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1.  Effects of pretesting implicit self-determined motivation on behavioral engagement: evidence for the mere measurement effect at the implicit level 
Research into individuals’ intended behavior and performance has traditionally adopted explicitly measured, self-report constructs, and outcomes. More recently, research has shown that completing explicit self-report measures of constructs may effect subsequent behavior, termed the “mere measurement” effect. The aim of the present experiment was to investigate whether implicit measures of motivation showed a similar mere measurement effect on subsequent behavior. It may be the case that measuring the implicit systems affects subsequent implicit interventions (e.g., priming), observable on subsequent behavior. Priming manipulations were also given to participants in order to investigate the interaction between measurement and priming of motivation. Initially, a 2 [implicit association test (IAT: present vs. absent) ×2 (Prime: autonomous vs. absent) and a 2 (IAT: present vs. absent) × 2 (Prime: controlled vs. absent)] between participants designs were conducted, these were them combined into a 2 (IAT: present vs. absent) ×3 (Prime: autonomous vs. controlled vs. absent) between participants design, with attempts at a novel task taken as the outcome measure. Implicit measure completion significantly decreased behavioral engagement. Priming autonomous motivation significantly facilitated, and controlled motivation significantly inhibited performance. Finally, there was a significant implicit measurement × priming interaction, such that priming autonomous motivation only improved performance in the absence of the implicit measure. Overall, this research provides an insight into the effects of implicit measurement and priming of motivation and the combined effect of completing both tasks on behavior.
PMCID: PMC3923262  PMID: 24592249
implicit measurement; mere measurement effect; self-determination theory; implicit association test; priming
2.  The INSPIRED study: a randomised controlled trial of the Whole Person Model of disease self-management for people with type 2 diabetes 
BMC Public Health  2014;14:134.
The prevalence of type 2 diabetes has increased dramatically in the last decade, and is continuing to rise. It is a chronic condition, often related to obesity, diet and sedentary lifestyles, and can lead to significant health complications, disability and early death. Diabetes is commonly associated with depression, which can impact significantly on a person’s ability to manage their illness and, consequently, on disease outcomes. Disease self-management is fundamental in diabetes and requires support from multiple health professionals and the active participation of the person, including in maintaining a healthy lifestyle. The Whole Person Model was developed in order to integrate emotional and behavioural aspects into a self-management program for people with type 2 diabetes. Here we describe a study designed to test the efficacy of the Whole Person Model of disease self-management in type 2 diabetes.
In a parallel-group randomised trial, 180 people with type 2 diabetes of between 2–10 years duration will be recruited via invitation through the Australian National Diabetes Services Scheme. Participants will undergo baseline assessment, followed by randomisation to either intervention or control condition. Control participants will receive fact sheets containing key information about diabetes self-management. The intervention group will receive the INSPIRED (Individual Support & Resources for Diabetes) Manual and be assigned a Health Coach. The INSPIRED Manual consists of six modules that provide key information about diabetes and disease management using the Whole Person Model. Engagement is facilitated by interactive tasks and contact with a Health Coach over seven weeks – an introductory face-to-face session, and six subsequent contacts by phone following each module. Follow-up assessments occur at 13 weeks (post-intervention) and 26 weeks. Primary outcomes include blood glucose management (HbA1c), weight and mood. Secondary outcomes include level of exercise, confidence to manage diabetes, and psychosocial well-being.
The Whole Person Model is designed to enable health professionals to address mood disturbance without pathologizing any disorders and, in the context of the chronic illness, empowering behavior change and self-management. If proven effective, this model will strengthen capacity of the healthcare workforce to foster and support effective diabetes self-management.
Trial registration
Australia and New Zealand Clinical Trials Register, ACTRN12613000391774
PMCID: PMC3933337  PMID: 24507417
Type 2 diabetes mellitus; Diabetes; Self-management; Depression; Support; Resources
3.  The Expression of stlA in Photorhabdus luminescens Is Controlled by Nutrient Limitation 
PLoS ONE  2013;8(11):e82152.
Photorhabdus is a genus of Gram-negative entomopathogenic bacteria that also maintain a mutualistic association with nematodes from the family Heterorhabditis. Photorhabdus has an extensive secondary metabolism that is required for the interaction between the bacteria and the nematode. A major component of this secondary metabolism is a stilbene molecule, called ST. The first step in ST biosynthesis is the non-oxidative deamination of phenylalanine resulting in the production of cinnamic acid. This reaction is catalyzed by phenylalanine-ammonium lyase, an enzyme encoded by the stlA gene. In this study we show, using a stlA-gfp transcriptional fusion, that the expression of stlA is regulated by nutrient limitation through a regulatory network that involves at least 3 regulators. We show that TyrR, a LysR-type transcriptional regulator that regulates gene expression in response to aromatic amino acids in E. coli, is absolutely required for stlA expression. We also show that stlA expression is modulated by σS and Lrp, regulators that are implicated in the regulation of the response to nutrient limitation in other bacteria. This work is the first that describes pathway-specific regulation of secondary metabolism in Photorhabdus and, therefore, our study provides an initial insight into the complex regulatory network that controls secondary metabolism, and therefore mutualism, in this model organism.
PMCID: PMC3838401  PMID: 24278476
World journal for pediatric & congenital heart surgery  2013;4(1):10.1177/2150135112461924.
Several distinct definitions of postoperative death have been used in various quality reporting programs. Some have defined a postoperative mortality as a patient who expires while still in the hospital, while others have considered all deaths occurring within a predetermined, standardized time interval after surgery. While if continues to collect mortality data using both these individual definitions, the Society of Thoracic Surgeons (STS) believes that either alone may be inadequate. Accordingly, the STS prefers a more encompassing metric, Operative Mortality, which is defined as (1) all deaths occurring during the hospitalization in which the operation was performed, even if after 30 days; and (2) all deaths occurring after discharge from the hospital, but before the end of the thirtieth postoperative day. This manuscript provides clarification for some uncommon but important scenarios where the correct application of this definition may be problematic.
PMCID: PMC3828200  PMID: 23799748
cardiac disease; thoracic disease; congenital heart disease; outcomes analysis; quality improvement; database; mortality
5.  An empirically based tool for analyzing morbidity associated with operations for congenital heart disease 
The Journal of thoracic and cardiovascular surgery  2012;145(4):10.1016/j.jtcvs.2012.06.029.
Congenital heart surgery outcomes analysis requires reliable methods of estimating the risk of adverse outcomes. Contemporary methods focus primarily on mortality or rely on expert opinion to estimate morbidity associated with different procedures. We created an objective, empirically based index that reflects statistically estimated risk of morbidity by procedure.
Morbidity risk was estimated using data from 62,851 operations in the Society of Thoracic Surgeons Congenital Heart Surgery Database (2002-2008). Model-based estimates with 95% Bayesian credible intervals were calculated for each procedure’s average risk of major complications and average postoperative length of stay. These 2 measures were combined into a composite morbidity score. A total of 140 procedures were assigned scores ranging from 0.1 to 5.0 and sorted into 5 relatively homogeneous categories.
Model-estimated risk of major complications ranged from 1.0% for simple procedures to 38.2% for truncus arteriosus with interrupted aortic arch repair. Procedure-specific estimates of average postoperative length of stay ranged from 2.9 days for simple procedures to 42.6 days for a combined atrial switch and Rastelli operation. Spearman rank correlation between raw rates of major complication and average postoperative length of stay was 0.82 in procedures with n greater than 200. Rate of major complications ranged from 3.2% in category 1 to 30.0% in category 5. Aggregate average postoperative length of stay ranged from 6.3 days in category 1 to 34.0 days in category 5.
Complication rates and postoperative length of stay provide related but not redundant information about morbidity. The Morbidity Scores and Categories provide an objective assessment of risk associated with operations for congenital heart disease, which should facilitate comparison of outcomes across cohorts with differing case mixes.
PMCID: PMC3824389  PMID: 22835225
6.  Initial application in the EACTS and STS Congenital Heart Surgery Databases of an empirically derived methodology of complexity adjustment to evaluate surgical case mix and results† 
Outcomes evaluation is enhanced by assignment of operative procedures to appropriate categories based upon relative average risk. Formal risk modelling is challenging when a large number of operation types exist, including relatively rare procedures. Complexity stratification provides an alternative methodology. We report the initial application in the Congenital Heart Surgery Databases of the Society of Thoracic Surgeons (STS) and the European Association for Cardio-thoracic Surgery (EACTS) of an empirically derived system of complexity adjustment to evaluate surgical case mix and results.
Complexity stratification is a method of analysis in which the data are divided into relatively homogeneous groups (called strata). A complexity stratification tool named the STS–EACTS Congenital Heart Surgery Mortality Categories (STAT Mortality Categories) was previously developed based on the analysis of 77 294 operations entered in the Congenital Heart Surgery Databases of EACTS (33 360 operations) and STS (43 934 patients). Procedure-specific mortality rate estimates were calculated using a Bayesian model that adjusted for small denominators. Operations were sorted by increasing risk and grouped into five categories (the STAT Mortality Categories) that were designed to minimize within-category variation and maximize between-category variation. We report here the initial application of this methodology in the EACTS Congenital Heart Surgery Database (47 187 operations performed over 4 years: 2006–09) and the STS Congenital Heart Surgery Database (64 307 operations performed over 4 years: 2006–09).
In the STS Congenital Heart Surgery Database, operations classified as STAT Mortality Categories 1–5 were (1): 17332, (2): 20114, (3): 9494, (4): 14525 and (5): 2842. Discharge mortality was (1): 0.54%, (2): 1.6%, (3): 2.4%, (4): 7.5% and (5): 17.8%. In the EACTS Congenital Heart Surgery Database, operations classified as STAT Mortality Categories 1–5 were (1): 19874, (2): 12196, (3): 5614, (4): 8287 and (5): 1216. Discharge mortality was (1): 0.99%, (2): 2.9%, (3): 5.0%, (4): 10.3% and (5): 25.0%.
The STAT Mortality Categories facilitate analysis of outcomes across the wide spectrum of distinct congenital heart surgery operations including infrequently performed procedures.
PMCID: PMC3858079  PMID: 22700597
Database; Outcomes; Quality assessment; Quality improvement
7.  Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding 
PLoS ONE  2013;8(9):e74200.
Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.
PMCID: PMC3774629  PMID: 24066121
8.  Implementing a training intervention to support caregivers after stroke: a process evaluation examining the initiation and embedding of programme change 
Medical Research Council (MRC) guidance identifies implementation as a key element of the development and evaluation process for complex healthcare interventions. Implementation is itself a complex process involving the mobilization of human, material, and organizational resources to change practice within settings that have pre-existing structures, historical patterns of relationships, and routinized ways of working. Process evaluations enable researchers and clinicians to understand how implementation proceeds and what factors impact on intended program change. A qualitative process evaluation of the pragmatic cluster randomized controlled trial; Training Caregivers after Stroke was conducted to examine how professionals were engaged in the work of delivering training; how they reached and involved caregivers for whom the intervention was most appropriate; how did those on whom training was targeted experience and respond to it. Normalization Process Theory, which focuses attention on implementing and embedding program change, was used as a sensitizing framework to examine selected findings.
Contextual factors including organizational history and team relationships, external policy, and service development initiatives, impinged on implementation of the caregiver training program in unintended ways that could not have been predicted through focus on mechanisms of individual and collective action at unit level. Factors that facilitated or impeded the effectiveness of the cascade training model used, whether and how stroke unit teams made sense of and engaged individually and collectively with a complex caregiver training intervention, and what impact these factors had on embedding the intervention in routine stroke unit practice were identified.
Where implementation of complex interventions depends on multiple providers, time needs to be invested in reaching agreement on who will take responsibility for delivery of specific components and in determining how implementation and its effectiveness will be monitored. This goes beyond concern with intervention fidelity; explicit consideration also needs to be given to the implementation process in terms of how program change can be effected at organizational, practice, and service delivery levels. Normalization Process Theory’s constructs help identify vulnerable features of implementation processes in respect of the work involved in embedding complex interventions.
PMCID: PMC3765868  PMID: 23972027
Process evaluation; Implementation theory; Stroke; Caregiver training; Normalization process theory
9.  30 Days in the Life: Daily Nutrient Balancing in a Wild Chacma Baboon 
PLoS ONE  2013;8(7):e70383.
For most animals, the ability to regulate intake of specific nutrients is vital to fitness. Recent studies have demonstrated nutrient regulation in nonhuman primates over periods of one observation day, though studies of humans indicate that such regulation extends to longer time frames. Little is known about longer-term regulation in nonhuman primates, however, due to the challenges of multiple-day focal follows. Here we present the first detailed study of nutrient intake across multiple days in a wild nonhuman primate. We conducted 30 consecutive all day follows on one female chacma baboon (Papio hamadryas ursinus) in the Cape Peninsula of South Africa. We documented dietary composition, compared the nutritional contribution of natural and human-derived foods to the diet, and quantified nutrient intake using the geometric framework of nutrition. Our focus on a single subject over consecutive days allowed us to examine daily dietary regulation within an individual over time. While the amounts varied daily, our subject maintained a strikingly consistent balance of protein to non-protein (fat and carbohydrate) energy across the month. Human-derived foods, while contributing a minority of the diet, were higher in fat and lower in fiber than naturally-derived foods. Our results demonstrate nutrient regulation on a daily basis in our subject, and demonstrate that she was able to maintain a diet with a constant proportional protein content despite wide variation in the composition of component foods. From a methodological perspective, the results of this study suggest that nutrient intake is best estimated over at least an entire day, with longer-term regulatory patterns (e.g., during development and reproduction) possibly requiring even longer sampling. From a management and conservation perspective, it is notable that nearly half the subject’s daily energy intake derived from exotic foods, including those currently being eradicated from the study area for replacement by indigenous vegetation.
PMCID: PMC3722187  PMID: 23894645
10.  Thiorhodamines Containing Amide and Thioamide Functionality as Inhibitors of the ATP-Binding Cassette Drug Transporter P-glycoprotein (ABCB1) 
Bioorganic & medicinal chemistry  2012;20(14):4290-4302.
Twelve thiorhodamine derivatives have been examined for their ability to stimulate the ATPase activity of purified human P-glycoprotein (P-gp)-His10, to promote uptake of calcein AM and vinblastine into multidrug-resistant, P-gp-overexpressing MDCKII-MDR1 cells, and for their rates of transport in monolayers of multidrug-resistant, P-gp-overexpressing MDCKII-MDR1 cells. The thiorhodamine derivatives have structural diversity from amide and thioamide functionality (N,N-diethyl and N-piperidyl) at the 5-position of a 2-thienyl substituent on the thiorhodamine core and from diversity at the 3-amino substituent with N,N-dimethylamino, fused azadecalin (julolidyl), and fused N-methylcyclohexylamine (half-julolidyl) substituents. The julolidyl and half-julolidyl derivatives were more effective inhibitors of P-gp than the dimethylamino analogues. Amide-containing derivatives were transported much more rapidly than thioamide-containing derivatives.
PMCID: PMC3400123  PMID: 22727780
Multidrug resistance; P-glycoprotein; thiorhodamine; transport inhibition; ATPase stimulation
11.  Inhibition of the PLP-dependent enzyme serine palmitoyltransferase by cycloserine: evidence for a novel decarboxylative mechanism of inactivation 
Molecular bioSystems  2010;6(9):1682-1693.
Cycloserine (CS, 4-amino-3-isoxazolidone) is a cyclic amino acid mimic that is known to inhibit many essential pyridoxal 5′-phosphate (PLP)-dependent enzymes. Two CS enantiomers are known; d-cycloserine (DCS, also known as Seromycin), is a natural product that is used to treat resistant Mycobacterium tuberculosis infections as well as neurological disorders since it is a potent NMDA receptor agonist, and l-cycloserine (LCS), is a synthetic enantiomer whose usefulness as a drug has been hampered by its inherent toxicity arising through inhibition of sphingolipid metabolism. Previous studies on various PLP-dependent enzymes revealed a common mechanism of inhibition by both enantiomers of CS; the PLP cofactor is disabled by forming a stable 3-hydroxyisoxazole/pyridoxamine 5′-phosphate (PMP) adduct at the active site where the cycloserine ring remains intact. Here we describe a novel mechanism of CS inactivation of the PLP-dependent enzyme serine palmitoyltransferase (SPT) from Sphingomonas paucimobilis. SPT catalyses the condensation of l-serine and palmitoyl-CoA, the first step in the de novo sphingolipid biosynthetic pathway. We have used a range of kinetic, spectroscopic and structural techniques to postulate that both LCS and DCS inactivate SPT by transamination to form a free pyridoxamine 5′-phosphate (PMP) and β-aminooxyacetaldehyde that remain bound at the active site. We suggest this occurs by ring opening of the cycloserine ring followed by decarboxylation. Enzyme kinetics show that inhibition is reversed by incubation with excess PLP and that LCS is a more effective SPT inhibitor than DCS. UV-visible spectroscopic data, combined with site-directed mutagenesis, suggest that a mobile Arg378 residue is involved in cycloserine inactivation of SPT.
PMCID: PMC3670083  PMID: 20445930
12.  A review of the abuse potential assessment of atomoxetine: a nonstimulant medication for attention-deficit/hyperactivity disorder 
Psychopharmacology  2013;226(2):189-200.
Treatment of attention-deficit/hyperactivity disorder (ADHD) has for many years relied on psychostimulants, particularly various formulations of amphetamines and methylphenidate. These are central nervous system stimulants and are scheduled because of their abuse potential. Atomoxetine (atomoxetine hydrochloride; Strattera®) was approved in 2002 for treatment of ADHD, and was the first nonstimulant medication approved for this disorder. It was classified as an unscheduled medication indicating a low potential for abuse. However, the abuse potential of atomoxetine has not been reviewed.
In this article, we review the evidence regarding abuse potential of atomoxetine, a selective inhibitor of the presynaptic norepinephrine transporter, which is unscheduled/unrestricted in all countries where it is approved.
Results from receptor binding, in vitro electrophysiology, in vivo microdialysis, preclinical behavioral, and human laboratory studies have been reviewed.
Atomoxetine has no appreciable affinity for, or action at, central receptors through which drugs of abuse typically act, i.e., dopamine transporters, GABAA receptors, and opioid μ receptors. In behavioral experiments in rodents, atomoxetine does not increase locomotor activity, and in drug discrimination studies, its profile is similar to that of drugs without abuse potential. Atomoxetine does not serve as a reinforcer in monkey self-administration studies, and human laboratory studies suggest that atomoxetine does not induce subjective effects indicative of abuse.
Neurochemical, preclinical, and early clinical studies predicted and supported a lack of abuse potential of atomoxetine, which is consistent with the clinical trial and postmarketing spontaneous event data in the past 10 years.
PMCID: PMC3579642  PMID: 23397050
Atomoxetine (Strattera®); ADHD; Abuse potential; Nonstimulant
13.  Linking Proteomic and Transcriptional Data through the Interactome and Epigenome Reveals a Map of Oncogene-induced Signaling 
PLoS Computational Biology  2013;9(2):e1002887.
Cellular signal transduction generally involves cascades of post-translational protein modifications that rapidly catalyze changes in protein-DNA interactions and gene expression. High-throughput measurements are improving our ability to study each of these stages individually, but do not capture the connections between them. Here we present an approach for building a network of physical links among these data that can be used to prioritize targets for pharmacological intervention. Our method recovers the critical missing links between proteomic and transcriptional data by relating changes in chromatin accessibility to changes in expression and then uses these links to connect proteomic and transcriptome data. We applied our approach to integrate epigenomic, phosphoproteomic and transcriptome changes induced by the variant III mutation of the epidermal growth factor receptor (EGFRvIII) in a cell line model of glioblastoma multiforme (GBM). To test the relevance of the network, we used small molecules to target highly connected nodes implicated by the network model that were not detected by the experimental data in isolation and we found that a large fraction of these agents alter cell viability. Among these are two compounds, ICG-001, targeting CREB binding protein (CREBBP), and PKF118–310, targeting β-catenin (CTNNB1), which have not been tested previously for effectiveness against GBM. At the level of transcriptional regulation, we used chromatin immunoprecipitation sequencing (ChIP-Seq) to experimentally determine the genome-wide binding locations of p300, a transcriptional co-regulator highly connected in the network. Analysis of p300 target genes suggested its role in tumorigenesis. We propose that this general method, in which experimental measurements are used as constraints for building regulatory networks from the interactome while taking into account noise and missing data, should be applicable to a wide range of high-throughput datasets.
Author Summary
The ways in which cells respond to changes in their environment are controlled by networks of physical links among the proteins and genes. The initial signal of a change in conditions rapidly passes through these networks from the cytoplasm to the nucleus, where it can lead to long-term alterations in cellular behavior by controlling the expression of genes. These cascades of signaling events underlie many normal biological processes. As a result, being able to map out how these networks change in disease can provide critical insights for new approaches to treatment. We present a computational method for reconstructing these networks by finding links between the rapid short-term changes in proteins and the longer-term changes in gene regulation. This method brings together systematic measurements of protein signaling, genome organization and transcription in the context of protein-protein and protein-DNA interactions. When used to analyze datasets from an oncogene expressing cell line model of human glioblastoma, our approach identifies key nodes that affect cell survival and functional transcriptional regulators.
PMCID: PMC3567149  PMID: 23408876
14.  (E)-5-[3-Cyano-2-(dicyano­methyl­ene)-1-oxaspiro­[4.5]dec-3-en-4-yl]-3-(1-methyl-1,4-dihydro­pyridin-4-yl­idene)pent-4-en-1-yl 3,5-bis­(benz­yloxy)benzoate 
In the title compound, C45H40N4O5, the cyclo­hexane entity on the (3-cyano-2,5-dihydro­furan-2-yl­idene)propane­dinitrile group, which replaces the usual dimethyl substituents, has not perturbed the delocalization geometry significantly. Weak inter­molecular inter­actions, viz. C—H⋯N(cyano), C—H⋯O(ether), C—H⋯π and π–π [between the aromatic rings with the shortest centroid–centroid distance of 3.603 (3) Å], consolidate the crystal packing, which exhibits voids of 57 Å3.
PMCID: PMC3588224  PMID: 23476366
15.  Psychometric properties of the cardiac depression scale in patients with coronary heart disease 
BMC Psychiatry  2012;12:216.
This study examined the psychometric properties of the Cardiac Depression Scale (CDS) in a sample of coronary heart disease (CHD) patients.
A total of 152 patients were diagnosed with coronary heart disease and were administered the CDS along with the Beck Depression Inventory- 2 (BDI-2) and the State Trait Anxiety Inventory (STAI) 3.5-months after cardiac hospitalization.
The CDS’s factorial composition in the current sample was similar to that observed in the original scale. Varimax-rotated principal-components analyses extracted six factors, corresponding to mood, anhedonia, cognition, fear, sleep and suicide. Reliability analyses yielded internal consistency α - coefficients for the six subscales ranging from 0.62 to 0.82. The CDS showed strong concurrent validity with the BDI-II (r = 0.64). More patients were classified as severely depressed using the CDS. Both the CDS and the BDI-2 displayed significantly strong correlations with the STAI (r = 0.61 and r = 0.64), respectively.
These findings encourage the use of the CDS for measuring the range of depressive symptoms in those with CHD 3.5 months after cardiac hospitalization.
PMCID: PMC3534230  PMID: 23199307
Cardiac depression scale; Depression; Coronary heart disease; Validity; Reliability; Psychometric properties
16.  Normalization and Statistical Analysis of Multiplexed Bead-based Immunoassay Data Using Mixed-effects Modeling* 
Multiplexed bead-based flow cytometric immunoassays are a powerful experimental tool for investigating cellular communication networks, yet their widespread adoption is limited in part by challenges in robust quantitative analysis of the measurements. Here we report our application of mixed-effects modeling for the normalization and statistical analysis of bead-based immunoassay data. Our data set consisted of bead-based immunoassay measurements of 16 phospho-proteins in lysates of HepG2 cells treated with ligands that regulate acute-phase protein secretion. Mixed-effects modeling provided estimates for the effects of both the technical and biological sources of variance, and normalization was achieved by subtracting the technical effects from the measured values. This approach allowed us to detect ligand effects on signaling with greater precision and sensitivity and to more accurately characterize the HepG2 cell signaling network using constrained fuzzy logic. Mixed-effects modeling analysis of our data was vital for ascertaining that IL-1α and TGF-α treatment increased the activities of more pathways than IL-6 and TNF-α and that TGF-α and TNF-α increased p38 MAPK and c-Jun N-terminal kinase (JNK) phospho-protein levels in a synergistic manner. Moreover, we used mixed-effects modeling-based technical effect estimates to reveal the substantial variance contributed by batch effects along with the absence of loading order and assay plate position effects. We conclude that mixed-effects modeling enabled additional insights to be gained from our data than would otherwise be possible and we discuss how this methodology can play an important role in enhancing the value of experiments employing multiplexed bead-based immunoassays.
PMCID: PMC3536905  PMID: 23071098
17.  A Systematic Investigation of Differential Effects of Cell Culture Substrates on the Extent of Artifacts in Single-Molecule Tracking 
PLoS ONE  2012;7(9):e45655.
Single-molecule techniques are being increasingly applied to biomedical investigation, notwithstanding the numerous challenges they pose in terms of signal-to-noise ratio issues. Non-specific binding of probes to glass substrates, in particular, can produce experimental artifacts due to spurious molecules on glass, which can be particularly deleterious in live-cell tracking experiments. In order to resolve the issue of non-specific probe binding to substrates, we performed systematic testing of a range of available surface coatings, using three different proteins, and then extended our assessment to the ability of these coatings to foster cell growth and retain non-adhesive properties. Linear PEG, a passivating agent commonly used both in immobilized-molecule single-molecule techniques and in tissue engineering, is able to both successfully repel non-specific adhesion of fluorescent probes and to foster cell growth when functionalized with appropriate adhesive peptides. Linear PEG treatment results in a significant reduction of tracking artifacts in EGFR tracking with Affibody ligands on a cell line expressing EGFR-eGFP. The findings reported herein could be beneficial to a large number of experimental situations where single-molecule or single-particle precision is required.
PMCID: PMC3458086  PMID: 23049831
18.  Activation of fast skeletal muscle troponin as a potential therapeutic approach for treating neuromuscular diseases 
Nature medicine  2012;18(3):452-455.
Limited neuromuscular input results in muscle weakness in neuromuscular disease either because of a reduction in the density of muscle innervation, the rate of neuromuscular junction activation or the efficiency of synaptic transmission1. We developed a small molecule fast skeletal troponin activator, CK-2017357, as a means to increase muscle strength by amplifying the response of muscle when neuromuscular input is diminished secondary to a neuromuscular disease. Binding selectively to the fast skeletal troponin complex, CK-2017357 slows the rate of calcium release from troponin C and sensitizes muscle to calcium. As a consequence, the force-calcium relationship of muscle fibers shifts leftwards as does the force-frequency relationship of a nerve-muscle pair. In vitro and in vivo, CK-2017357 increases the production of force at sub-maximal stimulation rates. Importantly, we show that sensitization of the fast skeletal troponin complex to calcium improves muscle force and grip strength immediately after single doses of CK-2017357 in a model of neuromuscular disease, myasthenia gravis. Troponin activation may provide a new therapeutic approach to improve physical activity in diseases where neuromuscular function is compromised.
PMCID: PMC3296825  PMID: 22344294
19.  Rhodamine Inhibitors of P-glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity 
Journal of Medicinal Chemistry  2009;52(10):3328-3341.
We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His10, for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave KM of 0.087 μM in human P-gp. Small changes in structure among this set of compounds affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive, and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide 31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC50’s of ~2 μM in MDCKII-MDR1 cells.
PMCID: PMC3400110  PMID: 19402665
Multidrug resistance; P-glycoprotein; rhodamines; rosamines; inhibition
20.  Guidelines for the Psychosocial and Bereavement Support of Family Caregivers of Palliative Care Patients 
Journal of Palliative Medicine  2012;15(6):696-702.
Support for family caregivers, including bereavement follow-up, is a core function of palliative care. Many caregivers acknowledge positive aspects associated with the role; however a considerable proportion will experience poor psychological, social, financial, spiritual, and physical well-being and some will suffer from complicated grief. Many family caregivers have unmet needs and would like more information, preparation, and support to assist them in the caregiving role. There is a shortage of evidence-based strategies to guide health professionals in providing optimal support while the caregiver is providing care and after the patient's death.
To develop clinical practice guidelines for the psychosocial and bereavement support of family caregivers of palliative care patients.
(1) Literature review; (2) focus groups and structured interviews with key stakeholders within Australia; (3) national and international expert opinion to further develop and refine the guidelines using a modified Delphi process; and (4) endorsement of the guidelines from key palliative care, caregiver, and bereavement organizations (national and international).
The guidelines were developed for multidisciplinary health care professionals and clinical services commonly involved in caring for adult patients receiving palliative care in a variety of care sites throughout Australia. These consensus-based guidelines have been endorsed key Australian and international organizations.
The guidelines may prove valuable for the international palliative care community and for generalist health care providers who occasionally care for palliative care patients. Research is recommended to explore the uptake, implementation, and effectiveness of the guidelines.
PMCID: PMC3362953  PMID: 22385026
21.  Amelioration of Acute Mercury Toxicity by a Novel, Non-Toxic Lipid Soluble Chelator N,N′bis-(2-mercaptoethyl)isophthalamide: Effect on Animal Survival, Health, Mercury Excretion and Organ Accumulation 
The toxic effects of mercury are known to be complex with specific enzyme inhibitions and subsequent oxidative stress adding to the damaging effects. There are likely other factors involved, such as the development of impaired metal ion homeostasis and depletion of thiol and selenium based metabolites such as cysteine and selenium. Much of the toxicity of mercury occurs at the intracellular level via binding of Hg2+ to thiol groups in specific proteins. Therefore, amelioration of mercury toxicity by the use of chelation would likely be enhanced by the use of a chelator that could cross the cell membrane and the blood brain barrier. It would be most favorable if this compound was of low toxicity, had appropriate pharmacokinetics, bound and rendered mercury cation non-toxic and had antioxidant properties. Herein we report on such a chelator, N,N′-bis(2-mercaptoethyl)isophthalamide (NBMI), and, using an animal model, show that it prevented the toxic effects associated with acute exposure induced by injected mercury chloride.
PMCID: PMC3346673  PMID: 22573916
Chelation; mercury toxicity; NBMI; metal excretion; oxidative stress; antioxidant
22.  Multicolour Single Molecule Imaging in Cells with Near Infra-Red Dyes 
PLoS ONE  2012;7(4):e36265.
The autofluorescence background of biological samples impedes the detection of single molecules when imaging. The most common method of reducing the background is to use evanescent field excitation, which is incompatible with imaging beyond the surface of biological samples. An alternative would be to use probes that can be excited in the near infra-red region of the spectrum, where autofluorescence is low. Such probes could also increase the number of labels that can be imaged in multicolour single molecule microscopes. Despite being widely used in ensemble imaging, there is a currently a shortage of information available for selecting appropriate commercial near infra-red dyes for single molecule work. It is therefore important to characterise available near infra-red dyes relevant to multicolour single molecule imaging.
Methodology/Principal Findings
A range of commercially available near infra-red dyes compatible with multi-colour imaging was screened to find the brightest and most photostable candidates. Image series of immobilised samples of the brightest dyes (Alexa 700, IRDye 700DX, Alexa 790 and IRDye 800CW) were analysed to obtain the mean intensity of single dye molecules, their photobleaching rates and long period blinking kinetics. Using the optimum dye pair, we have demonstrated for the first time widefield, multi-colour, near infra-red single molecule imaging using a supercontinuum light source in MCF-7 cells.
We have demonstrated that near infra-red dyes can be used to avoid autofluorescence background in samples where restricting the illumination volume of visible light fails or is inappropriate. We have also shown that supercontinuum sources are suited to single molecule multicolour imaging throughout the 470–1000 nm range. Our measurements of near infra-red dye properties will enable others to select optimal dyes for single molecule imaging.
PMCID: PMC3338497  PMID: 22558412
23.  Complete Genome Sequence of the Crohn's Disease-Associated Adherent-Invasive Escherichia coliStrain HM605▿  
Journal of Bacteriology  2011;193(17):4540.
Adherent-invasive Escherichia colistrains are increasingly being associated with intestinal pathologies. Here we present the genome sequence of E. coliHM605, a strain isolated from colonic biopsy specimens of a patient with Crohn's disease.
PMCID: PMC3165516  PMID: 21705601
24.  Single Molecule Fluorescence Detection and Tracking in Mammalian Cells: The State-of-the-Art and Future Perspectives 
Insights from single-molecule tracking in mammalian cells have the potential to greatly contribute to our understanding of the dynamic behavior of many protein families and networks which are key therapeutic targets of the pharmaceutical industry. This is particularly so at the plasma membrane, where the method has begun to elucidate the mechanisms governing the molecular interactions that underpin many fundamental processes within the cell, including signal transduction, receptor recognition, cell-cell adhesion, etc. However, despite much progress, single-molecule tracking faces challenges in mammalian samples that hinder its general application in the biomedical sciences. Much work has recently focused on improving the methods for fluorescent tagging of target molecules, detection and localization of tagged molecules, which appear as diffraction-limited spots in charge-coupled device (CCD) images, and objectively establishing the correspondence between moving particles in a sequence of image frames to follow their diffusive behavior. In this review we outline the state-of-the-art in the field and discuss the advantages and limitations of the methods available in the context of specific applications, aiming at helping researchers unfamiliar with single molecules methods to plan out their experiments.
PMCID: PMC3509608  PMID: 23203092
single molecule tracking; mammalian cells; experimental methods; fluorescent labels; feature detection
25.  The treatment of patients with medically unexplained symptoms in primary care: a review of the literature 
Mental Health in Family Medicine  2010;7(4):209-221.
Medically unexplained symptoms (MUS) are among the most common and frustrating in primary care. Our goal was to review published evidence to guide busy general practitioners working with a culturally diverse, challenging patient population coping with MUS. A search of PubMed and PsycINFO from 1985 to the present was conducted using MUS and related terms.
The literature was then organised into subcategories based on its relevance to primary care. We conclude with a description of gaps in the literature based on the literature review and the clinical experience of the authors.
PMCID: PMC3083260  PMID: 22477945
general practice; medically unexplained symptoms; primary care

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