.01). BMI z-score modestly decreased by 0.2 (p=0.01). Despite these reductions, issues with programme referral, attrition and long-term effectiveness were evident. Further investigation into strategies which reduce paediatric overweight is warranted.

A targeted proteomic multiplex CSF assay identifies increased malate dehydrogenase and other neurodegenerative biomarkers in individuals with Alzheimer's disease pathology.

Alzheimer's disease (AD) is the most common cause of dementia. Biomarkers are required to identify individuals in the preclinical phase, explain phenotypic diversity, measure progression and estimate prognosis. The development of assays to validate candidate biomarkers is costly and time-consuming. Targeted proteomics is an attractive means of quantifying novel proteins in cerebrospinal and other fluids, and has potential to help overcome this bottleneck in biomarker development. We used a previously validated multiplexed 10-min, targeted proteomic assay to assess 54 candidate cerebrospinal fluid (CSF) biomarkers in two independent cohorts comprising individuals with neurodegenerative dementias and healthy controls. Individuals were classified as 'AD' or 'non-AD' on the basis of their CSF T-tau and amyloid Aß1-42 profile measured using enzyme-linked immunosorbent assay; biomarkers of interest were compared using univariate and multivariate analyses. In all, 35/31 individuals in Cohort 1 and 46/36 in Cohort 2 fulfilled criteria for AD/non-AD profile CSF, respectively. After adjustment for multiple comparisons, five proteins were elevated significantly in AD CSF compared with non-AD CSF in both cohorts: malate dehydrogenase; total APOE; chitinase-3-like protein 1 (YKL-40); osteopontin and cystatin C. In an independent multivariate orthogonal projection to latent structures discriminant analysis (OPLS-DA), these proteins were also identified as major contributors to the separation between AD and non-AD in both cohorts. Independent of CSF Aß1-42 and tau, a combination of these biomarkers differentiated AD and non-AD with an area under curve (AUC)=0.88. This targeted proteomic multiple reaction monitoring (MRM)-based assay can simultaneously and rapidly measure multiple candidate CSF biomarkers. Applying this technique to AD we demonstrate differences in proteins involved in glucose metabolism and neuroinflammation that collectively have potential clinical diagnostic utility.

Inter-laboratory study of human in vitro toxicogenomics-based tests as alternative methods for evaluating chemical carcinogenicity: a bioinformatics perspective.

The assessment of the carcinogenic potential of chemicals with alternative, human-based in vitro systems has become a major goal of toxicogenomics. The central read-out of these assays is the transcriptome, and while many studies exist that explored the gene expression responses of such systems, reports on robustness and reproducibility, when testing them independently in different laboratories, are still uncommon. Furthermore, there is limited knowledge about variability induced by the data analysis protocols. We have conducted an inter-laboratory study for testing chemical carcinogenicity evaluating two human in vitro assays: hepatoma-derived cells and hTERT-immortalized renal proximal tubule epithelial cells, representing liver and kidney as major target organs. Cellular systems were initially challenged with thirty compounds, genome-wide gene expression was measured with microarrays, and hazard classifiers were built from this training set. Subsequently, each system was independently established in three different laboratories, and gene expression measurements were conducted using anonymized compounds. Data analysis was performed independently by two separate groups applying different protocols for the assessment of inter-laboratory reproducibility and for the prediction of carcinogenic hazard. As a result, both workflows came to very similar conclusions with respect to (1) identification of experimental outliers, (2) overall assessment of robustness and inter-laboratory reproducibility and (3) re-classification of the unknown compounds to the respective toxicity classes. In summary, the developed bioinformatics workflows deliver accurate measures for inter-laboratory comparison studies, and the study can be used as guidance for validation of future carcinogenicity assays in order to implement testing of human in vitro alternatives to animal testing.

Growth and nutritional risk in children with developmental delay.

INTRODUCTION: Nutritional risk impacts outcome in developmental delay. The main objectives were to identify the incidence and factors contributing to growth faltering. METHODS: Clinical data review was completed for 500 patients with developmental delay accepted to an Early Intervention service. Data was collected using the standardised parent nutrition screening checklist prior to and at time of initial dietary assessment. Data was compared to nutritional assessment data, GOSH and FSAI dietary guidelines. Weight category was determined using RCPCH growth and BMI charts. Statistical analysis was conducted using Statistical Package for the Social Sciences version 20 (IBM Statistics 20.0). RESULTS: Nutritional risk was identified in 48.6 %. Weight categories were growth faltering (13.5 %), underweight (7.7 %), overweight (8.4 %) and obesity (4.3 %) at initial assessment. Growth faltering was correlated with age <1 year (p = 0.000) and with gestational age (p = 0.017) with highest rates identified in those born 32-36 weeks (3.6 %). Weight category was associated with introduction of solids pre 17 weeks recommendation (10.1 %), ANOVA demonstrating significance (P = 0.013). There was poor parental recognition of nutritional risk in 22.7 % of those assessed. Nutritional difficulties were common: 4.2 % were enterally fed, 7.7 % were on prescribed nutritional supplements, 29.1 % (n = 121) had feeding difficulties and 13.9 % (n = 58) had behavioural feeding difficulties. Iron intake did not meet the recommended intake in 20.9 % (n = 87), calcium in 4.5 % (n = 19). The prevalence of constipation was 21.6, 11.8 % of whom required medical management. CONCLUSIONS: Developmental delay predisposes to nutritional deficits which influence outcome. Screening, assessment and timely interventions are warranted to prevent poorer developmental outcomes.

γ-Secretase inhibition promotes fibrotic effects of albumin in proximal tubular epithelial cells.

BACKGROUND AND PURPOSE: Albuminuria is an important biomarker of renal dysfunction and is a major mediator of renal damage and fibrosis during kidney disease. The mechanisms underlying albumin-induced renal fibrosis remain unclear. There has been significant interest in γ-secretase activity in tubular epithelial cells in recent times; however, its potential role in albumin-induced fibrosis has not been investigated. EXPERIMENTAL APPROACH: The primary aim of this study was to examine the role of γ-secretase in albumin-induced fibrotic effects in proximal tubular cells. The effects of increasing albumin concentrations on fibrosis indicators and mediators in the human HK-2 cell line were examined in the presence and absence of a γ-secretase inhibitor, compound E. KEY RESULTS: Treatment with albumin resulted in a number of pro-fibrotic effects, including up-regulation of fibronectin, TGF-ß1 and the EGF-R. Interestingly, similar effects were observed in response to treatment with the γ-secretase inhibitor, compound E. Co-treatment of cells with albumin and an EGF-R inhibitor, AG-1478, resulted in significant inhibition of the observed pro-fibrotic effects, suggesting a major role for the EGF-R in albumin-induced fibrotic events. Albumin-induced effects on the EGF-R appeared to be mediated through inhibition of γ-secretase activity and were dependent on ERK-MAPK signalling. CONCLUSIONS AND IMPLICATIONS: These results provide novel insights into the mechanisms of albumin-induced fibrotic effects in tubular epithelial cells, suggesting important roles for the γ-secretase and the EGF-R. These results suggest that the proposed use of γ-secretase inhibitors as anti-fibrotic agents requires further investigation.

Sustained benefits of a community dietetics intervention designed to improve oral nutritional supplement prescribing practices.

BACKGROUND: Healthcare professionals working in the community do not always prescribe oral nutritional supplements (ONS) according to best practice guidelines for the management of malnutrition. The present study aimed to determine the impact of a community dietetics intervention on ONS prescribing practices and expenditure 1 year later. METHODS: The intervention involved general practitioners (GPs), practice nurses, nurses in local nursing homes and community nurses. It comprised an education programme together with the provision of a new community dietetics service. Changes in health care professionals' nutrition care practices were determined by examining community dietetics records. ONS prescribing volume and expenditure on ONS were assessed using data from the Primary Care Reimbursement Service of the Irish Health Service Executive. RESULTS: Seven out of 10 principal GPs participated in the nutrition education programme. One year later, screening for malnutrition risk was better, dietary advice was provided more often, referral to the community dietetics service improved and ONS were prescribed for a greater proportion of patients at 'high risk' of malnutrition than before (88% versus 37%; P < 0.001). There was a trend towards fewer patients being prescribed ONS (18% reduction; P = 0.074) and there was no significant change in expenditure on ONS by participating GPs (3% reduction; P = 0.499), despite a 28% increase nationally by GPs on ONS. CONCLUSIONS: The community dietetics intervention improved ONS prescribing practices by GPs and nurses, in accordance with best practice guidelines, without increasing expenditure on ONS during the year after intervention.

An evaluation of a community dietetics intervention on the management of malnutrition for healthcare professionals.

BACKGROUND: Healthcare professionals working in the community setting have limited knowledge of the evidence-based management of malnutrition. The present study aimed to evaluate a community dietetics intervention, which included an education programme for healthcare professionals in conjunction with the introduction of a community dietetics service for patients 'at risk' of malnutrition. Changes in nutritional knowledge and the reported management of malnourished patients were investigated and the acceptability of the intervention was explored. METHODS: An education programme, incorporating 'Malnutrition Universal Screening Tool (MUST)' training, was implemented in eight of 10 eligible primary care practices (14 general practitioners and nine practice nurses attended), in seven private nursing homes (20 staff nurses attended) and two health centres (53 community nurses attended) in conjunction with a community dietetics service for patients at risk of malnutrition. Nutritional knowledge was assessed before, immediately after, and 6 months after the intervention using self-administered, multiple-choice questionnaires. Reported changes in practice and the acceptability of the education programme were considered using self-administered questionnaires 6 months after the intervention. RESULTS: A significant increase in nutritional knowledge 6 months after the intervention was observed (P < 0.001). The management of malnutrition was reported to be improved, with 69% (38/55) of healthcare professionals reporting to weigh patients 'more frequently', whereas 80% (43/54) reported giving dietary advice to prevent or treat malnutrition. Eighty-percent (44/55) of healthcare professionals stated that 'MUST' was an acceptable nutrition screening tool. CONCLUSION: An education programme supported by a community dietetics service for patients 'at risk' of malnutrition increased the nutritional knowledge and improved the reported management of malnourished patients in the community by healthcare professionals.

The use of oral nutritional supplements in an Irish community setting.

BACKGROUND: The frequency of oral nutritional supplement (ONS) prescribing has been increasing steadily in the Republic of Ireland (ROI). Available evidence indicates that health professionals in the community setting in the ROI have a poor level of knowledge about ONS. The objectives of the present study were to investigate ONS prescribing practices and to identify the types of patient who were prescribed these products. METHODS: Ten of 17 eligible general practitioners were recruited and asked to refer all patients (aged > 16 years) who were prescribed ONS during a 3-month period. Patients were interviewed by a community dietitian, using a questionnaire incorporating the Malnutrition Universal Screening Tool (MUST). ONS prescriptions were judged either to fulfil or not to fulfil a set of criteria developed for ONS prescribing in the community. RESULTS: The majority of patients were female (62/78). Their mean (SD) age was 79 (10.5) years. According to MUST criteria, 31 of 78 patients were at 'low risk', 18 of 78 were at 'medium risk' and 29 of 78 were at 'high risk' of malnutrition. Less than half of the patients (36/78) had a body mass index of < 20 kg m(-2). Only 21 of 78 patients reported having received dietary advice in addition to their ONS prescription. Almost one-third (31%) of ONS prescriptions did not fulfil the criteria. Social factors, such as living alone, and difficulties with cooking and shopping, influenced the need for ONS in almost 70% of cases. CONCLUSIONS: ONS were prescribed in accordance with the prescribing criteria in the majority of cases; however, some patients who were prescribed ONS were not 'at risk' of malnutrition. Social circumstances played an important part in determining the need for ONS prescriptions.

Quality of life and economic impact of switching from established infliximab therapy to adalimumab in patients with rheumatoid arthritis.

OBJECTIVE: To evaluate the quality of life and economic impact of switching therapy from infliximab to adalimumab in patients with rheumatoid arthritis (RA). METHODS: In this open-label study, patients demonstrating a clinical response to infliximab were switched to treatment with adalimumab and followed for 16 weeks. Both generic (Health Assessment Questionnaire and Short Form 36 Physical Component Summary and Mental Component Summary) and specific (Rheumatoid Arthritis Quality of Life questionnaire) assessment instruments of physical function and of quality of life were employed. An economic analysis of treatment-related costs was also performed. Disease activity was assessed by the composite 28-joint count Disease Activity Score (DAS28). C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) were measured as acute phase markers. RESULTS: Nineteen patients were enrolled and completed the study. No changes in functional and quality-of-life measures were observed. One-year extrapolation data showed potential reductions in costs following switching to adalimumab that could be attributed primarily to reductions in patient- and staff-related costs. Safety and tolerability were similar for both treatments. Although there was a significant reduction in DAS28 (P < 0.005) and CRP (P < 0.001) after switching to adalimumab, there were no significant changes in individual DAS28 components, including swollen and tender joint counts and ESR. CONCLUSIONS: A switch from infliximab to adalimumab in patients with RA who have responded to infliximab is a feasible, well-tolerated treatment option, with the potential for direct and indirect economic advantages.

Kappa-casein interactions in the suspension of the two major calcium-sensitive human beta-caseins.

The possible effects of both the beta-casein (beta-CN) phosphorylation level and the kappa-CN glycosylation level on micelle formation were studied using the doubly-phosphorylated form (beta-CN-2P) and the quadruply-phosphorylated form (beta-CN-4P) of human beta-CN, along with bovine kappa-CN to compare with previous studies using the more highly glycosylated human kappa-CN. Addition of bovine kappa-CN to human beta-CN-2P, beta-CN-4P, or a 1/1 (wt/wt) mixture of the two was at kappa/beta molar ratios from 0.0 to approximately 0.6 and micelles were reconstituted by addition of Ca+2 either directly at 37 degrees C for determination of the fraction suspended or at an initial temperature of 4 degrees that was gradually increased to 37 degrees C with the change in particle size monitored by turbidity measurements. Analysis of the data indicates that the 4P form requires more kappa-CN for stabilization than the 2P form but that the mixture of the two is more like the 4P form in that lateral kappa-kappa interactions may enhance beta-kappa interactions and micelle formation. Above a kappa/beta molar ratio of about 0.2, the caseins were fully suspended into reconstituted micelles. However, micelle size decreased at a higher ratio, indicating that the kappa-CN probably occupies a surface position and may regulate micelle size by its relative abundance. A comparison with published results suggests that the higher glycosylation level of human kappa-CN may protect a larger surface area and result in smaller micelles. Changes in reconstituted micelle size with pH indicate that positively charged groups in the kappa-CN may interact with the negatively charged phosphate esters in the beta-CN moieties in addition to kappa-beta hydrophobic interactions.

The use of lithium chloride to study human milk micelles.

Various methods have been used to study the dissociation of milk micelles in attempts to determine their structure and the interactions that stabilize them. These include the addition of urea, cooling to alter hydrophobic bonding, the addition of EDTA to sequester calcium, and changes in pH to alter molecular charge. For this study, the mild chaotropic agent LiCl was added to human milk micelles, and measurements were made on the relative percentages of the six different phosphorylation levels of beta-casein (CN) at various LiCl concentrations for different lengths of time and at different temperatures. Added LiCl had little effect at 37 degrees C but caused maximal dissociation, mainly of the beta-CN species with higher phosphorylation levels, at 23 degrees C and 4 degrees C between 1 and 2 M concentration. Comparison was made with 2-M additions of NaCl, MgCl2, and KCl at 4 degrees C, with LiCl showing the only appreciable change. The results suggest that Li+ may displace Ca2+ in protein-Ca2+-protein or protein-colloidal calcium phos+ phate-protein salt bridges and that the nonphosphorylated form of human beta-CN may change its conformation and mode of interaction upon phosphorylation. Lithium chloride may be useful to study the dissociation of the different CN in bovine milk micelles.

Formation of reconstituted casein micelles with human beta-caseins and bovine kappa-casein.

Human beta-casein (CN) is the major protein of the human milk casein fraction (approximately 80%) and exists in six calcium-sensitive forms, having zero to five organic phosphates per molecule. The major forms are the doubly-phosphorylated (beta-CN-2P; approximately 30%) and the quadruply phosphorylated (beta-CN-4P; approximately 35%) forms. Although calcium-insensitive, kappa-CN is known for its role in preventing the precipitation of beta-CN in the presence of Ca+2, but it is not known how the different levels of phosphorylation may affect this. In the present investigation, turbidity, measured at 400 nm, was determined at increasing temperatures (4 up to 37 degrees C) for solutions of beta-CN-2P and beta-CN-4P (3 mg/ml in 0.02 M NaCl, 0.01 M imidazole, pH 7) individually and also mixed with bovine kappa-CN in 6/1 and 3/1 weight ratios of beta/kappa and containing 0, 5, and 10 mM Ca+2. The results indicate that the first step of micelle formation probably leads to polymers of limited size, the only complexes available to beta-CN-2P under most conditions. With beta-CN-4P, these polymers aggregate further to give reconstituted micelles, probably because of the ability to form crosslinks at this phosphorylation level. The formation of reconstituted micelles under various conditions of pH, Ca+2 concentration and kappa-CN content indicates that both hydrophobic interactions and Ca+2 bridges or crosslinks may contribute to protein aggregation and micelle building.

Association of mixtures of the two major forms of beta-casein from human milk.

Human milk beta-casein (CN) is unique in that it may be phosphorylated at any level from zero (beta-CN-0P) to five (beta-CN-5P) organic phosphates per molecule. The 2P and 4P forms are the major components, with about 30 to 35% each. Here, we present the association properties of mixtures of these two moieties of human beta-CN. The aggregation patterns, as functions of temperature and ionic strength of these mixtures, generally follow those for the individual components. However, the mixtures yielded polymers with slightly different properties, which indicates extensive interaction between the two. Some properties of the mixtures were more like those for the 2P form, such as association in low salt buffer to give a peak with a sedimentation coefficient, s20,w, of approximately 11 S, in contrast to the 2P form alone with a peak of approximately 13 S and 4P alone with only a small amount of material with s20,w greater than 2 S at 27 degrees C. The solubility and interactions in the presence of Ca2+ ions were intermediate but more like the 4P form. A protein-concentration dependence for s20,w was seen, and laser light scattering indicated that there was an increase in size and/or a change in shape as the protein concentration increased. From the results, it is apparent that submicellar oligomers are probably formed by rapidly established equilibrium association reactions. The presence of an equal amount of the 2P form along with the 4P form does not appear to be a disadvantage in casein micelle formation and function.

Analysis of allosteric effector binding sites of potato ADP-glucose pyrophosphorylase through reverse genetics.

ADP-glucose pyrophosphorylase (AGPase) is a key regulatory enzyme of bacterial glycogen and plant starch synthesis as it controls carbon flux via its allosteric regulatory behavior. Unlike the bacterial enzyme that is composed of a single subunit type, the plant AGPase is a heterotetrameric enzyme (alpha2beta2) with distinct roles for each subunit type. The large subunit (LS) is involved mainly in allosteric regulation through its interaction with the catalytic small subunit (SS). The LS modulates the catalytic activity of the SS by increasing the allosteric regulatory response of the hetero-oligomeric enzyme. To identify regions of the LS involved in binding of effector molecules, a reverse genetics approach was employed. A potato (Solanum tuberosum L.) AGPase LS down-regulatory mutant (E38A) was subjected to random mutagenesis using error-prone polymerase chain reaction and screened for the capacity to form an enzyme capable of restoring glycogen production in glgC(-) Escherichia coli. Dominant mutations were identified by their capacity to restore glycogen production when the LS containing only the second site mutations was co-expressed with the wild-type SS. Sequence analysis showed that most of the mutations were decidedly nonrandom and were clustered at conserved N- and C-terminal regions. Kinetic analysis of the dominant mutant enzymes indicated that the K(m) values for cofactor and substrates were comparable with the wild-type AGPase, whereas the affinities for activator and inhibitor were altered appreciably. These AGPase variants displayed increased resistance to P(i) inhibition and/or greater sensitivity toward 3-phosphoglyceric acid activation. Further studies of Lys-197, Pro-261, and Lys-420, residues conserved in AGPase sequences, by site-directed mutagenesis suggested that the effectors 3-phosphoglyceric acid and P(i) interact at two closely located binding sites.

In nonhepatic cells, cholesterol 7alpha-hydroxylase induces the expression of genes regulating cholesterol biosynthesis, efflux, and homeostasis.

CHO cells expressing the liver-specific gene product cholesterol-7alpha-hydroxylase showed a 6-fold increase in the biosynthesis of [(14)C]cholesterol from [(14)C]acetate, as well as increased enzymatic activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and squalene synthase. Cells expressing cholesterol-7alpha-hydroxylase contained less sterol response element-binding protein 1 (SREBP1) precursor, whereas the cellular content of mature SREBP1, as well as the mRNAs of cholesterol biosynthetic genes (HMG-CoA reductase and squalene synthase), were all increased approximately 3-fold. Cells expressing cholesterol-7alpha-hydroxylase displayed greater activities of luciferase reporters containing the SREBP-dependent promoter elements derived from HMG-CoA reductase and farnesyl diphosphate synthase, in spite of accumulating significantly more free and esterified cholesterol and 7alpha-hydroxycholesterol. While cells expressing cholesterol-7alpha-hydroxylase displayed increased SREBP-dependent transcription, sterol-mediated repression of SREBP-dependent transcription by LDL-cholesterol and exogenous oxysterols was similar in both cell types. Cells expressing cholesterol-7alpha-hydroxylase displayed greater rates of secretion of cholesterol as well as increased expression of the ABC1 cassette protein mRNA. Adding 25-hydroxycholesterol to the culture medium of both cell types increased the expression of ABC1 cassette protein mRNA. The combined data suggest that in nonhepatic CHO cells multiple regulatory processes sensitive to cellular sterols act independently to coordinately maintain cellular cholesterol homeostasis.