An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

Intravitreal AAV2.COMP-Ang1 prevents neurovascular degeneration in a murine model of diabetic retinopathy

Diabetic retinopathy (DR) is the leading cause of blindness in the working-age population in the United States. The vision-threatening processes of neuroglial and vascular dysfunction in DR occur in concert, driven by hyperglycemia and propelled by a pathway of inflammation, ischemia, vasodegeneration, and breakdown of the blood retinal barrier. Currently, no therapies exist for normalizing the vasculature in DR. Here we show that a single intravitreal dose of adeno-associated virus serotype 2 encoding a more stable, soluble, and potent form of angiopoietin 1 (AAV2.COMP-Ang1) can ameliorate the structural and functional hallmarks of DR in Ins2Akita mice, with sustained effects observed through six months. In early DR, AAV2.COMP-Ang1 restored leukocyte-endothelial interaction, retinal oxygenation, vascular density, vascular marker expression, vessel permeability, retinal thickness, inner retinal cellularity, and retinal neurophysiological response to levels comparable to non-diabetic controls. In late DR, AAV2.COMP-Ang1 enhanced the therapeutic benefit of intravitreally-delivered endothelial colony-forming cells by promoting their integration into the vasculature and thereby stemming further visual decline. AAV2.COMP-Ang1 single-dose gene therapy can prevent neurovascular pathology, support vascular regeneration, and stabilize vision in DR.

GTPase activity of dynamin and resulting conformation change are essential for endocytosis

Dynamin is a large GTPase with a relative molecular mass of 96,000 (Mr 96K) that is involved in clathrin-mediated endocytosis and other vesicular trafficking processes. Although its function is apparently essential for scission of newly formed vesicles from the plasma membrane, the nature of dynamin's role in the scission process is still unclear. It has been proposed that dynamin is a regulator (similar to classical G proteins) of downstream effectors. Here we report the analysis of several point mutants of dynamin's GTPase effector (GED) and GTPase domains. We show that oligomerization and GTP binding alone, by dynamin, are not sufficient for endocytosis in vivo. Rather, efficient GTP hydrolysis and an associated conformational change are also required. These data argue that dynamin has a mechanochemical function in vesicle scission.

Treatment of Unexplained Chronic Cough: CHEST Guideline and Expert Panel Report

Background: Unexplained chronic cough (UCC) causes significant quality of life impairment. There is a need to identify effective assessment and treatment approaches for UCC.

Methods: This systematic review of randomized controlled clinical trials asked: What is the efficacy of treatment compared to usual care on cough severity, cough frequency, and cough-related quality of life in patients with unexplained chronic cough (UCC)? Studies of adults and adolescents >12 years with a chronic cough of >8 weeks duration that was unexplained after systematic investigation and treatment were included and assessed for relevance and quality. Based upon the systematic review, guideline suggestions were developed and voted upon using CHEST organization methodology.

Results: 11 RCTs and 5 systematic reviews were included. The 11 RCTs reported data on 570 participants with chronic cough who received a variety of interventions. Study quality was high in 10 RCTs. The studies used a variety of descriptors and assessments to identify unexplained chronic cough. While gabapentin and morphine showed positive effects on cough-related quality of life, only gabapentin was supported as a treatment recommendation. Studies of inhaled corticosteroids (ICS) suffered from intervention fidelity bias, and when this was addressed, ICS were not found to be effective for UCC. Esomeprazole was not effective for UCC without features of gastroesophageal acid reflux. Studies addressing non-acid gastroesophageal reflux were not identified. A multimodality speech pathology intervention improved cough severity.

Conclusions: The evidence supporting the diagnosis and management of UCC is limited. UCC requires further study to establish agreed terminology and the optimal methods of investigation using established criteria for intervention fidelity. Speech pathology based cough suppression is suggested as a treatment option for UCC. This guideline presents suggestions for diagnosis and treatment based on the best available evidence and identifies gaps in our knowledge and areas for future research.

Regulating Digital Financial Services Agents in Developing Countries to Promote Financial Inclusion

Limited access to bank branches excludes over one billion people from accessing financial services in developing countries. Digital financial services offered by banks and mobile money providers through agents can solve this problem without the need for complex and costly physical banking infrastructures. Delivering digital financial services through agents requires a legal framework to regulate liability. This article analyses whether vicarious liability of the principal is a more efficient regulatory approach than personal liability of the agent. Agent liability in Kenya, Fiji, and Malawi is analysed to demonstrate that vicarious liability of the principal, coupled to an explicit agreement as to agent rewards and penalties, is the more efficient regulatory approach.

Glycosylation of Vitamin D Binding Protein Reduced in Juvenile Idiopathic Arthritis Patients At Risk of Disease Extension.

Background/Purpose:Juvenile idiopathic arthritis (JIA) comprises a poorly understood group of chronic, childhood onset, autoimmune diseases with variable clinical outcomes. We investigated whether profiling of the synovial fluid (SF) proteome by a fluorescent dye based, two-dimensional gel (DIGE) approach could distinguish the subset of patients in whom inflammation extends to affect a large number of joints, early in the disease process. The post-translational modifications to candidate protein markers were verified by a novel deglycosylation strategy.Methods:SF samples from 57 patients were obtained around time of initial diagnosis of JIA. At 1 year from inclusion patients were categorized according to ILAR criteria as oligoarticular arthritis (n=26), extended oligoarticular (n=8) and polyarticular disease (n=18). SF samples were labeled with Cy dyes and separated by two-dimensional electrophoresis. Multivariate analyses were used to isolate a panel of proteins which distinguish patient subgroups. Proteins were identified using MALDI-TOF mass spectrometry with vitamin D binding protein (VDBP) expression and siaylation further verified by immunohistochemistry, ELISA test and immunoprecipitation. Candidate biomarkers were compared to conventional inflammation measure C-reactive protein (CRP). Sialic acid residues were enzymatically cleaved from immunopurified SF VDBP, enriched by hydrophilic interaction liquid chromatography (HILIC) and analysed by mass spectrometry.Results:Hierarchical clustering based on the expression levels of a set of 23 proteins segregated the extended-to-be oligoarticular from the oligoarticular patients. A cleaved isoform of VDBP, spot 873, is present at significantly reduced levels in the SF of oligoarticular patients at risk of disease extension, relative to other subgroups (p<0.05). Conversely total levels of vitamin D binding protein are elevated in plasma and ROC curves indicate an improved diagnostic sensitivity to detect patients at risk of disease extension, over both spot 873 and CRP levels. Sialysed forms of intact immunopurified VDBP were more prevalent in persistent oligoarticular patient synovial fluids.Conclusion:The data indicate that a subset of the synovial fluid proteome may be used to stratify patients to determine risk of disease extension. Reduced conversion of VDBP to a macrophage activation factor may represent a novel pathway contributing to increased risk of disease extension in JIA patients.

Are older people prescribed antidepressants on the basis of fewer symptoms of depression, and for longer periods of time? A survey of 1825 New Zealanders

Aims: To determine whether older people are prescribed antidepressants at lower levels of depression and with fewer symptoms, and whether they are more likely to engage in chronic usage. Methods: An online survey about experiences with, and opinions about, depression and antidepressants, was completed by 1,825 New Zealand adults who had been prescribed antidepressants in the preceding five years. Results: Participants over 55 were prescribed antidepressants with significantly fewer symptoms and were significantly less likely to meet DSM criteria for depression. They were also significantly more likely to have used the drugs for three years and still be using them. Conclusions: Prescribing physicians and their older patients might benefit from discussing the pros and cons of antidepressants (including the additional risk factors with this age group) and the alternatives; and, if prescription does occur, careful monitoring to avoid unnecessary, potentially damaging, long-term use is recommended.

Understanding the non-pharmacological correlates of self-reported efficacy of antidepressants

Objective: To explore the non-pharmacological correlates of the perceived effectiveness of antidepressants (ADs), thereby enhancing understanding of the mechanisms involved in recovery from depression while taking ADs. Method: An online survey was completed by 1781 New Zealand adults who had taken ADs in the previous 5 years. Results: All 18 psychosocial variables measured were associated with depression reduction, and 16 with improved quality of life (QoL). Logistic regression models revealed that the quality of the relationship with the prescriber was related to both depression reduction and improved QoL. In addition, depression reduction was related to younger age, higher income, being fully informed about ADs by the prescriber, fewer social causal beliefs for depression and not having lost a loved one in the 2 months prior to prescription. Furthermore, both outcome measures were positively related to belief in ‘chemical’ rather than ‘placebo’ effects. Conclusion: There are multiple non-pharmacological processes involved in recovery while taking ADs. Enhancing them, for example focusing on the prescriber–patient relationship and giving more information, may enhance recovery rates, with or without ADs.

Complex Interplay Between Determinants of Pacing and Performance During 20 km Cycle Time Trials

Purpose This study examined the determinants of pacing strategy and performance during self paced maximal exercise. Methods Eight well trained cyclists completed two 20 km time trials. Power output, RPE, positive and negative affect, and iEMG activity of the active musculature were recorded every 0.5km, confidence in achieving pre-exercise goals was assessed every 5 km, and blood lactate and pH were measured post-exercise. Differences in all parameters were assessed between fastest (FAST) and slowest (SLOW) trials performed. Results Mean power output was significantly higher during the initial 90% of FAST, but not the final 10%, and blood lactate concentration was significantly higher and pH significantly lower following FAST. Mean iEMG activity was significantly higher throughout SLOW. RPE was similar throughout both trials, but participants had significantly more positive affect and less negative affect throughout FAST. Participants grew less confident in their ability to achieve their goals throughout SLOW. Conclusions The results suggest that affect may be the primary psychological regulator of pacing strategy and that higher levels of positivity and lower levels of negativity may have been associated with a more aggressive strategy during FAST. Although the exact mechanisms through which affect acts to influence performance are unclear, it may determine the degree of physiological disruption that can be tolerated, or be reflective of peripheral physiological status in relation to the still to be completed exercise task.

Influence of Different Performance Levels on Pacing Strategy During the Women's World Championship Marathon Race.

Purpose To analyse pacing strategies displayed by athletes achieving differing levels of performance during an elite level marathon race. Methods Competitors in the 2009 IAAF Women’s Marathon Championship were split into Groups 1, 2, 3, and 4 comprising the first, second, third, and fourth 25% of finishers respectively. Final, intermediate, and personal best (PB) times of finishers were converted to mean speeds, and relative speed (% of PB speed) was calculated for intermediate segments. Results Mean PB speed decreased from Group 1 to 4 and speed maintained in the race was 98.5 + 1.8%, 97.4 + 3.2%, 95.0 + 3.1% and 92.4 + 4.4% of PB speed for Groups 1-4 respectively. Group 1 was fastest in all segments and differences in speed between groups increased throughout the race. Group 1 ran at lower relative speeds than other groups for the first two 5 km segments, but higher relative speeds after 35km. Significant differences (P<0.01) in the percentage of PB speed maintained were observed between Groups 1 and 4, and 2 and 4 in all segments after 20 km, and Groups 3 and 4 from 20-25 km and 30-35 km. Conclusions Group 1 athletes achieved superior finishing times relative to their PB than athletes in other Groups who selected unsustainable initial speeds resulting in subsequent significant losses of speed. It is suggested that psychological factors specific to a major competitive event influenced decision making by athletes and poor decisions resulted in final performances inferior to those expected based on PB times.

Common Ragweed: A Threat to Environmental Health in Europe

Common or short ragweed (Ambrosia artemisiifolia L.) is an annual herb belonging to the Asteraceae family that was described by Carl Linnaeus in the 18th century. It is a noxious invasive species that is an important weed in agriculture and a source of highly allergenic pollen. The importance placed on A. artemisiifolia is reflected by the number of international projects that have now been launched by the European Commission and the increasing number of publications being produced on this topic. This review paper examines existing knowledge about ragweed ecology, distribution and flowering phenology and the environmental health risk that this noxious plant poses in Europe. The paper also examines control measures used in the fight against it and state of the art methods for modelling atmospheric concentrations of this important aeroallergen. Common ragweed is an environmental health threat, not only in its native North America but also in many parts of the world where it has been introduced. In Europe, where the plant has now become naturalised and frequently forms part of the flora, the threat posed by ragweed has been identified and steps are being taken to reduce further geographical expansion and limit increases in population densities of the plant in order to protect the allergic population. This is particularly important when one considers possible range shifts, changes in flowering phenology and increases in the amount of pollen and allergenic potency that could be brought about by changes in climate.

Application of Decision-Making Theory to the Regulation of Muscular Work Rate During Self-Paced Competitive Endurance Activity.

Successful participation in competitive endurance activities requires continual regulation of muscular work rate in order to maximise physiological performance capacities, meaning that individuals must make numerous decisions with regards to the muscular work rate selected at any point in time. Decisions relating to the setting of appropriate goals and the overall strategic approach to be utilised are made prior to the commencement of an event, whereas tactical decisions are made during the event itself. This review examines current theories of decision-making in an attempt to explain the manner in which regulation of muscular work is achieved during athletic activity. We describe rational and heuristic theories, and relate these to current models of regulatory processes during self-paced exercise in an attempt to explain observations made in both laboratory and competitive environments. Additionally, we use rational and heuristic theories in an attempt to explain the influence of the presence of direct competitors on the quality of the decisions made during these activities. We hypothesise that although both rational and heuristic models can plausibly explain many observed behaviours in competitive endurance activities, the complexity of the environment in which such activities occur would imply that effective rational decision-making is unlikely. However, at present, many proposed models of the regulatory process share similarities with rational models. We suggest enhanced understanding of the decision-making process during self-paced activities is crucial in order to improve the ability to understand regulation of performance and performance outcomes during athletic activity.

Risk Perception Influences Athletic Pacing Strategy.

PURPOSE: To examine risk-taking and risk-perception associations with perceived exertion, pacing and performance in athletes. METHODS: Two experiments were conducted in which risk-perception was assessed using the domain-specific risk-taking (DOSPERT) scale in 20 novice cyclists (Experiment 1) and 32 experienced ultra-marathon runners (Experiment 2). In Experiment 1, participants predicted their pace and then performed a 5 km maximum effort cycling time-trial on a calibrated KingCycle mounted bicycle. Split-times and perceived exertion were recorded every kilometer. In experiment 2, each participant predicted their split times before running a 100 km ultra-marathon. Split-times and perceived exertion were recorded at 7 check-points. In both experiments, higher and lower risk-perception groups were created using median split of DOSPERT scores. RESULTS: In experiment 1, pace during the first km was faster among lower compared to higher risk-perceivers, t(18)=2.0 P=0.03, and faster among higher compared lower risk-takers, t(18)=2.2 P=0.02. Actual pace was slower than predicted pace during the first km in both the higher risk perceivers, t(9)=-4.2 P=0.001, and lower risk-perceivers, t(9)=-1.8 P=0.049. In experiment 2, pace during the first 36 km was faster among lower compared to higher risk-perceivers, t(16)=2.0 P=0.03. Irrespective of risk-perception group, actual pace was slower than predicted pace during the first 18 km, t(16)=8.9 P<0.001, and from 18 to 36 km, t(16)=4.0 P<0.001. In both experiments there was no difference in performance between higher and lower risk-perception groups. CONCLUSIONS: Initial pace is associated with an individual's perception of risk, with low perceptions of risk being associated with a faster starting pace. Large differences between predicted and actual pace suggests the performance template lacks accuracy, perhaps indicating greater reliance on momentary pacing decisions rather than pre-planned strategy.