719 research outputs found
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Revealing nascent proteomics in signaling pathways and cell differentiation.
Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in technologies that can quantitatively capture the immediate proteomic changes that allow cells to respond to specific stimuli. Here, we present a method to capture and identify nascent proteomes in situ across different cell types without disturbing normal growth conditions, using O-propargyl-puromycin (OPP). Cell-permeable OPP rapidly labels nascent elongating polypeptides, which are subsequently conjugated to biotin-azide, using click chemistry, and captured with streptavidin beads, followed by digestion and analysis, using liquid chromatography-tandem mass spectrometry. Our technique of OPP-mediated identification (OPP-ID) allows detection of widespread proteomic changes within a short 2-hour pulse of OPP. We illustrate our technique by recapitulating alterations of proteomic networks induced by a potent mammalian target of rapamycin inhibitor, MLN128. In addition, by employing OPP-ID, we identify more than 2,100 proteins and uncover distinct protein networks underlying early erythroid progenitor and differentiation states not amenable to alternative approaches such as amino acid analog labeling. We present OPP-ID as a method to quantitatively identify nascent proteomes across an array of biological contexts while preserving the subtleties directing signaling in the native cellular environment
Making the user more efficient: Design for sustainable behaviour
User behaviour is a significant determinant of a product’s environmental impact; while engineering advances permit increased efficiency of product operation, the user’s decisions and habits ultimately have a major effect on the energy or other resources used by the product. There is thus a need to change users’ behaviour. A range of design techniques developed in diverse contexts suggest opportunities for engineers, designers and other stakeholders working in the field of sustainable innovation to affect users’ behaviour at the point of interaction with the product or system, in effect ‘making the user more efficient’. Approaches to changing users’ behaviour from a number of fields are reviewed and discussed, including: strategic design of affordances and behaviour-shaping constraints to control or affect energyor other resource-using interactions; the use of different kinds of feedback and persuasive technology techniques to encourage or guide users to reduce their environmental impact; and context-based systems which use feedback to adjust their behaviour to run at optimum efficiency and reduce the opportunity for user-affected inefficiency. Example implementations in the sustainable engineering and ecodesign field are suggested and discussed
Impact of The Daily Mile on children's physical and mental health, and educational attainment in primary schools: iMprOVE cohort study protocol
INTRODUCTION: School-based active mile initiatives such as The Daily Mile (TDM) are widely promoted to address shortfalls in meeting physical activity recommendations. The iMprOVE Study aims to examine the impact of TDM on children's physical and mental health and educational attainment throughout primary school. METHODS AND ANALYSIS: iMprOVE is a longitudinal quasi-experimental cohort study. We will send a survey to all state-funded primary schools in Greater London to identify participation in TDM. The survey responses will be used for non-random allocation to either the intervention group (Daily Mile schools) or to the control group (non-Daily Mile schools). We aim to recruit 3533 year 1 children (aged 5-6 years) from 77 primary schools and follow them up annually until the end of their primary school years. Data collection taking place at baseline (children in school year 1) and each primary school year thereafter includes device-based measures of moderate-to-vigorous physical activity (MVPA) and questionnaires to measure mental health (Strengths and Difficulties Questionnaire) and educational attainment (ratings from 'below expected' to 'above expected levels'). The primary outcome is the mean change in MVPA minutes from baseline to year 6 during the school day among the intervention group compared with controls. We will use multilevel linear regression models adjusting for sociodemographic data and participation in TDM. The study is powered to detect a 10% (5.5 min) difference between the intervention and control group which would be considered clinically significant. ETHICS AND DISSEMINATION: Ethics has been approved from Imperial College Research Ethics Committee, reference 20IC6127. Key findings will be disseminated to the public through research networks, social, print and media broadcasts, community engagement opportunities and schools. We will work with policy-makers for direct application and impact of our findings
Elucidating drivers of oral epithelial dysplasia formation and malignant transformation to cancer using RNAseq
Oral squamous cell carcinoma (OSCC) is a prevalent cancer with poor prognosis. Most OSCC progresses via a non-malignant stage called dysplasia. Effective treatment of dysplasia prior to potential malignant transformation is an unmet clinical need. To identify markers of early disease, we performed RNA sequencing of 19 matched HPV negative patient trios: normal oral mucosa, dysplasia and associated OSCC. We performed differential gene expression, principal component and correlated gene network analysis using these data. We found differences in the immune cell signatures present at different disease stages and were able to distinguish early events in pathogenesis, such as upregulation of many HOX genes, from later events, such as down-regulation of adherens junctions. We herein highlight novel coding and non-coding candidates for involvement in oral dysplasia development and malignant transformation, and speculate on how our findings may guide further translational research into the treatment of oral dysplasia
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Reframing physically active learning as movement-centred pedagogy: a European priority action framework
YesPhysically active learning (PAL) has emerged as a promising way of eliciting health and education-based outcomes for pupils. Concurrently, research suggests large variability in how PAL is perceived, operationalized, and prioritized in practice across Europe. Therefore, this study aimed to co-develop a framework for action to support the adoption and implementation of PAL.
Adopting a design thinking approach, 40 international stakeholders representing 13 countries engaged in an idea generation workshop during a two-day PAL international conference. Participants included professionals from research (n = 20), practice (n = 4) and policy (n = 1) or a combination (n = 15). Their experience with PAL ranged from none to 19 years (with an average of 3.9 years). Participants were allocated into one of six heterogeneous and multidisciplinary groups and led through interactive tasks to identify: the landscape for PAL across Europe, barriers to the adoption and implementation of PAL, and key objectives for research, policy and practice to improve the adoption and implementation of PAL. All discussions were audio recorded and prioritized objectives were transcribed verbatim and analysed using inductive qualitative content analysis.
Five interlinked and mutually reinforcing themes were identified: (1) Integration of the health and education paradigms (2) Coherent national policy and decision making (3) Building confident and competent teachers (4) Adopting a whole school approach for PAL (5) Strengthening the evidence base for PAL.
The priority action framework identifies five key areas for action to facilitate PAL adoption and implementation across Europe. Central to the success of border uptake of PAL is the integration of the health and education paradigms. To achieve this aim, reframing PAL as movement-centered pedagogy would provide a more holistic and inclusive perspective.The authors of this manuscript were supported and funded by the European Union ERASMUS + Strategic Partnership Fund as part of the Activating Classroom Teachers project, ACTivate (Grant no 2019-1-N001-KA203-063024)
Electron Capture Dissociation Mass Spectrometry of Tyrosine Nitrated Peptides
In vivo protein nitration is associated with many disease conditions that involve oxidative stress and inflammatory response. The modification involves addition of a nitro group at the position ortho to the phenol group of tyrosine to give 3-nitrotyrosine. To understand the mechanisms and consequences of protein nitration, it is necessary to develop methods for identification of nitrotyrosine-containing proteins and localization of the sites of modification.Here, we have investigated the electron capture dissociation (ECD) and collision-induced association (CID) behavior of 3-nitrotyrosine-containing peptides. The presence of nitration did not affect the CID behavior of the peptides. For the doubly-charged peptides, addition of nitration severely inhibited the production of ECD sequence fragments. However, ECD of the triply-charged nitrated peptides resulted in some singly-charged sequence fragments. ECD of the nitrated peptides is characterized by multiple losses of small neutral species including hydroxyl radicals, water and ammonia. The origin of the neutral losses has been investigated by use of activated ion (AI) ECD. Loss of ammonia appears to be the result of non-covalent interactions between the nitro group and protonated lysine side-chains
The Effect of Using an Inappropriate Protein Database for Proteomic Data Analysis
A recent study by Bromenshenk et al., published in PLoS One (2010), used proteomic analysis to identify peptides purportedly of Iridovirus and Nosema origin; however the validity of this finding is controversial. We show here through re-analysis of a subset of this data that many of the spectra identified by Bromenshenk et al. as deriving from Iridovirus and Nosema proteins are actually products from Apis mellifera honey bee proteins. We find no reliable evidence that proteins from Iridovirus and Nosema are present in the samples that were re-analyzed. This article is also intended as a learning exercise for illustrating some of the potential pitfalls of analysis of mass spectrometry proteomic data and to encourage authors to observe MS/MS data reporting guidelines that would facilitate recognition of analysis problems during the review process
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