Therapeutic Rescue of Misfolded Mutants: Validation of Primary High Throughput Screens for Identification of Pharmacoperone Drugs

Functional rescue of misfolded mutant receptors by small non-peptide molecules has been demonstrated. These small, target-specific molecules (pharmacological chaperones or "pharmacoperones") serve as molecular templates, promote correct folding and allow otherwise misfolded mutants to pass the scrutiny of the cellular quality control system (QCS) and be expressed at the plasma membrane (PM) where they function similarly to wild type (WT) proteins. In the case of the gonadotropin releasing hormone receptor (GnRHR), drugs that rescue one mutant typically rescue many mutants, even if the mutations are located at distant sites (extracellular loops, intracellular loops, transmembrane helices). This increases the value of these drugs. These drugs are typically identified, post hoc, from "hits" in screens designed to detect antagonists or agonists. The therapeutic utility of pharmacoperones has been limited due to the absence of screens that enable identification of pharmacoperones per se.We describe a generalizable primary screening approach for pharmacoperone drugs based on measurement of gain of activity in stable HeLa cells stably expressing the mutants of two different model G-protein coupled receptors (GPCRs) (hGnRHR[E(90)K] or hV2R[L(83)Q]). These cells turn off expression of the receptor mutant gene of interest in the presence of tetracycline and its analogs, which provides a convenient means to identify false positives.The methods described and characterized here provide the basis of novel primary screens for pharmacoperones that detect drugs that rescue GPCR mutants of specific receptors. This approach will identify structures that would have been missed in screens that were designed to select only agonists or antagonists. Non-antagonistic pharmacoperones have a therapeutic advantage since they will not compete for endogenous agonists and may not have to be washed out once rescue has occurred and before activation by endogenous or exogenous agonists

Selecting and implementing overview methods: implications from five exemplar overviews

This is the final version of the article. Available from BioMed Central via the DOI in this record.Background Overviews of systematic reviews are an increasingly popular method of evidence synthesis; there is a lack of clear guidance for completing overviews and a number of methodological challenges. At the UK Cochrane Symposium 2016, methodological challenges of five overviews were explored. Using data from these five overviews, practical implications to support methodological decision making of authors writing protocols for future overviews are proposed. Methods Methods, and their justification, from the five exemplar overviews were tabulated and compared with areas of debate identified within current literature. Key methodological challenges and implications for development of overview protocols were generated and synthesised into a list, discussed and refined until there was consensus. Results Methodological features of three Cochrane overviews, one overview of diagnostic test accuracy and one mixed methods overview have been summarised. Methods of selection of reviews and data extraction were similar. Either the AMSTAR or ROBIS tool was used to assess quality of included reviews. The GRADE approach was most commonly used to assess quality of evidence within the reviews. Eight key methodological challenges were identified from the exemplar overviews. There was good agreement between our findings and emerging areas of debate within a recent published synthesis. Implications for development of protocols for future overviews were identified. Conclusions Overviews are a relatively new methodological innovation, and there are currently substantial variations in the methodological approaches used within different overviews. There are considerable methodological challenges for which optimal solutions are not necessarily yet known. Lessons learnt from five exemplar overviews highlight a number of methodological decisions which may be beneficial to consider during the development of an overview protocol.The overview conducted by Pollock [19] was supported by a project grant from the Chief Scientist Office of the Scottish Government. The overview conducted by McClurg [21] was supported by a project grant by the Physiotherapy Research Foundation. The overview by Hunt [22] was supported as part of doctoral programme funding by the National Institute for Health Research (NIHR) Collaboration for Leadership in Applied Health Research and Care South West Peninsula (PenCLAHRC). The overview conducted by Estcourt [20] was supported by an NIHR Cochrane Programme Grant for the Safe and Appropriate Use of Blood Components. The overview conducted by Brunton [23] was commissioned by the Department of Health as part of an ongoing programme of work on health policy research synthesis. Alex Pollock is employed by the Nursing, Midwifery and Allied Health Professions (NMAHP) Research Unit, which is supported by the Chief Scientist Office of the Scottish Government. Pauline Campbell is supported by the Chief Nurses Office of the Scottish Government

Walks4work: Rationale and study design to investigate walking at lunchtime in the workplace setting

Background: Following recruitment of a private sector company, an 8week lunchtime walking intervention was implemented to examine the effect of the intervention on modifiable cardiovascular disease risk factors, and further to see if walking environment had any further effect on the cardiovascular disease risk factors. Methods. For phase 1 of the study participants were divided into three groups, two lunchtime walking intervention groups to walk around either an urban or natural environment twice a week during their lunch break over an 8week period. The third group was a waiting-list control who would be invited to join the walking groups after phase 1. In phase 2 all participants were encouraged to walk during their lunch break on self-selecting routes. Health checks were completed at baseline, end of phase 1 and end of phase 2 in order to measure the impact of the intervention on cardiovascular disease risk. The primary outcome variables of heart rate and heart rate variability were measured to assess autonomic function associated with cardiovascular disease. Secondary outcome variables (Body mass index, blood pressure, fitness, autonomic response to a stressor) related to cardiovascular disease were also measured. The efficacy of the intervention in increasing physical activity was objectively monitored throughout the 8-weeks using an accelerometer device. Discussion. The results of this study will help in developing interventions with low researcher input with high participant output that may be implemented in the workplace. If effective, this study will highlight the contribution that natural environments can make in the reduction of modifiable cardiovascular disease risk factors within the workplace. © 2012 Brown et al.; licensee BioMed Central Ltd

Annual variations in the number of malaria cases related to two different patterns of Anopheles darlingi transmission potential in the Maroni area of French Guiana

<p>Abstract</p> <p>Background</p> <p>With an Annual Parasite Incidence (API) of 132.1, in the high and moderate risks zones, the Maroni area of French Guiana has the second highest malaria incidence of South-America after Guyana (API = 183.54) and far above Brazil (API = 28.25). Malaria transmission is occurring despite strong medical assistance and active vector control, based on general WHO recommendations. This situation is generated by two main factors that are the social and cultural characteristics of this border area, where several ethnic groups are living, and the lack of understanding of transmission dynamics of the main mosquito vector, <it>Anopheles darlingi.</it> In this context, entomological data collected in two villages belonging to two different ethnic groups of the French border of the Maroni River, were retrospectively analysed to find out how the mosquito bionomics are related to the malaria transmission patterns.</p> <p>Methods</p> <p>Data were provided by human landing catches of mosquitoes carried out each month for two years in two villages belonging to two ethnic groups, the Amerindians Wayanas and the Aloukous of African origin. The mosquitoes were sorted by species, sex, date, hour and place of collection and processed for <it>Plasmodium sp</it>. parasite detection. The data were compiled to provide the following variables: human biting rates (HBR), parity rates (PR), numbers of infective bites (IB), entomological inoculation rates (EIR) and numbers of infected mosquitoes surviving enough to transmit (IMT). Spatial and temporal differences of variables between locations and during the night were tested by the Kruskall-Wallis analysis of variance to find out significant variations.</p> <p>Results</p> <p>The populations of the main mosquito vector <it>An. darlingi </it>showed significant variations in the spatial and temporal HBR/person/night and HBR/person/hour, IB/person/month and IB/person/hour, and IMT/village/night and IMT/village/hour. In the village of Loca (Aloukous), the IMT peaked from June to August with a very low transmission during the other months. The risks were higher during the first part of the night and an EIR of 10 infective bites per person and per year was estimated. In the village of Twenke (Wayanas), high level of transmission was reported all year with small peaks in March and October. The risk was higher during the second part of the night and an EIR of 5 infective bites per person and per year was estimated.</p> <p>Conclusion</p> <p>For the first time in the past 40 years, the mosquito bionomics was related to the malaria transmission patterns in French Guiana. The peak of malaria cases reported from August to October in the Maroni region is concomitant with the significant peak of <it>An. darlingi </it>IMT, reported from the village of Loca where transmission is higher. However, the persistent number of cases reported all year long may also be related to the transmission in the Amerindian villages. The <it>An. darlingi </it>bionomics for these two close populations were found significantly different and may explain why a uniform vector control method is inadequate. Following these findings, malaria prevention measures adapted to the local conditions are needed. Finally, the question of the presence of <it>An. darlingi </it>sub-species is raised.</p

Eliciting Dirichlet and Gaussian copula prior distributions for multinomial models

In this paper, we propose novel methods of quantifying expert opinion about prior distributions for multinomial models. Two different multivariate priors are elicited using median and quartile assessments of the multinomial probabilities. First, we start by eliciting a univariate beta distribution for the probability of each category. Then we elicit the hyperparameters of the Dirichlet distribution, as a tractable conjugate prior, from those of the univariate betas through various forms of reconciliation using least-squares techniques. However, a multivariate copula function will give a more flexible correlation structure between multinomial parameters if it is used as their multivariate prior distribution. So, second, we use beta marginal distributions to construct a Gaussian copula as a multivariate normal distribution function that binds these marginals and expresses the dependence structure between them. The proposed method elicits a positive-definite correlation matrix of this Gaussian copula. The two proposed methods are designed to be used through interactive graphical software written in Java

Improving access to emergent spinal care through knowledge translation : an ethnographic study

Background: For patients and family members, access to timely specialty medical care for emergent spinal conditions is a significant stressor to an already serious condition. Timing to surgical care for emergent spinal conditions such as spinal trauma is an important predictor of outcome. However, few studies have explored ethnographically the views of surgeons and other key stakeholders on issues related to patient access and care for emergent spine conditions. The primary study objective was to determine the challenges to the provision of timely care as well as to identify areas of opportunities to enhance care delivery. Methods: An ethnographic study of key administrative and clinical care providers involved in the triage and care of patients referred through CritiCall Ontario was undertaken utilizing standard methods of qualitative inquiry. This comprised 21 interviews with people involved in varying capacities with the provision of emergent spinal care, as well as qualitative observations on an orthopaedic/neurosurgical ward, in operating theatres, and at CritiCall Ontario’s call centre. Results: Several themes were identified and organized into categories that range from inter-professional collaboration through to issues of hospital-level resources and the role of relationships between hospitals and external organizations at the provincial level. Underlying many of these issues is the nature of the medically complex emergent spine patient and the scientific evidentiary base upon which best practice care is delivered. Through the implementation of knowledge translation strategies facilitated from this research, a reduction of patient transfers out of province was observed in the one-year period following program implementation. Conclusions: Our findings suggest that competing priorities at both the hospital and provincial level create challenges in the delivery of spinal care. Key stakeholders recognized spinal care as aligning with multiple priorities such as emergent/critical care, medical through surgical, acute through rehabilitative, disease-based (i.e. trauma, cancer), and wait times initiatives. However, despite newly implemented strategies, there continues to be increasing trends over time in the number of spinal CritiCall Ontario referrals. This reinforces the need for ongoing inter-professional efforts in care delivery that take into account the institutional contexts that may constrain individual or team efforts

Low-Volume High-Intensity Interval Training in a Gym Setting Improves Cardio-Metabolic and Psychological Health.

BACKGROUND: Within a controlled laboratory environment, high-intensity interval training (HIT) elicits similar cardiovascular and metabolic benefits as traditional moderate-intensity continuous training (MICT). It is currently unclear how HIT can be applied effectively in a real-world environment. PURPOSE: To investigate the hypothesis that 10 weeks of HIT, performed in an instructor-led, group-based gym setting, elicits improvements in aerobic capacity (VO2max), cardio-metabolic risk and psychological health which are comparable to MICT. METHODS: Ninety physically inactive volunteers (42±11 y, 27.7±4.8 kg.m-2) were randomly assigned to HIT or MICT group exercise classes. HIT consisted of repeated sprints (15-60 seconds, >90% HRmax) interspersed with periods of recovery cycling (≤25 min.session-1, 3 sessions.week-1). MICT participants performed continuous cycling (~70% HRmax, 30-45 min.session-1, 5 sessions.week-1). VO2max, markers of cardio-metabolic risk, and psychological health were assessed pre and post-intervention. RESULTS: Mean weekly training time was 55±10 (HIT) and 128±44 min (MICT) (p<0.05), with greater adherence to HIT (83±14% vs. 61±15% prescribed sessions attended, respectively; p<0.05). HIT improved VO2max, insulin sensitivity, reduced abdominal fat mass, and induced favourable changes in blood lipids (p<0.05). HIT also induced beneficial effects on health perceptions, positive and negative affect, and subjective vitality (p<0.05). No difference between HIT and MICT was seen for any of these variables. CONCLUSIONS: HIT performed in a real-world gym setting improves cardio-metabolic risk factors and psychological health in physically inactive adults. With a reduced time commitment and greater adherence than MICT, HIT offers a viable and effective exercise strategy to target the growing incidence of metabolic disease and psychological ill-being associated with physical inactivity

Discovery of Dual-Action Membrane-Anchored Modulators of Incretin Receptors

The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptors are considered complementary therapeutic targets for type 2 diabetes. Using recombinant membrane-tethered ligand (MTL) technology, the present study focused on defining optimized modulators of these receptors, as well as exploring how local anchoring influences soluble peptide function.Serial substitution of residue 7 in membrane-tethered GIP (tGIP) led to a wide range of activities at the GIP receptor, with [G(7)]tGIP showing enhanced efficacy compared to the wild type construct. In contrast, introduction of G(7) into the related ligands, tGLP-1 and tethered exendin-4 (tEXE4), did not affect signaling at the cognate GLP-1 receptor. Both soluble and tethered GIP and GLP-1 were selective activators of their respective receptors. Although soluble EXE4 is highly selective for the GLP-1 receptor, unexpectedly, tethered EXE4 was found to be a potent activator of both the GLP-1 and GIP receptors. Diverging from the pharmacological properties of soluble and tethered GIP, the newly identified GIP-R agonists, (i.e. [G(7)]tGIP and tEXE4) failed to trigger cognate receptor endocytosis. In an attempt to recapitulate the dual agonism observed with tEXE4, we conjugated soluble EXE4 to a lipid moiety. Not only did this soluble peptide activate both the GLP-1 and GIP receptors but, when added to receptor expressing cells, the activity persists despite serial washes.These findings suggest that conversion of a recombinant MTL to a soluble membrane anchored equivalent offers a means to prolong ligand function, as well as to design agonists that can simultaneously act on more than one therapeutic target

Molecular Analysis of Serum and Bronchoalveolar Lavage in a Mouse Model of Influenza Reveals Markers of Disease Severity That Can Be Clinically Useful in Humans

Background: Management of influenza, a major contributor to the worldwide disease burden, is complicated by lack of reliable methods for early identification of susceptible individuals. Identification of molecular markers that can augment existing diagnostic tools for prediction of severity can be expected to greatly improve disease management capabilities. Methodology/Principal Findings: We have analyzed cytokines, proteome flux and protein adducts in bronchoalveolar lavage (BAL) and sera from mice infected with influenza A virus (PR8 strain) using a previously established non-lethal model of influenza infection. Through detailed cytokine and protein adduct measurements of murine BAL, we first established the temporal profile of innate and adaptive responses as well as macrophage and neutrophil activities in response to influenza infection. A similar analysis was also performed with sera from a longitudinal cohort of influenza patients. We then used an iTRAQ-based, comparative serum proteome analysis to catalog the proteome flux in the murine BAL during the stages correlating with “peak viremia,” “inflammatory damage,” as well as the “recovery phase.” In addition to activation of acute phase responses, a distinct class of lung proteins including surfactant proteins was found to be depleted from the BAL coincident with their “appearance” in the serum, presumably due to leakage of the protein following loss of the integrity of the lung/epithelial barrier. Serum levels of at least two of these proteins were elevated in influenza patients during the febrile phase of infection compared to healthy controls or to the same patients at convalescence. Conclusions/Significance: The findings from this study provide a molecular description of disease progression in a mouse model of influenza and demonstrate its potential for translation into a novel class of markers for measurement of acute lung injury and improved case management.Singapore. National Research FoundationSingapore-MIT Alliance for Research and Technology (ID-IRG research program