Recent genetic and clinical evidence has implicated glucokinase regulatory protein (GKRP) in the pathogenesis of type 2 diabetes and related traits. The primary role of GKRP is to bind and inhibit ...hepatic glucokinase (GCK), a critically important protein in human health and disease that exerts a significant degree of control over glucose metabolism. As activation of GCK has been associated with improved glucose tolerance, perturbation of the GCK-GKRP interaction represents a potential therapeutic target for pharmacological modulation. Recent structural and kinetic advances are beginning to provide insight into the interaction of these two proteins. However, tools to comprehensively assess the GCK-GKRP interaction, particularly in the context of small molecules, would be a valuable resource. We therefore developed three robust and miniaturized assays for assessing the interaction between recombinant human GCK and GKRP: an HTRF assay, a diaphorase-coupled assay, and a luciferase-coupled assay. The assays are complementary, featuring distinct mechanisms of detection (luminescence, fluorescence, FRET). Two assays rely on GCK enzyme activity modulation by GKRP while the FRET-based assay measures the GCK-GKRP protein-protein interaction independent of GCK enzymatic substrates and activity. All three assays are scalable to low volumes in 1536-well plate format, with robust Z' factors (>0.7). Finally, as GKRP sequesters GCK in the hepatocyte nucleus at low glucose concentrations, we explored cellular models of GCK localization and translocation. Previous findings from freshly isolated rat hepatocytes were confirmed in cryopreserved rat hepatocytes, and we further extended this study to cryopreserved human hepatocytes. Consistent with previous reports, there were several key differences between the rat and human systems, with our results suggesting that human hepatocytes can be used to interrogate GCK translocation in response to small molecules. The assay panel developed here should help direct future investigation of the GCK-GKRP interaction in these or other physiologically relevant human systems.
Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ∼65% are missense. In many cases ...co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113-1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) -mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC(50) 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively p<0.03). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively p<0.001). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.
Most studies seeking common variant associations with type 2 diabetes (T2D) have focused on individuals of European ancestry. These discoveries need to be evaluated in other major ancestral groups, ...to understand ethnic differences in predisposition, and establish whether these contribute to variation in T2D prevalence and presentation. This study aims to establish whether common variants conferring T2D-risk in Europeans contribute to T2D-susceptibility in the South Asian population of Sri Lanka.
Lead single nucleotide polymorphism (SNPs) at 37 T2D-risk loci attaining genome-wide significance in Europeans were genotyped in 878 T2D cases and 1523 normoglycaemic controls from Sri Lanka. Association testing was performed by logistic regression adjusting for age and sex and by the Cochran-Mantel-Haenszel test after stratifying according to self-identified ethnolinguistic subgroup. A weighted genetic risk score was generated to examine the combined effect of these SNPs on T2D-risk in the Sri Lankan population.
Of the 36 SNPs passing quality control, sixteen showed nominal (p<0.05) association in Sri Lankan samples, fifteen of those directionally-consistent with the original signal. Overall, these association findings were robust to analyses that accounted for membership of ethnolinguistic subgroups. Overall, the odds ratios for 31 of the 36 SNPs were directionally-consistent with those observed in Europeans (p = 3.2×10(-6)). Allelic odds ratios and risk allele frequencies in Sri Lankan subjects were not systematically different to those reported in Europeans. Genetic risk score and risk of T2D were strongly related in Sri Lankans (per allele OR 1.10 95%CI 1.08-1.13, p = 1.2×10(-17)).
Our data indicate that most T2D-risk variants identified in Europeans have similar effects in South Asians from Sri Lanka, and that systematic difference in common variant associations are unlikely to explain inter-ethnic differences in prevalence or presentation of T2D.
Background
This study aimed to develop and provide a psychometric and feasibility pilot evaluation of the Heart Failure (HF) Symptom Tracker (HFaST), a new patient-reported tool designed to ...facilitate communication between patients and health care providers (HCPs) in routine clinical care. The HFaST enables patients to identify worsening HF symptoms, with a long-term goal of preventing hospitalizations or emergency room visits.
Methods
The HFaST was developed drawing on evidence from the literature, qualitatively with cognitive interviews (12 patient/caregiver and 8 HCPs), and evaluated quantitatively (psychometric, feasibility assessment). The HFaST was administered for 7 consecutive days to 100 individuals diagnosed with HF during a multisite, non-interventional US pilot study. Health care providers then completed a survey assessing the feasibility and importance of the HFaST in clinical practice.
Qualitative development included a literature review and cognitive interviews with patients, caregivers, and HCPs. The psychometric properties of the HFaST were evaluated using classical test theory methods. Descriptive statistics provided insight into HCPs’ perceptions of the feasibility of using the HFaST in clinical practice.
Results
A preliminary set of 40 items was developed for the symptom tracker and iteratively reduced to 10 items based on the qualitative phase. Test-retest reliability (weighted kappa 0.71–0.97), discriminating validity, and construct validity of the HFaST were acceptable. HCPs rated the HFaST as a good (70%) or excellent (30%) means of tracking HF symptoms. Six HFaST items were ultimately retained, covering concepts of fatigue, shortness of breath (3 items), swelling, and rapid weight gain.
Conclusions
The 6-item HFaST is an easy-to-use tool designed to raise patients’ awareness of HF symptoms and facilitate communication with HCPs. Future research should evaluate HFaST implementation in clinical practice and effectiveness as an intervention to potentially prevent hospitalizations and emergency room visits.
Over the years, some have lamented the Supreme Court's willingness to overrule itself and have urged the Court to abandon its weak presumption of stare decisis in constitutional cases in favor of a ...more stringent rule. This article points out that one virtue of the weak presumption is that it promotes doctrinal stability while still accommodating pluralism on the Court. It explains the general contours of stare decisis. It develops the thesis that, at least in controversial constitutional cases, an overlooked function of stare decisis is mediating jurisprudential disagreement. The article observes, however, that less rides on the strength of stare decisis than is commonly supposed. Discussions of stare decisis tend to proceed as if horizontal stare decisis -- the Court's obligation to follow its own precedent -- is the only mechanism for maintaining doctrinal stability.
The advent of affinity-based proteomics technologies for global protein profiling provides the prospect of finding new molecular biomarkers for common, multifactorial disorders. The molecular ...phenotypes obtained from studies on such platforms are driven by multiple sources, including genetic, environmental, and experimental components. In characterizing the contribution of different sources of variation to the measured phenotypes, the aim is to facilitate the design and interpretation of future biomedical studies employing exploratory and multiplexed technologies. Thus, biometrical genetic modelling of twin or other family data can be used to decompose the variation underlying a phenotype into biological and experimental components.
Using antibody suspension bead arrays and antibodies from the Human Protein Atlas, we study unfractionated serum from a longitudinal study on 154 twins. In this study, we provide a detailed description of how the variation in a molecular phenotype in terms of protein profile can be decomposed into familial i.e. genetic and common environmental; individual environmental, short-term biological and experimental components. The results show that across 69 antibodies analyzed in the study, the median proportion of the total variation explained by familial sources is 12% (IQR 1-22%), and the median proportion of the total variation attributable to experimental sources is 63% (IQR 53-72%).
The variability analysis of antibody arrays highlights the importance to consider variability components and their relative contributions when designing and evaluating studies for biomarker discoveries with exploratory, high-throughput and multiplexed methods.
Relying on something it calls "supervisory power" or "supervisory authority, " the Supreme Court regularly prescribes rules of procedure and evidence for inferior courts. Both scholars and the Court ...have treated the Court's exercises of this authority as unexceptional exercises of the inherent authority that Article III grants every federal court to regulate procedure in the course of adjudication. Article III's grant of inherent authority, however, is conventionally understood as permitting a federal court to regulate its own proceedings. When the Supreme Court exercises supervisory power, it regulates the proceedings of other federal courts. More than a reference to every court's inherent authority, therefore, is required to justify the Court's action. If the Supreme Court possesses a unique ability to regulate federal court procedure, it must be because of some unique attribute of the Supreme Court. This Article explores a justification that may well animate the Court's assertions of supervisory power: the notion that the Court possesses supervisory power by virtue of its constitutional supremacy. Analyzing this justification requires pursuit of two questions that are wholly unexplored in the literature and case law. Does Article III's distinction between supreme and inferior courts operate only as a limit on the way that Congress can structure the judicial department, or does it also operate as a source of inherent authority for the Supreme Court? And assuming that the Court's supremacy grants it inherent authority over inferior courts, is supervisory power over procedure part of the authority granted?
The purpose of this study was to investigate the relationship between sleep problems, gastrointestinal symptoms, social functioning, autism traits, and social support on quality of life (QoL) in 107 ...adults with autism spectrum disorder (ASD).
Questionnaires included the Autism Spectrum Quotient-10 (Adult), Multidimensional Scale of Perceived Social Support, Social Functioning Questionnaire, Pittsburgh Sleep Quality Index, Gastrointestinal Symptom Inventory, and World Health Organization Quality of Life-BREF.
GI symptoms were a common comorbidity with 86 % of participants presenting with them. Sleep problems were also frequent issues with 89 % of participants being classified as poor sleepers. Greater sleep problems were correlated with poorer QoL in the physical health and environment domains. Specifically, the sleep problem of daytime dysfunction was correlated with poorer QoL in physical health. Daytime dysfunction and sleep duration were correlated with poorer QoL in the environment domain. Better social support was correlated with greater QoL in the psychological, social and environment domains. Poorer social functioning was correlated with poorer QoL in each of the four domains.
This research indicated that GI symptoms and sleep problems are common comorbid conditions in the adult ASD population. This paper expanded upon the existing literature by highlighting unexplored factors influencing QoL in adults with ASD.
Patient-reported outcome measures (PROMs) provide an important complement to physician-assessed clinical outcome measures in dermatologic diseases such as atopic dermatitis (AD) and chronic hand ...eczema (CHE). AD and CHE are chronic and relapsing inflammatory skin conditions that often co-occur. While both diseases result in various signs and symptoms that are burdensome and can negatively affect patients’ lives, there may be distinct differences in the signs, symptoms, burden, and health-related quality of life (HRQOL) impact of these diseases. The objective of this study was to identify and evaluate PROMs used in studies of AD and CHE. The aim was to explore the assessment of key symptoms and impacts, and identify any gaps in the measures in use. A structured review of the PubMed database was conducted to identify PROMs used or developed for use in AD or CHE. The Dermatology Life Quality Index (DLQI), the Pruritus/Itch Numeric Rating Scale (NRS), the Patient-Oriented Eczema Measure (POEM), and the Quality of Life in Hand Eczema Questionnaire (QOLHEQ) were identified and reviewed in detail. With these measures, the AD and CHE symptoms and impacts most commonly evaluated in the literature include dermatology-related HRQOL in the domains of symptoms and feelings, daily activities, leisure, work and school, personal relationships, and adverse effects; pruritus; sleep disturbance; AD-specific symptoms (dryness, itching, flaking, cracking, bleeding, and weeping/oozing); and CHE-specific symptoms (pain, itch, fissuring, redness, bleeding, and dryness). A review of regulatory labels of drugs approved for AD by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) found that, among the four measures reviewed, the Pruritus NRS was included in the FDA and EMA labels for dupilumab, the DLQI was included in the EMA labels for dupilumab and tacrolimus, and the POEM was included in the EMA label for dupilumab. Key symptoms of AD (e.g. itching, flaking, cracking) and CHE (e.g. pain, itching, fissuring) are increasingly being assessed with PROMs; however, primary endpoints in clinical trials are often based on clinician-reported outcome measures. As therapeutic strategies in dermatology are targeted at specific dermatologic symptoms and diseases affecting specific sites (e.g. CHE), future research should explore patients’ experiences with these symptoms and sites and the changes with treatment that are most meaningful to them.
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