Interactions of Viral and Cellular Helicases

The innate immune system is a part of the first line of defense against virus infection. An important subset of the innate immune system consists of a group of intracellular pattern recognition receptors (PRRs) which recognize conserved features of bacteria and viruses and initiate an interferon response. The RIG-I like receptors (RLRs) are PRRs that bind to RNA viruses (such as hepatitis c virus) and signal through the adaptor mitochondrial anti-viral signaling protein (MAVS). Hepatitis C virus (HCV) is a small enveloped RNA virus that belongs to the flaviviridae family of viruses. HCV infects hepatocytes and can cause a persistent infection. If a chronic infection is established, progressive liver damage along with cirrhosis and sometimes hepatocellular carcinoma may occur. The multi-functional HCV non-structural-3 (NS3) protein is essential for HCV replication and contains covalently linked protease and helicase/ATPase domains. A covalently linked protease and helicase is unique to the flaviviridae family of viruses and it is unclear why the two functions are linked. There are multiple effective direct acting anti-virals which target the protease, but none currently approved which inhibit the helicase. In addition to aiding in viral replication, the NS3 protease assists HCV in establishing a persistent infection through cleaving the innate immune RIG-I adaptor protein, MAVS. The purpose of the studies contained in this thesis is to gain a greater understanding of the function and purpose of the covalently linked HCV NS3 protease and helicase. Förster resonance energy transfer (FRET) is used to explore the interaction of NS3 with RIG-I like receptor proteins and to use FRET to look at the interaction of the RLRs with themselves. The interaction of the NS3 protease and helicase domain was probed through the exploration of the mechanism of action of a NS3 inhibitor (HPI) which is able to inhibit both the protease and helicase functions of NS3, while not disrupting the ATPase activity. The activity of HPI was determined in vitro using a fluorescent protease cleavage assay and a fluorescent helicase unwinding assay. HPI can inhibit both functions with low micro-molar EC50. Next, analysis of HPI to inhibit peptide hydrolysis by wild-type NS3 and a set of NS3 mutants with mutations in the protease domain, helicase domain, and the allosteric groove between the protease and helicase domain suggested that HPI forms a bridge between the NS3 helicase RNA-binding site and the allosteric groove between the protease and helicase domains. The activity of HPI was measured in cells using an HCV sub-genomic replicon tagged with a luciferase reporter. The inhibition of HPI alone and in the presence of other protease inhibitors was tested. HPI can inhibit the HCV genotype 1b sub-genomic replicon and when applied in conjunction with first generation protease inhibitors, telaprevir and boceprevir, the inhibition was additive, as defined by the Bliss Independence Model of additive inhibition. However, when HPI was used in conjunction with macro-cyclic protease inhibitors, danoprevir and grazoprevir, modest synergy was observed. To look at the protein:protein interactions of the NS3 helicase and the RIG-I like receptor helicases in live cells, a series of quantitative FRET spectrometry studies were employed. Quantitative micro-spectroscopic imaging (Q-MSI) is a technique which uses a fluorescent dye or fluorescent protein to identify sub-cellular regions and then calculates Förster Resonance Energy Transfer (FRET) efficiency and the concentrations of the donor and acceptor proteins. The technique was first applied in vitro with a fluorescently tagged NS3 helicase and fluorescently tagged DNA molecules. Next, the technique was applied to combinations of recombinant fluorescently tagged helicases expressed in HEK293T cells. The NS3 helicase, RIG-I like receptor helicases, DDX1, DDX3, and DDX5 helicases, and MAVS were all designed to express off plasmids which also encode and attach a fluorescent protein. The fluorescent proteins used were either cyan fluorescent protein (CFP), enhanced green fluorescent protein-2 (GFP2), yellow fluorescent protein (YFP) or Venus fluorescent protein and each combination included a donor (CFP or GFP2) and an acceptor (YFP or Venus) fluorescent protein. The combinations were tested in presence or absence of polyinosinic-polyctyidlic acid (poly I:C) which is a synthetic RNA analog capable of eliciting an RLR response. To localize the interaction to the mitochondria, the mitochondrial stain, Mito-Tracker-Red, was used in some experiments. The experiments revealed a previously unknown interaction between NS3 and the RLR protein, laboratory of genetics and physiology protein-2 (LGP2) which may be biologically relevant. In addition, the relocation of LGP2 cytoplasmic foci in cells over-expressing DDX3 was observed. Q-MSI was used to visualize previously known interactions of RLRs at the mitochondria and in conjunction with MAVS

Music Performance Anxiety in Adolescent Student Singers

This project seeks to sidestep the debilitating effects of music performance anxiety by cross-referencing knowledge from the areas of adolescent psychology with literature on MPA in singers in general in order to target adolescent singers early in their training. As well as considering the causes, symptoms and treatment of music performance anxiety, the project examines the role of the natural anxieties of adolescence in triggering music performance anxiety and seeks to chart a way through. Its intended readership is the classical singing teacher

Evaluation of a Workplace Treadmill Desk Intervention: A Randomized Controlled Trial

OBJECTIVE: To evaluate the effectiveness of a 3-month treadmill desk intervention in eliciting changes in physical activity and sedentary behavior among overweight/obese office workers. METHODS: A randomized controlled trial was conducted among overweight/obese office workers (n=41; mean age = 40.1 ± 10.1 years) at a private workplace. Participants were randomly assigned to a shared-treadmill desk intervention (n=21) or a usual working condition control group (n=20). Accelerometer-determined physical activity and sedentary behavior were measured before and after the intervention. RESULTS: Compared with the control group, the intervention group increased daily steps (1622 steps/day; p=0.013) and light physical activity (1.6 minutes/hour; p=0.008), and decreased sedentary time (−3.6 minutes/hour; p=0.047) during working hours. CONCLUSIONS: Shared-treadmill desks in the workplace can be effective at promoting favorable changes in light physical activity (specifically 40-99 steps/minute) and sedentary behavior among overweight/obese office workers.This is a non-final version of an article. The article is copyrighted by the American College of Occupational and Environmental Medicine and published by Lippincott Williams & Wilkins. It can be found as published in final form at: http://journals.lww.com/joem/pages/default.asp

Calorie restriction improves lipid-related emerging cardiometabolic risk factors in healthy adults without obesity: Distinct influences of BMI and sex from CALERIE™ a multicentre, phase 2, randomised controlled trial

Background: For many cardiovascular risk factors there is no lower limit to which further reduction will result in decreased disease risk; this includes values within ranges considered normal for healthy adults. This seems to be true for new emerging metabolic risk factors identified by innovative technological advances. Further, there seems to be ever evolving evidence of differential responses to lifestyle interventions by sex and body compositions in the normal range. In this secondary analysis, we had the opportunity to test these principles for newly identified molecular biomarkers of cardiometabolic risk in a young (21–50 years), normal weight healthy population undergoing calorie restriction for two years. Methods: The Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE™) was a 24-month, multicenter, randomized controlled trial (May 2007-November 2012) in healthy, adults without obesity to evaluate the potential for calorie restriction (CR) to promote anti-aging adaptations, including those associated with disease risk. 218 participants (age 37.9 ± 7.2 years and body mass index (BMI) 25.1 ± 1.7 kg/m2, mean±SD) were randomized 2:1 to 24 months of CR (prescribed as 25% reduction from baseline calorie intake) versus ad libitum (AL). Fasting plasma from baseline, 12, and 24 months was used for assessments of lipoproteins, metabolites, and inflammatory markers using nuclear magnetic resonance spectroscopy. Findings: Averaging 11.9% CR, the CR group had reductions at 12 and 24 months in the cardiovascular disease risk markers, apolipoprotein B and GlycA, and risks for insulin resistance and type 2 diabetes—Lipoprotein Insulin Resistance Index and Diabetes Risk Index (all PCRvsAL≤0.0009). Insulin resistance and diabetes risk improvements resulted from CR-induced alterations in lipoproteins, specifically reductions in triglyceride-rich lipoprotein particles and low-density lipoprotein particles, a shift to larger high-density lipoprotein particles (more effective cholesterol transporters), and reductions in branched chain amino acids (BCAAs) (all PCRvsAL≤0.004). These CR responses were more pronounced in overweight than normal weight participants and greater in men than women. Interpretation: In normal to slightly overweight adults without overt risk factors or disease, 12 months of ∼12% CR improved newly identified risk markers for atherosclerotic cardiovascular disease, insulin resistance and type 2 diabetes. These markers suggest that CR improves risks by reducing inflammation and BCAAs and shifting lipoproteins from atherogenic to cholesterol transporting. Additionally, these improvements are greater for men and for those with greater BMIs indicating sex and BMI-influences merit attention in future investigations of lifestyle-mediated improvements in disease risk factors

Protocol to evaluate the effectiveness and cost-effectiveness of an environmental nutrition and physical activity intervention in nurseries (Nutrition and Physical Activity Self Assessment for Child Care - NAP SACC UK): a multicentre cluster randomised controlled trial

Background: One in seven UK children have obesity when starting school, with higher prevalence associated with deprivation. Most pre-school children do not meet UK recommendations for physical activity and nutrition. Formal childcare settings provide opportunities to deliver interventions to improve nutritional quality and physical activity to the majority of 3–4-year-olds. The nutrition and physical activity self-assessment for childcare (NAP SACC) intervention has demonstrated effectiveness in the USA with high acceptability in the UK. The study aims to evaluate the effectiveness and cost-effectiveness of the NAP SACC UK intervention to increase physical activity, reduce sedentary time and improve nutritional intake. Methods: Multi-centre cluster RCT with process and economic evaluation. Participants are children aged 2 years or over, attending UK early years settings (nurseries) for ≥ 12 h/week or ≥ 15 h/week during term time and their parents, and staff at participating nurseries. The 12-month intervention involves nursery managers working with a Partner (public health practitioner) to self-assess policies and practices relating to physical activity and nutrition; nursery staff attending one physical activity and one nutrition training workshop and setting goals to be achieved within 6 months. The Partner provides support and reviews progress. Nursery staff receive a further workshop and new goals are set, with Partner support for a further 6 months. The comparator is usual practice. Up to 56 nurseries will be stratified by area and randomly allocated to intervention or comparator arm with minimisation of differences in level of deprivation. Primary outcomes: accelerometer-assessed mean total activity time on nursery days and average total energy (kcal) intake per eating occasion of lunch and morning/afternoon snacks consumed within nurseries. Secondary outcomes: accelerometer-assessed mean daily minutes of moderate-to-vigorous physical activity and sedentary time per nursery day, total physical activity on nursery days compared to non-nursery days, average serving size of lunch and morning/afternoon snacks in nursery per day, average percentage of core and non-core food in lunch and morning/afternoon snacks, zBMI, proportion of children who are overweight/obese and child quality-of-life. A process evaluation will examine fidelity, acceptability, sustainability and context. An economic evaluation will compare costs and consequences from the perspective of the local government, nursery and parents. Trial registration: ISRCTN33134697, 31/10/2019

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Music performance anxiety in adolescent student singers : a thesis submitted to the Victoria University of Wellington in fulfilment of the requirements for the degree of Master of Music [in Performance]

This project seeks to sidestep the debilitating effects of music performance anxiety by cross-referencing knowledge from the areas of adolescent psychology with literature on MPA in singers in general in order to target adolescent singers early in their training. As well as considering the causes, symptoms and treatment of music performance anxiety, the project examines the role of the natural anxieties of adolescence in triggering music performance anxiety and seeks to chart a way through. Its intended readership is the classical singing teacher

Music Performance Anxiety in Adolescent Student Singers

This project seeks to sidestep the debilitating effects of music performance anxiety by cross-referencing knowledge from the areas of adolescent psychology with literature on MPA in singers in general in order to target adolescent singers early in their training. As well as considering the causes, symptoms and treatment of music performance anxiety, the project examines the role of the natural anxieties of adolescence in triggering music performance anxiety and seeks to chart a way through. Its intended readership is the classical singing teacher.</p

Octopamine mobilizes lipids from honey bee (Apis mellifera) hypopharyngeal glands

Recent widespread honey bee (Apis mellifera) colony loss is attributed to a variety of stressors, including parasites, pathogens, pesticides and poor nutrition. In principle, we can reduce stress-induced declines in colony health by either removing the stressor or increasing the bees' tolerance to the stressor. This latter option requires a better understanding than we currently have of how honey bees respond to stress. Here, we investigated how octopamine, a stress-induced hormone that mediates invertebrate physiology and behavior, influences the health of young nurse-aged bees. Specifically, we asked whether octopamine induces abdominal lipid and hypopharyngeal gland (HG) degradation, two physiological traits of stressed nurse bees. Nurse-aged workers were treated topically with octopamine and their abdominal lipid content, HG size and HG autophagic gene expression were measured. Hemolymph lipid titer was measured to determine whether tissue degradation was associated with the release of nutrients from these tissues into the hemolymph. The HGs of octopamine-treated bees were smaller than control bees and had higher levels of HG autophagy gene expression. Octopamine-treated bees also had higher levels of hemolymph lipid compared with control bees. Abdominal lipids did not change in response to octopamine. Our findings support the hypothesis that the HGs are a rich source of stored energy that can be mobilized during periods of stress.12 month embargo; published 16 April 2020This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Effect of calorie restriction on the free-living physical activity levels of nonobese humans: results of three randomized trials

The objective of this study was to evaluate the influence of calorie restriction (CR) on free-living physical activity levels among humans. Data were from three CALERIE phase I site-specific protocols. Participants were nonobese (body mass index = 23.5–29.9 kg/m2) adults randomly assigned to 25% CR, low-calorie diet (LCD, 890 kcal/day supplement diet until 15% weight loss, then weight maintenance), or control at Pennington Biomedical Research Center (PBRC); 30% or 10% CR at Tufts University; and 20% CR or control at Washington University School of Medicine (WUSM). Activity was measured at months 0, 3, and 6 (PBRC) and at months 0, 3, 6, 9, and 12 (WUSM and Tufts). Total daily energy expenditure (TEE) by doubly labeled water and resting metabolic rate (RMR) were used to compute activity energy expenditure: AEE = TEE − RMR − 0.1 * TEE. Accelerometry and 7-day recall categorized activities by intensity. At Tufts, the 10% and 30% CR groups experienced significant decreases in AEE at months 6, 9, and 12. At month 6, a larger decrease in AEE was observed in the CR than the control group at WUSM. At months 3 and 6, larger decreases in AEE were observed in the CR and LCD groups than the control group at PBRC. Accelerometry and 7-day PAR did not consistently detect changes in activity categories. CR-associated changes in AEE were variable but, generally, reduced the energy deficit, which would reduce the expected rate of weight loss. Accelerometry and recall did not consistently explain reduced AEE, suggesting that increased muscle efficiency and/or decreased fidgeting accounted for decreased AEE. Inaccuracy of accelerometry and recall also likely negatively affected sensitivity