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1.  Quantitative and qualitative preparations of bacterial outer membrane vesicles 
Gram-negative bacterial outer membrane vesicle production and function have been studied using a variety of quantitative and qualitative methods. These types of analyses can be hampered by the use of impure vesicle preparations. Here we describe a set of techniques that are useful for the quantitative analysis of vesicle production and for preparative yields of highly purified vesicles for studies of vesicle function or composition. Procedures and advice are also included for the purification of vesicles from encapsulated and low-yield strains.
PMCID: PMC4317262  PMID: 23299740
Gram-negative bacteria; outer membrane; vesicle; bleb; Escherichia coli; Pseudomonas aeruginosa; Klebsiella pneumoniae
2.  Exercise Training during Normobaric Hypoxic Confinement Does Not Alter Hormonal Appetite Regulation 
PLoS ONE  2014;9(6):e98874.
Both exposure to hypoxia and exercise training have the potential to modulate appetite and induce beneficial metabolic adaptations. The purpose of this study was to determine whether daily moderate exercise training performed during a 10-day exposure to normobaric hypoxia alters hormonal appetite regulation and augments metabolic health.
Fourteen healthy, male participants underwent a 10-day hypoxic confinement at ∼4000 m simulated altitude (FIO2 = 0.139±0.003%) either combined with daily moderate intensity exercise (Exercise group; N = 8, Age = 25.8±2.4 yrs, BMI = 22.9±1.2 kg·m−2) or without any exercise (Sedentary group; N = 6 Age = 24.8±3.1 yrs, BMI = 22.3±2.5 kg·m−2). A meal tolerance test was performed before (Pre) and after the confinement (Post) to quantify fasting and postprandial concentrations of selected appetite-related hormones and metabolic risk markers. 13C-Glucose was dissolved in the test meal and 13CO2 determined in breath samples. Perceived appetite ratings were obtained throughout the meal tolerance tests.
While body mass decreased in both groups (−1.4 kg; p = 0.01) following the confinement, whole body fat mass was only reduced in the Exercise group (−1.5 kg; p = 0.01). At Post, postprandial serum insulin was reduced in the Sedentary group (−49%; p = 0.01) and postprandial plasma glucose in the Exercise group (−19%; p = 0.03). Fasting serum total cholesterol levels were reduced (−12%; p = 0.01) at Post in the Exercise group only, secondary to low-density lipoprotein cholesterol reduction (−16%; p = 0.01). No differences between groups or testing periods were noted in fasting and/or postprandial concentrations of total ghrelin, peptide YY, and glucagon-like peptide-1, leptin, adiponectin, expired 13CO2 as well as perceived appetite ratings (p>0.05).
These findings suggest that performing daily moderate intensity exercise training during continuous hypoxic exposure does not alter hormonal appetite regulation but can improve the lipid profile in healthy young males.
PMCID: PMC4041840  PMID: 24887106
3.  A prospective randomised study comparing oral 13C-bicarbonate tracer technique versus indirect calorimetry for measurement of energy expenditure in adults 
e-SPEN journal  2012;7(1):e1-e4.
Background and Aims
Accurate assessment of energy expenditure (EE) is important in guiding nutritional therapy but current methods are unsatisfactory. This study compared the oral 13C-bicarbonate tracer (BT) technique using the IRIS® system (Wagner, Germany) against indirect calorimetry (IC, ventilated-hood) to measure CO2 output (VCO2) and thus estimate EE.
Ten overnight-fasted healthy male volunteers were randomised to studies at rest or mild exercise in a crossover manner. During each study BT-IRIS® and IC were used simultaneously to measure VCO2 and thus EE. Participants ingested a drink labelled with 50mg 13C-bicarbonate and breath samples were collected every 5 min for 180 min and analysed using IRIS®. Bland-Altman plots were used to assess agreement between the two techniques in measurements of VCO2 (L/day) and estimates of EE (kJ/day).
Mean ± SE age and BMI of participants were 21.1 ± 1.1 yrs and 23.6 ± 0.6 kg/m2. Both at rest and exercise, there was small bias but overall poor agreement between the two techniques as evident by the wide 95% limits of agreement in measurements of VCO2 and EE: rest VCO2 (bias 1.4, SD 93, 95% limits of agreement −180 to 183), rest EE (−8.3, 1830, −3595 to 3578), exercise VCO2 (49.3, 66.1, −80.4 to 178.9) and exercise EE (1083, 1944, −2727 to 4893). Furthermore, there was also evidence of systematic error in these measurements.
Prior to clinical application, further optimisation of the BT-IRIS® system should be undertaken, given the poor agreement with IC in measuring VCO2 and estimating EE.
PMCID: PMC4018572  PMID: 24834380
energy expenditure; bicarbonate; tracer; calorimetry; exercise; rest; adult
4.  Stress-Induced Outer Membrane Vesicle Production by Pseudomonas aeruginosa 
Journal of Bacteriology  2013;195(13):2971-2981.
As an opportunistic Gram-negative pathogen, Pseudomonas aeruginosa must be able to adapt and survive changes and stressors in its environment during the course of infection. To aid survival in the hostile host environment, P. aeruginosa has evolved defense mechanisms, including the production of an exopolysaccharide capsule and the secretion of a myriad of degradative proteases and lipases. The production of outer membrane-derived vesicles (OMVs) serves as a secretion mechanism for virulence factors as well as a general bacterial response to envelope-acting stressors. This study investigated the effect of sublethal physiological stressors on OMV production by P. aeruginosa and whether the Pseudomonas quinolone signal (PQS) and the MucD periplasmic protease are critical mechanistic factors in this response. Exposure to some environmental stressors was determined to increase the level of OMV production as well as the activity of AlgU, the sigma factor that controls MucD expression. Overexpression of AlgU was shown to be sufficient to induce OMV production; however, stress-induced OMV production was not dependent on activation of AlgU, since stress caused increased vesiculation in strains lacking algU. We further determined that MucD levels were not an indicator of OMV production under acute stress, and PQS was not required for OMV production under stress or unstressed conditions. Finally, an investigation of the response of P. aeruginosa to oxidative stress revealed that peroxide-induced OMV production requires the presence of B-band but not A-band lipopolysaccharide. Together, these results demonstrate that distinct mechanisms exist for stress-induced OMV production in P. aeruginosa.
PMCID: PMC3697536  PMID: 23625841
5.  Molecular genetic diagnostic techniques in choroideremia 
Molecular Vision  2014;20:535-544.
To optimize and streamline molecular genetics techniques in diagnosing choroideremia (CHM).
PCR primers were designed for exons 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 15 of the CHM gene. Each PCR protocol was optimized so that all exons could be amplified with the same component ratio and PCR conditions. Sense and antisense primers were tested for their ability to be used as sequencing primers. Fibroblast cells were cultured, and an immunoblot analysis was performed to detect the presence or absence of Rab escort protein 1 (REP-1) in a suspected CHM patient sample when no mutation was detected with sequencing. Multiplex ligation-dependent probe amplification (MLPA) of the CHM gene was performed and used to detect deletions and duplications in affected males and female carriers. RNA analysis using cDNA was used to detect the presence or absence of the CHM transcript and to search for splice defects.
The newly designed PCR primers allow for more efficient PCR preparation and sequencing to detect point mutations in affected males and female carriers. Immunoblot successfully detects the absence of REP-1 in a CHM patient. MLPA identifies deletions and duplications spanning multiple exons in the CHM gene. RNA analysis aids in detecting splice variants.
The development of new molecular biology techniques and ongoing optimization of existing methods allows for an improved integrated approach to confirm CHM diagnosis and carrier status in consideration of patient family history and available patient sample materials. CHM can be confirmed with an immunoblot assay. To detect the molecular cause of CHM, an examination of the genomic DNA or the mRNA must be performed. Presymptomatic carriers with no identifiable fundus signs can be identified only through molecular analysis of genomic DNA or through quantitative assays.
PMCID: PMC4000712  PMID: 24791138
6.  Offense and defense: microbial membrane vesicles play both ways 
Research in microbiology  2012;163(9-10):607-618.
Microbes have evolved over millennia to become adapted and specialized to the environments that they occupy. These environments may include water or soil, extreme environments such as hydrothermal vents, and can even include a host organism. To become adapted to these locations, microbes have evolved specific tools to mediate interactions with the environment. One such tool that prokaryotes have evolved includes the production of membrane vesicles (MVs). MVs are 10–300 nm spherical blebs derived from the outermost membrane and have known functions in protein secretion, immune activation and suppression, stress response, attachment, internalization and virulence. In this review, we consider the highly conserved role of membrane vesicles derived from Gram-negative, Gram-positive and archaeal species as a mechanism to facilitate intermicrobial and microbe-host interaction. We examine both the offensive and defensive capabilities of MVs in regard to the interaction of MVs with both host and microbial cells in their environment.
PMCID: PMC3518640  PMID: 23123555
OMV; MV; Bleb; Bacterial pathogenesis; Archaea; biofilm; Antibiotic resistance; Envelope stress response
7.  High-Resolution Images of Retinal Structure in Patients with Choroideremia 
To study retinal structure in choroideremia patients and carriers using high-resolution imaging techniques.
Subjects from four families (six female carriers and five affected males) with choroideremia (CHM) were characterized with best-corrected visual acuity (BCVA), kinetic and static perimetry, full-field electroretinography, and fundus autofluorescence (FAF). High-resolution macular images were obtained with adaptive optics scanning laser ophthalmoscopy (AOSLO) and spectral domain optical coherence tomography (SD-OCT). Coding regions of the CHM gene were sequenced.
Molecular analysis of the CHM gene identified a deletion of exons 9 to 15 in family A, a splice site mutation at position 79+1 of exon 1 in family B, deletion of exons 6 to 8 in family C, and a substitution at position 106 causing a premature stop in family D. BCVA ranged from 20/16 to 20/63 in carriers and from 20/25 to 5/63 in affected males. FAF showed abnormalities in all subjects. SD-OCT showed outer retinal layer loss, outer retinal tubulations at the margin of outer retinal loss, and inner retinal microcysts. Patchy cone loss was present in two symptomatic carriers. In two affected males, cone mosaics were disrupted with increased cone spacing near the fovea but more normal cone spacing near the edge of atrophy.
High-resolution retinal images in CHM carriers and affected males demonstrated RPE and photoreceptor cell degeneration. As both RPE and photoreceptor cells were affected, these cell types may degenerate simultaneously in CHM. These findings provide insight into the effect of CHM mutations on macular retinal structure, with implications for the development of treatments for CHM. ( number, NCT00254605.)
High-resolution retinal images in choroideremia carriers and affected males demonstrated degeneration of retinal pigment epithelial and photoreceptor cells. The findings illustrate the effect of CHM mutations on macular cone structure, with implications for the development of treatments for CHM.
PMCID: PMC3564452  PMID: 23299470
8.  Effect of (S)-3,5-DHPG on MicroRNA Expression in Mouse Brain 
Experimental Neurology  2012;235(2):497-507.
MicroRNAs are small non-coding RNAs that regulate post-transcriptional gene expression. In the short time since the discovery of microRNAs, the literature has burgeoned with studies focused on the biosynthesis of microRNAs, target prediction and binding, and mechanisms of translational repression by microRNAs. Given the prominent role of microRNAs in all areas of cell biology, it is not surprising that microRNAs are also linked to human diseases, including those of the nervous system. One of the least-studied areas of microRNA research is how their expression is regulated outside of development and cancer. Thus, we examined a role for regulation of microRNAs by neurotransmitter receptor activation in mouse brain. We focused on the group I metabotropic glutamate receptors by using intracerebroventricular injection of the selective agonist, (S)-3,5-dihydroxyphenylglycine (DHPG) in mouse brain. We then examined the expression of microRNAs in the cerebral cortex by Ambion and Invitrogen microarrays, and the expression of mature microRNA sequences by SABiosciences qPCR arrays, at 4, 8 and 24 hours after DHPG injection. These studies revealed that the largest number of significantly regulated microRNAs was detected 8 hours after DHPG injection in the microarrays and qPCR arrays. We then used RNA blots to quantify microRNA expression, and in situ hybridization to examine cellular distribution of the microRNAs regulated by DHPG. Bioinformatic analysis of the microRNAs regulated 8 hours after DHPG in all three arrays revealed KEGG pathways that are known to correlate with group I mGluR effects, as well as recently described and novel pathways. These studies are the first to show that DHGP regulates the expression of microRNAs in mouse cerebral cortex, and support the hypothesis that group I mGluRs may regulate microRNA expression in mouse brain.
PMCID: PMC3345043  PMID: 22309833
9.  Social laughter is correlated with an elevated pain threshold 
Although laughter forms an important part of human non-verbal communication, it has received rather less attention than it deserves in both the experimental and the observational literatures. Relaxed social (Duchenne) laughter is associated with feelings of wellbeing and heightened affect, a proximate explanation for which might be the release of endorphins. We tested this hypothesis in a series of six experimental studies in both the laboratory (watching videos) and naturalistic contexts (watching stage performances), using change in pain threshold as an assay for endorphin release. The results show that pain thresholds are significantly higher after laughter than in the control condition. This pain-tolerance effect is due to laughter itself and not simply due to a change in positive affect. We suggest that laughter, through an endorphin-mediated opiate effect, may play a crucial role in social bonding.
PMCID: PMC3267132  PMID: 21920973
laughter; positive affect; pain threshold; endorphins; social bonding
10.  You Spin Me Right Round: Cross-Relationship Variability in Interpersonal Emotion Regulation 
Individuals use a range of interpersonal emotion regulation strategies to influence the feelings of others, e.g., friends, family members, romantic partners, work colleagues. But little is known about whether people vary their strategy use across these different relational contexts. We characterize and measure this variability as “spin,” i.e., the extent of dispersion in a person’s interpersonal emotion regulation strategy use across different relationships, and focus on two key questions. First, is spin adaptive or maladaptive with regard to personal well-being and relationship quality? Second, do personality traits that are considered important for interpersonal functioning (i.e., empathy, attachment style) predict spin? The data used in this study is drawn from a large online survey. A key contribution of this study is to reveal that people who varied the type of strategies they used across relationships (i.e., those with high spin) had lower positive mood, higher emotional exhaustion, and less close relationships. A further key contribution is to show that spin was associated with low empathic concern and perspective taking and high anxious attachment style. High variability in interpersonal emotion regulation strategies across relationships therefore appears to be maladaptive both personally and socially.
PMCID: PMC3465984  PMID: 23060849
interpersonal emotion regulation; emotion regulation; interpersonal behavior; spin; relationships
11.  Mutations in RLBP1 associated with fundus albipunctatus in consanguineous Pakistani families 
The British journal of ophthalmology  2011;95(7):1019-1024.
To identify disease-causing mutations in two consanguineous Pakistani families with fundus albipunctatus.
Affected individuals in both families underwent a thorough clinical examination including funduscopy and electroretinography. Blood samples were collected from all participating members and genomic DNA was extracted. Exclusion analysis was completed with microsatellite short tandem repeat markers that span all reported loci for fundus albipunctatus. Two-point logarithm of odds (LOD) scores were calculated, and coding exons and exon–intron boundaries of RLBP1 were sequenced bi-directionally.
The ophthalmic examination of affected patients in both families was consistent with fundus albipunctatus. The alleles of markers on chromosome 15q flanking RLBP1 segregated with the disease phenotype in both families and linkage was further confirmed by two-point LOD scores. Bi-directional sequencing of RLBP1 identified a nonsense mutation (R156X) and a missense mutation (G116R) that segregated with the disease phenotype in their respective families.
These results strongly suggest that mutations in RLBP1 are responsible for fundus albipunctatus in the affected individuals of these consanguineous Pakistani families.
PMCID: PMC3459316  PMID: 21447491
12.  Splenectomy as an effective debulking therapy for disseminated mould infection in acute myeloid leukaemia following adjuvant therapy with interferon gamma and liposomal amphotericin 
Medical Mycology Case Reports  2012;1(1):82-84.
Invasive fungal infection is a major cause of morbidity and mortality in patients receiving treatment for Acute Myeloid Leukaemia (AML). Herein, we report a case of a 21 year old woman with an extremely resistant Fusarium species that responded to the addition of interferon gamma to her medical therapy, subsequently allowing definitive debulking surgery of her invasive Fusarium infection to be undertaken.
PMCID: PMC3854636  PMID: 24371746
Fusarium solani; Acute myeloid leukaemia; Interferon gamma
13.  TFAP2B Influences the Effect of Dietary Fat on Weight Loss under Energy Restriction 
PLoS ONE  2012;7(8):e43212.
Numerous gene loci are related to single measures of body weight and shape. We investigated if 55 SNPs previously associated with BMI or waist measures, modify the effects of fat intake on weight loss and waist reduction under energy restriction.
Methods and Findings
Randomized controlled trial of 771 obese adults. (Registration: ISRCTN25867281.) One SNP was selected for replication in another weight loss intervention study of 934 obese adults. The original trial was a 10-week 600 kcal/d energy-deficient diet with energy percentage from fat (fat%) in range of 20–25 or 40–45. The replication study used an 8-weeks diet of 880 kcal/d and 20 fat%; change in fat% intake was used for estimation of interaction effects. The main outcomes were intervention weight loss and waist reduction. In the trial, mean change in fat% intake was −12/+4 in the low/high-fat groups. In the replication study, it was −23/−12 among those reducing fat% more/less than the median. TFAP2B-rs987237 genotype AA was associated with 1.0 kg (95% CI, 0.4; 1.6) greater weight loss on the low-fat, and GG genotype with 2.6 kg (1.1; 4.1) greater weight loss on the high-fat (interaction p-value; p = 0.00007). The replication study showed a similar (non-significant) interaction pattern. Waist reduction results generally were similar. Study-strengths include (i) the discovery study randomised trial design combined with the replication opportunity (ii) the strict dietary intake control in both studies (iii) the large sample sizes of both studies. Limitations are (i) the low minor allele frequency of the TFAP2B polymorphism, making it hard to investigate non-additive genetic effects (ii) the different interventions preventing identical replication-discovery study designs (iii) some missing data for non-completers and dietary intake. No adverse effects/outcomes or side-effects were observed.
Under energy restriction, TFAP2B may modify the effect of dietary fat intake on weight loss and waist reduction.
PMCID: PMC3428346  PMID: 22952648
14.  High-Throughput Retina-Array for Screening 93 Genes Involved in Inherited Retinal Dystrophy 
To facilitate mutation screening in patients, a custom resequencing chip has been developed to detect sequence alterations of 267,550 bases of both sense and antisense sequences in 1,470 exons spanning 93 genes involved in inherited retinal dystrophy.
Retinal dystrophy (RD) is a broad group of hereditary disorders with heterogeneous genotypes and phenotypes. Current available genetic testing for these diseases is complicated, time consuming, and expensive. This study was conducted to develop and apply a microarray-based, high-throughput resequencing system to detect sequence alterations in genes related to inherited RD.
A customized 300-kb resequencing chip, Retina-Array, was developed to detect sequence alterations of 267,550 bases of both sense and antisense sequence in 1470 exons spanning 93 genes involved in inherited RD. Retina-Array was evaluated in 19 patient samples with inherited RD provided by the eyeGENE repository and four Centre d'Etudes du Polymorphisme Humaine reference samples through a high-throughput experimental approach that included an automated PCR assay setup and quantification, efficient post-quantification data processing, optimized pooling and fragmentation, and standardized chip processing.
The performance of the chips demonstrated that the average base pair call rate and accuracy were 93.56% and 99.86%, respectively. In total, 304 candidate variations were identified using a series of customized screening filters. Among 174 selected variations, 123 (70.7%) were further confirmed by dideoxy sequencing. Analysis of patient samples using Retina-Array resulted in the identification of 10 known mutations and 12 novel variations with high probability of deleterious effects.
This study suggests that Retina-Array might be a valuable tool for the detection of disease-causing mutations and disease severity modifiers in a single experiment. Retinal-Array may provide a powerful and feasible approach through which to study genetic heterogeneity in retinal diseases.
PMCID: PMC3231844  PMID: 22025579
15.  Cysteine Thioesters as Myelin Proteolipid Protein Analogues to Examine the Role of Butyrylcholinesterase in Myelin Decompaction 
ACS Chemical Neuroscience  2010;2(3):151-159.
Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disorder involving demyelination, axonal transection, and neuronal loss in the brain. Recent studies have indicated that active MS lesions express elevated levels of butyrylcholinesterase (BuChE). BuChE can hydrolyze a wide variety of esters, including fatty acid esters of protein. Proteolipid protein (PLP), an important transmembrane protein component of myelin, has six cysteine residues acylated, via thioester linkages, with fatty acids, usually palmitic, that contribute to the stability of myelin. Experimental chemical deacylation of PLP has been shown to lead to decompaction of myelin. Because of elevated levels of BuChE in active MS lesions and its propensity to catalyze the hydrolysis of acylated protein, we hypothesized that this enzyme may contribute to deacylation of PLP in MS, leading to decompaction of myelin and contributing to demyelination. To test this hypothesis, a series of increasing chain length (C2−C16) acyl thioester derivatives of N-acetyl-l-cysteine methyl ester were synthesized and examined for hydrolysis by human cholinesterases. All N-acetyl-l-cysteine fatty acyl thioester derivatives were hydrolyzed by BuChE but not by the related enzyme acetylcholinesterase. In addition, it was observed that the affinity of BuChE for the compound increased the longer the fatty acid chain, with the highest affinity for cysteine bound to palmitic acid. This suggests that the elevated levels of BuChE observed in active MS lesions could be related to the decompaction of myelin characteristic of the disorder.
PMCID: PMC3369730  PMID: 22778864
Cholinesterase; multiple sclerosis; donepezil; acylproteins; palmitic acid
16.  Genetic and phenotypic characteristics of three Mainland Chinese families with choroideremia 
Molecular Vision  2012;18:309-316.
To describe the phenotype and genotype of three Mainland Chinese families affected by choroideremia (CHM).
Complete ophthalmic examinations were conducted in three unrelated Chinese families with CHM. Peripheral blood samples were collected from the families for genetic and immunoblot analysis. All exons and flanking intronic regions of the gene encoding Rab escort protein-1 (Rep-1) were amplified with PCR and screened for mutations with Sanger sequencing. The three-dimensional structure of mutated Rep-1 was modeled using sequence homology with rat proteins to analyze the effect of the mutation detected in one family.
All affected males had characteristic signs and symptoms of CHM; however, central visual acuity impairment occurred earlier than expected. All female carriers older than 45 years had pigmentary changes, and one female carrier was symptomatic with vision loss. Three different mutations in Rep-1, c.1801–1G>A, c.1130 T>A, and c.612delAG, were detected in the three families.
In Mainland Chinese families, the central visual acuity of male patients with CHM can be affected at an early age (second decade), whereas female CHM carriers may manifest signs and symptoms at a later age (≥45 years). One previously reported and two novel Rep-1 mutations were detected in three Chinese patients with CHM.
PMCID: PMC3283217  PMID: 22355242
17.  Evidence of genetic heterogeneity in Alberta Hutterites with Usher syndrome type I 
Molecular Vision  2012;18:1379-1383.
To identify the genetic defect in a Hutterite population from northern Alberta with Usher syndrome type I.
Complete ophthalmic examinations were conducted on two boys and two girls from two related Hutterite families diagnosed with Usher syndrome type I. DNA from patients and their parents was first evaluated for a mutation in exon 10 of the protocadherin-related 15 (PCDH15) gene (c.1471delG), previously reported in southern Alberta Hutterite patients with Usher syndrome (USH1F). Single nucleotide polymorphic linkage analysis was then used to confirm another locus, and DNA was analyzed with the Usher Chip v4.0 platform.
Severe hearing impairment, unintelligible speech, and retinitis pigmentosa with varying degrees of visual acuity and visual field loss established a clinical diagnosis of Usher syndrome type I. The patients did not carry the exon 10 mutation in the PCDH15 gene; however, with microarray analysis, a previously reported mutation (c.52C>T; p.Q18X) in the myosin VIIA (MYO7A) gene was found in the homozygous state in the affected siblings.
The finding of a MYO7A mutation in two related Hutterite families from northern Alberta provides evidence of genetic heterogeneity in Hutterites affected by Usher syndrome type I.
PMCID: PMC3369897  PMID: 22690115
18.  Retinal Degenerations: Genetics, Mechanisms, and Therapies 
Journal of Ophthalmology  2011;2011:764873.
PMCID: PMC3216361  PMID: 22132314
19.  Evaluating the effectiveness of a schools-based programme to promote exercise self-efficacy in children and young people with risk factors for obesity: Steps to active kids (STAK) 
BMC Public Health  2011;11:830.
Low levels of physical activity in children have been linked to an increased risk of obesity, but many children lack confidence in relation to exercise (exercise self-efficacy). Factors which can impact on confidence include a chronic health condition such as asthma, poor motor skills and being overweight. Increasing levels of physical activity have obvious benefits for children with asthma and children who are overweight, but few activity interventions with children specifically target children with low exercise self-efficacy (ESE). This study aims to evaluate the efficacy and feasibility of a schools-based activity programme suitable for children with risk factors for adult obesity, including asthma, overweight and low exercise self-efficacy.
A clustered (at the level of school) RCT will be used to compare a targeted, 10 week, stepped activity programme (activity diary, dance DVD, circuit-training and motivational interviewing) designed to promote ESE. We will recruit 20 primary schools to participate in the intervention and 9-11 year old children will be screened for low levels of ESE, asthma and overweight. In order to provide sufficient power to detect a difference in primary outcomes (Body Mass Index-BMI & ESE at 12 month follow-up) between children in the intervention schools and control schools, the target sample size is 396. Assessments of BMI, ESE, waist circumference, peak flow, activity levels and emotional and behavioural difficulties will be made at baseline, 4 months and 12 month follow-up.
We aim to increase ESE and levels of physical activity in children with risk factors for adult obesity. The outcomes of this study will inform policy makers about the feasibility, acceptability and effectiveness of delivering targeted health interventions within a school setting.
Trial Registration
ISRCTN Register no. ISRCTN12650001
PMCID: PMC3212965  PMID: 22029547
20.  Hypertension and type 2 diabetes: What family physicians can do to improve control of blood pressure - an observational study 
BMC Family Practice  2011;12:86.
The prevalence of type 2 diabetes is rising, and most of these patients also have hypertension, substantially increasing the risk of cardiovascular morbidity and mortality. The majority of these patients do not reach target blood pressure levels for a wide variety of reasons. When a literature review provided no clear focus for action when patients are not at target, we initiated a study to identify characteristics of patients and providers associated with achieving target BP levels in community-based practice.
We conducted a practice- based, cross-sectional observational and mailed survey study. The setting was the practices of 27 family physicians and nurse practitioners in 3 eastern provinces in Canada. The participants were all patients with type 2 diabetes who could understand English, were able to give consent, and would be available for follow-up for more than one year. Data were collected from each patient's medical record and from each patient and physician/nurse practitioner by mailed survey. Our main outcome measures were overall blood pressure at target (< 130/80), systolic blood pressure at target, and diastolic blood pressure at target. Analysis included initial descriptive statistics, logistic regression models, and multivariate regression using hierarchical nonlinear modeling (HNLM).
Fifty-four percent were at target for both systolic and diastolic pressures. Sixty-two percent were at systolic target, and 79% were at diastolic target. Patients who reported eating food low in salt had higher odds of reaching target blood pressure. Similarly, patients reporting low adherence to their medication regimen had lower odds of reaching target blood pressure.
When primary care health professionals are dealing with blood pressures above target in a patient with type 2 diabetes, they should pay particular attention to two factors. They should inquire about dietary salt intake, strongly emphasize the importance of reduction, and refer for detailed counseling if necessary. Similarly, they should inquire about adherence to the medication regimen, and employ a variety of patient-oriented strategies to improve adherence.
PMCID: PMC3163533  PMID: 21834976
21.  Effects of Acute Insulin-Induced Hypoglycemia on Indices of Inflammation 
Diabetes Care  2010;33(7):1591-1597.
To examine the effects of acute insulin-induced hypoglycemia on inflammation, endothelial dysfunction, and platelet activation in adults with and without type 1 diabetes.
We studied 16 nondiabetic adults and 16 subjects with type 1 diabetes during euglycemia (blood glucose 4.5 mmol/l) and hypoglycemia (blood glucose 2.5 mmol/l). Markers of inflammation, thrombosis, and endothelial dysfunction (soluble P-selectin, interleukin-6, von Willebrand factor [vWF], tissue plasminogen activator [tPA], high-sensitivity C-reactive protein [hsCRP], and soluble CD40 ligand [sCD40L]) were measured; platelet-monocyte aggregation and CD40 expression on monocytes were determined using flow cytometry.
In nondiabetic participants, platelet activation occurred after hypoglycemia, with increments in platelet-monocyte aggregation and P-selectin (P ≤ 0.02). Inflammation was triggered with CD40 expression increasing maximally at 24 h (3.13 ± 2.3% vs. 2.06 ± 1.0%) after hypoglycemia (P = 0.009). Both sCD40L and hsCRP (P = 0.02) increased with a nonsignificant rise in vWF and tPA, indicating a possible endothelial effect. A reduction in sCD40L, tPA, and P-selectin occurred during euglycemia (P = 0.03, P ≤ 0.006, and P = 0.006, respectively). In type 1 diabetes, both CD40 expression (5.54 ± 4.4% vs. 3.65 ± 1.8%; P = 0.006) and plasma sCD40L concentrations increased during hypoglycemia (peak 3.41 ± 3.2 vs. 2.85 ± 2.8 ng/ml; P = 0.03). Platelet-monocyte aggregation also increased significantly at 24 h after hypoglycemia (P = 0.03). A decline in vWF and P-selectin occurred during euglycemia (P ≤ 0.04).
Acute hypoglycemia may provoke upregulation and release of vasoactive substances in adults with and without type 1 diabetes. This may be a putative mechanism for hypoglycemia-induced vascular injury.
PMCID: PMC2890364  PMID: 20587725
22.  Allelic Variants of Melanocortin 3 Receptor Gene (MC3R) and Weight Loss in Obesity: A Randomised Trial of Hypo-Energetic High- versus Low-Fat Diets 
PLoS ONE  2011;6(6):e19934.
The melanocortin system plays an important role in energy homeostasis. Mice genetically deficient in the melanocortin-3 receptor gene have a normal body weight with increased body fat, mild hypophagia compared to wild-type mice. In humans, Thr6Lys and Val81Ile variants of the melanocortin-3 receptor gene (MC3R) have been associated with childhood obesity, higher BMI Z-score and elevated body fat percentage compared to non-carriers. The aim of this study is to assess the association in adults between allelic variants of MC3R with weight loss induced by energy-restricted diets.
Subjects and Methods
This research is based on the NUGENOB study, a trial conducted to assess weight loss during a 10-week dietary intervention involving two different hypo-energetic (high-fat and low-fat) diets. A total of 760 obese patients were genotyped for 10 single nucleotide polymorphisms covering the single exon of MC3R gene and its flanking regions, including the missense variants Thr6Lys and Val81Ile. Linear mixed models and haplotype-based analysis were carried out to assess the potential association between genetic polymorphisms and differential weight loss, fat mass loss, waist change and resting energy expenditure changes.
No differences in drop-out rate were found by MC3R genotypes. The rs6014646 polymorphism was significantly associated with weight loss using co-dominant (p = 0.04) and dominant models (p = 0.03). These p-values were not statistically significant after strict control for multiple testing. Haplotype-based multivariate analysis using permutations showed that rs3827103–rs1543873 (p = 0.06), rs6014646–rs6024730 (p = 0.05) and rs3746619–rs3827103 (p = 0.10) displayed near-statistical significant results in relation to weight loss. No other significant associations or gene*diet interactions were detected for weight loss, fat mass loss, waist change and resting energy expenditure changes.
The study provided overall sufficient evidence to support that there is no major effect of genetic variants of MC3R and differential weight loss after a 10-week dietary intervention with hypo-energetic diets in obese Europeans.
PMCID: PMC3114803  PMID: 21695122
23.  Applications of multi-nuclear magnetic resonance spectroscopy at 7T 
World Journal of Radiology  2011;3(4):105-113.
AIM: To discuss the advantages of ultra-high field (7T) for 1H and 13C magnetic resonance spectroscopy (MRS) studies of metabolism.
METHODS: Measurements of brain metabolites were made at both 3 and 7T using 1H MRS. Measurements of glycogen and lipids in muscle were measured using 13C and 1H MRS respectively.
RESULTS: In the brain, increased signal-to-noise ratio (SNR) and dispersion allows spectral separation of the amino-acids glutamate, glutamine and γ-aminobutyric acid (GABA), without the need for sophisticated editing sequences. Improved quantification of these metabolites is demonstrated at 7T relative to 3T. SNR was 36% higher, and measurement repeatability (% coefficients of variation) was 4%, 10% and 10% at 7T, vs 8%, 29% and 21% at 3T for glutamate, glutamine and GABA respectively. Measurements at 7T were used to compare metabolite levels in the anterior cingulate cortex (ACC) and insula. Creatine and glutamate levels were found to be significantly higher in the insula compared to the ACC (P < 0.05). In muscle, the increased SNR and spectral resolution at 7T enables interleaved studies of glycogen (13C) and intra-myocellular lipid (IMCL) and extra-myocellular lipid (EMCL) (1H) following exercise and re-feeding. Glycogen levels were significantly decreased following exercise (-28% at 50% VO2 max; -58% at 75% VO2 max). Interestingly, levels of glycogen in the hamstrings followed those in the quadriceps, despite reduce exercise loading. No changes in IMCL and EMCL were found in the study.
CONCLUSION: The demonstrated improvements in brain and muscle MRS measurements at 7T will increase the potential for use in investigating human metabolism and changes due to pathologies.
PMCID: PMC3084434  PMID: 21532871
Magnetic resonance spectroscopy; 13C; 1H; 7 Tesla; Glutamate; Glutamine; γ-aminobutyric acid
24.  Global Patterns and Predictions of Seafloor Biomass Using Random Forests 
PLoS ONE  2010;5(12):e15323.
A comprehensive seafloor biomass and abundance database has been constructed from 24 oceanographic institutions worldwide within the Census of Marine Life (CoML) field projects. The machine-learning algorithm, Random Forests, was employed to model and predict seafloor standing stocks from surface primary production, water-column integrated and export particulate organic matter (POM), seafloor relief, and bottom water properties. The predictive models explain 63% to 88% of stock variance among the major size groups. Individual and composite maps of predicted global seafloor biomass and abundance are generated for bacteria, meiofauna, macrofauna, and megafauna (invertebrates and fishes). Patterns of benthic standing stocks were positive functions of surface primary production and delivery of the particulate organic carbon (POC) flux to the seafloor. At a regional scale, the census maps illustrate that integrated biomass is highest at the poles, on continental margins associated with coastal upwelling and with broad zones associated with equatorial divergence. Lowest values are consistently encountered on the central abyssal plains of major ocean basins The shift of biomass dominance groups with depth is shown to be affected by the decrease in average body size rather than abundance, presumably due to decrease in quantity and quality of food supply. This biomass census and associated maps are vital components of mechanistic deep-sea food web models and global carbon cycling, and as such provide fundamental information that can be incorporated into evidence-based management.
PMCID: PMC3012679  PMID: 21209928
25.  Current Concepts in the Treatment of Retinitis Pigmentosa 
Journal of Ophthalmology  2010;2011:753547.
Inherited retinal degenerations, including retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA), affect 1 in 4000 individuals in the general population. A majority of the genes which are mutated in these conditions are expressed in either photoreceptors or the retinal pigment epithelium (RPE). There is considerable variation in the clinical severity of these conditions; the most severe being autosomal recessive LCA, a heterogeneous retinal degenerative disease and the commonest cause of congenital blindness in children. Here, we discuss all the potential treatments that are now available for retinal degeneration. A number of therapeutic avenues are being explored based on our knowledge of the pathophysiology of retinal degeneration derived from research on animal models, including: gene therapy, antiapoptosis agents, neurotrophic factors, and dietary supplementation. Technological advances in retinal implant devices continue to provide the promise of vision for patients with end-stage disease.
PMCID: PMC2964907  PMID: 21048997

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