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BACKGROUND: Policymakers and researchers recommend supporting the capabilities of feedback recipients to increase the quality of care. There are different ways to support capabilities. We aimed to describe the content and delivery of feedback facilitation interventions delivered alongside audit and feedback within randomised controlled trials. METHODS: We included papers describing feedback facilitation identified by the latest Cochrane review of audit and feedback. The piloted extraction proforma was based upon a framework to describe intervention content, with additional prompts relating to the identification of influences, selection of improvement actions and consideration of priorities and implications. We describe the content and delivery graphically, statistically and narratively. RESULTS: We reviewed 146 papers describing 104 feedback facilitation interventions. Across included studies, feedback facilitation contained 26 different implementation strategies. There was a median of three implementation strategies per intervention and evidence that the number of strategies per intervention is increasing. Theory was used in 35 trials, although the precise role of theory was poorly described. Ten studies provided a logic model and six of these described their mechanisms of action. Both the exploration of influences and the selection of improvement actions were described in 46 of the feedback facilitation interventions; we describe who undertook this tailoring work. Exploring dose, there was large variation in duration (15-1800 min), frequency (1 to 42 times) and number of recipients per site (1 to 135). There were important gaps in reporting, but some evidence that reporting is improving over time. CONCLUSIONS: Heterogeneity in the design of feedback facilitation needs to be considered when assessing the intervention's effectiveness. We describe explicit feedback facilitation choices for future intervention developers based upon choices made to date. We found the Expert Recommendations for Implementing Change to be valuable when describing intervention components, with the potential for some minor clarifications in terms and for greater specificity by intervention providers. Reporting demonstrated extensive gaps which hinder both replication and learning. Feedback facilitation providers are recommended to close reporting gaps that hinder replication. Future work should seek to address the 'opportunity' for improvement activity, defined as factors that lie outside the individual that make care or improvement behaviour possible. REVIEW REGISTRATION: The study protocol was published at: https://www.protocols.io/private/4DA5DE33B68E11ED9EF70A58A9FEAC02 .
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Retroalimentación , Humanos , Ensayos Clínicos Controlados Aleatorios como Asunto , Mejoramiento de la Calidad/organización & administración , Retroalimentación Formativa , Ciencia de la ImplementaciónRESUMEN
BACKGROUND: Adaptation seeks to increase intervention fit with context, an important influence upon implementation. People with dementia in acute hospitals in Ireland do not routinely receive best care. To improve care in Ireland, we sought to adapt an existing quality improvement collaborative, to support the improvement capabilities of recipients of feedback from the Irish National Audit of Dementia. METHODS: The study followed a staged process to co-design adaptations to reflect contextual differences between the English and the Irish healthcare systems, and to explore feasibility of the adapted Quality Improvement Collaborative in Ireland. We used co-design group meetings involving dementia clinicians from three hospitals, delivered the intervention virtually and interviewed healthcare workers from seven hospitals to adapt and explore the fidelity, affective response and reported appropriateness of the intervention. RESULTS: The intervention required adaptation to reflect differences in strategic intention, ways of working and hospital social structures. There was evidence that the adapted intervention generated a positive affective response, was perceived as appropriate and led to fidelity of receipt and response. CONCLUSION: We describe implications for the adaptation of interventions to increase participants' quality improvement capabilities and highlight the importance of socio-adaptive work. We propose further work to explore antecedents of senior positional leader engagement, to describe the delivery of intra-participant behaviour change techniques and to adapt the intervention to other clinical domains.
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Demencia , Mejoramiento de la Calidad , Humanos , Estudios de Factibilidad , Irlanda , Personal de Salud , Demencia/terapiaRESUMEN
BACKGROUND: People with type 1 diabetes and raised glucose levels are at greater risk of retinopathy, nephropathy, neuropathy, cardiovascular disease, sexual health problems and foot disease. The UK National Institute for Health and Care Excellence (NICE) recommends continuous subcutaneous 'insulin pump' therapy for people with type 1 diabetes whose HbA1c is above 69 mmol/mol. Insulin pump use can improve quality of life, cut cardiovascular risk and increase treatment satisfaction. About 90,000 people in England and Wales meet NICE criteria for insulin pumps but do not use one. Insulin pump use also varies markedly by deprivation, ethnicity, sex and location. Increasing insulin pump use is a key improvement priority. Audit and feedback is a common but variably effective intervention. Limited capabilities of healthcare providers to mount effective responses to feedback from national audits, such as the National Diabetes Audit (NDA), undermines efforts to improve care. We have co-developed a theoretically and empirically informed quality improvement collaborative (QIC) to strengthen local responses to feedback with patients and carers, national audits and healthcare providers. We will evaluate whether the QIC improves the uptake of insulin pumps following NDA feedback. METHODS: We will undertake an efficient cluster randomised trial using routine data. The QIC will be delivered alongside the NDA to specialist diabetes teams in England and Wales. Our primary outcome will be the proportion of people with type 1 diabetes and an HbA1c above 69 mmol/mol who start and continue insulin pump use during the 18-month intervention period. Secondary outcomes will assess change in glucose control and duration of pump use. Subgroup analyses will explore impacts upon inequalities by ethnicity, sex, age and deprivation. A theory-informed process evaluation will explore diabetes specialist teams' engagement, implementation, fidelity and tailoring through observations, interviews, surveys and documentary analysis. An economic evaluation will micro-cost the QIC, estimate cost-effectiveness of NDA feedback with QIC and estimate the budget impact of NHS-wide QIC roll out. DISCUSSION: Our study responds to a need for more head-to-head trials of different ways of reinforcing feedback delivery. Our findings will have implications for other large-scale audit and feedback programmes. TRIAL REGISTRATION: ISRCTN82176651 Registered 18 October 2022.
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Diabetes Mellitus Tipo 1 , Insulinas , Humanos , Análisis Costo-Beneficio , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Hemoglobina Glucada , Mejoramiento de la Calidad , Calidad de Vida , Ensayos Clínicos Controlados Aleatorios como Asunto , Masculino , FemeninoRESUMEN
BACKGROUND: While simple Audit & Feedback (A&F) has shown modest effectiveness in reducing low-value care, there is a knowledge gap on the effectiveness of multifaceted interventions to support de-implementation efforts. Given the need to make rapid decisions in a context of multiple diagnostic and therapeutic options, trauma is a high-risk setting for low-value care. Furthermore, trauma systems are a favorable setting for de-implementation interventions as they have quality improvement teams with medical leadership, routinely collected clinical data, and performance-linked to accreditation. We aim to evaluate the effectiveness of a multifaceted intervention for reducing low-value clinical practices in acute adult trauma care. METHODS: We will conduct a pragmatic cluster randomized controlled trial (cRCT) embedded in a Canadian provincial quality assurance program. Level I-III trauma centers (n = 30) will be randomized (1:1) to receive simple A&F (control) or a multifaceted intervention (intervention). The intervention, developed using extensive background work and UK Medical Research Council guidelines, includes an A&F report, educational meetings, and facilitation visits. The primary outcome will be the use of low-value initial diagnostic imaging, assessed at the patient level using routinely collected trauma registry data. Secondary outcomes will be low-value specialist consultation, low-value repeat imaging after a patient transfer, unintended consequences, determinants for successful implementation, and incremental cost-effectiveness ratios. DISCUSSION: On completion of the cRCT, if the intervention is effective and cost-effective, the multifaceted intervention will be integrated into trauma systems across Canada. Medium and long-term benefits may include a reduction in adverse events for patients and an increase in resource availability. The proposed intervention targets a problem identified by stakeholders, is based on extensive background work, was developed using a partnership approach, is low-cost, and is linked to accreditation. There will be no attrition, identification, or recruitment bias as the intervention is mandatory in line with trauma center designation requirements, and all outcomes will be assessed with routinely collected data. However, investigators cannot be blinded to group allocation and there is a possibility of contamination bias that will be minimized by conducting intervention refinement only with participants in the intervention arm. TRIAL REGISTRATION: This protocol has been registered on ClinicalTrials.gov (February 24, 2023, # NCT05744154 ).
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Cuidados Críticos , Atención de Bajo Valor , Humanos , Adulto , Canadá , Cuidados Críticos/métodos , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
ABSTRACT: Clinical audit is commonly used to assess and improve the quality of care. The impact of clinical audit on practice could be improved by supporting the ability of people who receive feedback from audits to mount effective responses. This article, written for people who receive feedback, audit providers and quality improvement leads, describes important practices in the organisational response to feedback. The practices were identified through work to describe the current response to national audit and draw upon theory-informed hypotheses for enhancing audit and feedback. Both the content and implementation of the practices were co-designed with stakeholders and tested alongside different national audits. The identified quality improvement practices provide practical guidance for feedback recipients and enable providers and quality improvement leads to consider the capabilities required for such practices. The approach resonates with organisational readiness to change theory, proposing that informational appraisal and change commitment underpin effective improvement actions.
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Auditoría Médica , Mejoramiento de la Calidad , Humanos , Retroalimentación , Auditoría ClínicaRESUMEN
BACKGROUND: National audits are a common, but variably effective, intervention to improve services. This study aimed to design an intervention to increase the effectiveness of national audit. METHODS: We used interviews, documentary analysis, observations, co-design and stakeholder engagement methods. The intervention was described in an intervention manual and illustrated using a logic model. Phase 1 described the current hospital response to a national audit. Phase 2 identified potential enhancements. Phase 3 developed a strategy to implement the enhancements. Phase 4 explored the feasibility of the intervention alongside the National Audit of Dementia and refined the intervention. Phase 5 adapted the intervention to a second national audit (National Diabetes Audit). Phase 6 explored the feasibility and fidelity of the intervention alongside the National Diabetes Audit and used the findings to further refine the intervention. RESULTS: The developed intervention is a quality improvement collaborative (QIC), containing virtual educational workshop, virtual outreach for local team leads and virtual facilitation of a learning collaborative delivered after feedback has been received. The QIC aims to support national audit recipients to undertake improvement actions tailored to their local context. The target audience is clinical and clinical governance leaders. We found that actions from national audit were constrained by what the clinical lead perceived they deliver personally, these actions were not aligned to identified influences upon performance. We found that the hospital response could be enhanced by targeting low baseline performance, identifying and addressing influences upon to performance, developing trust and credibility, addressing recipient priorities, presenting meaningful comparisons, developing a conceptual model, involving stakeholders and considering the opportunity cost. Phase 3 found that an educational workshop and outreach strategy could support implementation of the enhancements through developing coherence and cognitive participation. We found feasibility could be increased by revising the content, re-naming the intervention, amending activities to address time commitment, incorporating a more structured analysis of influences, supporting collaboration and developing local feedback mechanisms. Phase 5 found adaptation to a second national audit involved reflecting differences in the clinical topic, context and contractual requirements. We found that the behaviour change techniques identified in the manual were delivered by facilitators. Participants reported positive attitudes towards the intervention and that the intervention was appropriate. CONCLUSIONS: The QIC supports local teams to tailor their actions to local context and develop change commitment. Future work will evaluate the effectiveness of the intervention as an adjunct to the National Diabetes Audit.
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BACKGROUND: Dissemination is an under-researched activity that is important to researchers and funders and may have a role in creating conditions for implementation. We aim to study the impact of two animations shared via social media upon dissemination. METHODS: We developed two short animations to increase the awareness of healthcare positional leaders of work undertaken to enhance a much-used implementation intervention. We measured both how frequently the related articles were accessed, and engagement with the research team, before and after the intervention. We analysed variation over time using statistical process control to identify both trend and periods of significant change. RESULTS: We found evidence that the animation increased how frequently the articles were accessed, with a significant increase (p = <0.01) during the two weeks after release of each animation. One animation was associated with an increase in positional leaders' engagement with the research team. CONCLUSIONS: Animations shared via social media can enhance dissemination. We describe lessons from the work to develop the intervention and support calls for work to increase the understanding and adoption of effective dissemination interventions. Our findings provide support for further work using randomised study designs.
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Ciencia de la Implementación , Medios de Comunicación Sociales , Atención a la Salud , HumanosRESUMEN
Co-methodological working is gaining increasing traction in healthcare, but studies with older people have been slower to develop. Our aim was to investigate how and how well older people have been engaged in healthcare intervention design, development or delivery using co-methodologies. We conducted a systematic search of four electronic databases to identify international literature published between 2009 and November 2019. We included peer-reviewed empirical research of any design. Three authors screened papers. Our review is reported in accordance with the Joanna Briggs Institute manual for scoping reviews, we have referred to the preferred reporting items for systematic reviews and meta-analyses statement. We data extracted to a bespoke spreadsheet and used the Co:Create Co-production Matrix to guide quality appraisal. Included studies (n = 48) were diverse in nature of interventions, co-methodologies and reporting. We offer a narrative summary of included papers. Establishing how older people were engaged in co-methodological work was largely straightforward. How well this was done was more challenging, however we have identified gems of good practice and offered directions for future practice. The Co:Create Co-Production Matrix was the best fit for evaluating papers, however it is not intended as a measure per se. In essence we argue that notions of 'best' and 'scores' are an oxymoron in co-methodological working, what is important that: (a) researchers embrace these methods, (b) incremental change is the way forward, (c) researchers need to do what is right for people and purpose and (d) have time to consider and articulate why they are choosing this approach and how best this can be achieved for their particular situation. Future evaluation of participant's experience of the process would enable others to learn about what works for who and in what circumstances.
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Atención a la Salud , Narración , Anciano , Instituciones de Salud , HumanosRESUMEN
BACKGROUND: Hospitals in many countries are encouraged to develop audits to assess and improve the quality of care. Ward audit is a specific form of audit and feedback that is commonly used but little studied. The aim of this study is to describe the content and application of hospital ward audit in order to identify potential enhancements to such audits. METHODS: Multiple qualitative methods were used to study a diversity sample of four English National Health Service organisations over a 16-month period. We undertook semi-structured interviews (n = 32), documentary analysis (n = 44) and 25 h of observations of healthcare workers involved in the design and implementation of ward audit. Data were analysed using framework analysis. Findings were presented iteratively to stakeholders who used them to develop a description of the content and delivery of ward audit. RESULTS: Ward audit consisted of seven stages: impetus; method; preparation of staff; assessing practice; analysis; feedback; and decide on action to improve. Two key stages were the monthly assessment of practice using case note data extraction, and the resulting feedback to clinical staff, ward managers, matrons and directors of nursing. At three organisations, the case note data were extracted by staff and there was evidence that this resulted in misrepresentation of the clinical performance audited. The misrepresentation appeared to be associated with the anticipation of punitive feedback from directors of nursing and matrons, as well as time pressures and a lack clarity about the method of audit data collection. Punitive feedback was reported to occur if no data were collected, if data demonstrated poor performance or if performance did not improve. CONCLUSIONS: Organisations invest considerable clinical resources in ward audit, but such audits may have unintended, potentially negative, consequences due to the impacts from punitive feedback. We discuss potential enhancements to ward audit (e.g. providing feedback recipients with suggested actions for improvement) and discuss implications for theory. There is a need to reduce the use of punitive feedback.
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Hospitales , Medicina Estatal , Auditoría Clínica , Retroalimentación , Humanos , Auditoría Médica , Investigación CualitativaRESUMEN
BACKGROUND: National audit is a key strategy used to improve care for patients with dementia. Audit and feedback has been shown to be effective, but with variation in how much it improves care. Both evidence and theory identify active ingredients associated with effectiveness of audit and feedback. It is unclear to what extent national audit is consistent with evidence- and theory-based audit and feedback best practice. METHODS: We explored how the national audit of dementia is undertaken in order to identify opportunities to enhance its impact upon the improvement of care for people with dementia. We undertook a multi-method qualitative exploration of the national audit of dementia at six hospitals within four diverse English National Health Service organisations. Inductive framework analysis of 32 semi-structured interviews, documentary analysis (n = 39) and 44 h of observations (n = 36) was undertaken. Findings were presented iteratively to a stakeholder group until a stable description of the audit and feedback process was produced. RESULTS: Each organisation invested considerable resources in the audit. The audit results were dependent upon the interpretation by case note reviewers who extracted the data. The national report was read by a small number of people in each organisation, who translated it into an internal report and action plan. The internal report was presented at specialty- and organisation-level committees. The internal report did not include information that was important to how committee members collectively decided whether and how to improve performance. Participants reported that the national audit findings may not reach clinicians who were not part of the specialty or organisation-level committees. CONCLUSIONS: There is considerable organisational commitment to the national audit of dementia. We describe potential evidence- and theory-informed enhancements to the enactment of the audit to improve the local response to performance feedback in the national audit. The enhancements relate to the content and delivery of the feedback from the national audit provider, support for the clinicians leading the organisational response to the feedback, and the feedback provided within the organisation.
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Demencia/terapia , Auditoría Médica/organización & administración , Mejoramiento de la Calidad/organización & administración , Medicina Estatal/organización & administración , Actitud del Personal de Salud , Inglaterra , Humanos , Auditoría Médica/normas , Motivación , Investigación Cualitativa , Medicina Estatal/normasRESUMEN
A disintegrin and metalloprotease 10 (ADAM10) is a transmembrane protein essential for embryonic development, and its dysregulation underlies disorders such as cancer, Alzheimer's disease, and inflammation. ADAM10 is a "molecular scissor" that proteolytically cleaves the extracellular region from >100 substrates, including Notch, amyloid precursor protein, cadherins, growth factors, and chemokines. ADAM10 has been recently proposed to function as six distinct scissors with different substrates, depending on its association with one of six regulatory tetraspanins, termed TspanC8s. However, it remains unclear to what degree ADAM10 function critically depends on a TspanC8 partner, and a lack of monoclonal antibodies specific for most TspanC8s has hindered investigation of this question. To address this knowledge gap, here we designed an immunogen to generate the first monoclonal antibodies targeting Tspan15, a model TspanC8. The immunogen was created in an ADAM10-knockout mouse cell line stably overexpressing human Tspan15, because we hypothesized that expression in this cell line would expose epitopes that are normally blocked by ADAM10. Following immunization of mice, this immunogen strategy generated four Tspan15 antibodies. Using these antibodies, we show that endogenous Tspan15 and ADAM10 co-localize on the cell surface, that ADAM10 is the principal Tspan15-interacting protein, that endogenous Tspan15 expression requires ADAM10 in cell lines and primary cells, and that a synthetic ADAM10/Tspan15 fusion protein is a functional scissor. Furthermore, two of the four antibodies impaired ADAM10/Tspan15 activity. These findings suggest that Tspan15 directly interacts with ADAM10 in a functional scissor complex.
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Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Tetraspaninas/metabolismo , Células A549 , Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Células HEK293 , Humanos , Células Jurkat , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Tetraspaninas/genéticaRESUMEN
BACKGROUND: Evidence-based care for people with dementia is a priority for patients, carers and clinicians and a policy priority. There is evidence that people with dementia do not always receive such care. Audit and feedback, also known as clinical audit, is an extensively-used intervention to improve care. However, there is uncertainty about the best way to use it. OBJECTIVES: To investigate whether audit and feedback is effective for improving health professionals' care of people with dementia. To investigate whether the content and delivery of audit and feedback affects its effectiveness in the context of health professionals' care for people with dementia. DESIGN: Systematic review DATA SOURCES: The Cochrane Central Register of Controlled Trials, Prospero, Medline (1946-December week 1 2016), PsycInfo (1967-January 2017), Cinahl (1982-January 2017), HMIC (1979-January 2017), Embase (1974-2017 week 1) databases and the Science Citation Index and Social Science Citation Index were searched combining terms for audit and feedback, health personnel, and dementia. REVIEW METHODS: Following screening, the data were extracted using the Template for Intervention Description and Replication (TIDieR), and synthesised graphically using harvest plots and narratively. RESULTS: Thirteen studies met the inclusion criteria. Published studies of audit and feedback in dementia rarely described more than one cycle. None of the included studies had a comparison group: 12 were before and after designs and one was an interrupted time series without a comparison group. The median absolute improvement was greater than in studies beyond dementia which have used stronger designs with fewer risks of bias. Included studies demonstrated large variation in the effectiveness of audit and feedback. CONCLUSIONS: Whilst methodological and reporting limitations in the included studies hinder the ability to draw strong conclusions on the effectiveness of audit and feedback in dementia care, the large interquartile range indicates further work is needed to understand the factors which affect the effectiveness of this much-used intervention.
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Demencia/terapia , Auditoría Médica , Retroalimentación , Humanos , IncertidumbreRESUMEN
BACKGROUND: Harm in mental health has traditionally been viewed as "unambiguous" and measured in terms of suicide, self-harm, self-neglect and violence. In order to develop an organisational patient safety strategy, one Trust engaged with service users, carers and senior clinicians and managers in order to understand how they define harm. AIM: To explore the meaning of harm in a mental health and learning disabilities setting. METHOD: This paper describes the outcome of service improvement work with service users, carers, senior clinicians and managers at one Trust to determine what harm meant to them. RESULTS: Harm is a concept which is broader than elements currently seen within organisational patient safety metrics and clinical risk assessments. CONCLUSIONS: Taking into account the diverse feedback received about what constitutes harm, a more inclusive definition emerges which could be incorporated into a new framework for risk management, balancing risk of harms across multiple dimensions. This approach has the potential to bring together consideration of the risk and recovery agendas.
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Servicios de Salud Mental/normas , Salud Mental , Seguridad del Paciente , Conducta Autodestructiva/epidemiología , Conducta Autodestructiva/prevención & control , Humanos , Medición de Riesgo , Factores de RiesgoRESUMEN
The assembly of ribosomes from a discrete set of components is a key aspect of the highly coordinated process of ribosome biogenesis. In this review, we present a brief history of the early work on ribosome assembly in Escherichia coli, including a description of in vivo and in vitro intermediates. The assembly process is believed to progress through an alternating series of RNA conformational changes and protein-binding events; we explore the effects of ribosomal proteins in driving these events. Ribosome assembly in vivo proceeds much faster than in vitro, and we outline the contributions of several of the assembly cofactors involved, including Era, RbfA, RimJ, RimM, RimP, and RsgA, which associate with the 30S subunit, and CsdA, DbpA, Der, and SrmB, which associate with the 50S subunit.
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Proteínas de Escherichia coli/metabolismo , Ribosomas/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , GTP Fosfohidrolasas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Bacteriano/metabolismo , ARN Ribosómico/química , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Ribosomas/química , Ribosomas/genéticaRESUMEN
Although high-resolution structures of the ribosome have been solved in a series of functional states, relatively little is known about how the ribosome assembles, particularly in vivo. Here, a general method is presented for studying the dynamics of ribosome assembly and ribosomal assembly intermediates. Since significant quantities of assembly intermediates are not present under normal growth conditions, the antibiotic neomycin is used to perturb wild-type Escherichia coli. Treatment of E. coli with the antibiotic neomycin results in the accumulation of a continuum of assembly intermediates for both the 30S and 50S subunits. The protein composition and the protein stoichiometry of these intermediates were determined by quantitative mass spectrometry using purified unlabeled and (15)N-labeled wild-type ribosomes as external standards. The intermediates throughout the continuum are heterogeneous and are largely depleted of late-binding proteins. Pulse-labeling with (15)N-labeled medium time-stamps the ribosomal proteins based on their time of synthesis. The assembly intermediates contain both newly synthesized proteins and proteins that originated in previously synthesized intact subunits. This observation requires either a significant amount of ribosome degradation or the exchange or reuse of ribosomal proteins. These specific methods can be applied to any system where ribosomal assembly intermediates accumulate, including strains with deletions or mutations of assembly factors. This general approach can be applied to study the dynamics of assembly and turnover of other macromolecular complexes that can be isolated from cells.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteoma/análisis , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Escherichia coli/efectos de los fármacos , Neomicina/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Proteoma/efectos de los fármacos , ARN Ribosómico/química , ARN Ribosómico/genética , Subunidades Ribosómicas Grandes Bacterianas/química , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , Subunidades Ribosómicas Pequeñas Bacterianas/química , Subunidades Ribosómicas Pequeñas Bacterianas/metabolismo , Ribosomas/química , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Relative levels of ribosomal proteins were quantified in crude cell lysates using mass spectrometry. A method for quantifying cellular protein levels using macromolecular standards is presented that does not require complex sample separation, identification of high-responding peptides, affinity purification, or postgrowth modifications. Perturbations in ribosomal protein levels by overexpression of individual proteins correlate to known autoregulatory mechanisms and extend the network of ribosomal protein regulation.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Espectrometría de Masas/métodos , Proteínas Ribosómicas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Espectrometría de Masas/economía , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Ribosómicas/genéticaRESUMEN
Escherichia coli DbpA is an ATP-dependent RNA helicase with specificity for hairpin 92 of 23S ribosomal RNA, an important part of the peptidyl transferase center. The R331A active site mutant of DbpA confers a dominant slow growth and cold sensitive phenotype when overexpressed in E. coli containing endogenous DbpA. Ribosome profiles from cells overexpressing DbpA R331A display increased levels of 50S and 30S subunits and decreased levels 70S ribosomes. Profiles run at low Mg(2+) exhibit fewer 50S subunits and accumulate a 45S particle that contains incompletely processed and undermodified 23S rRNA in addition to reduced levels of several ribosomal proteins that bind late in the assembly pathway. Unlike mature 50S subunits, these 45S particles can stimulate the ATPase activity of DbpA, indicating that hairpin 92 has not yet been sequestered within the 50S subunit. Overexpression of the inactive DbpA R331A mutant appears to block assembly at a late stage when the peptidyl transferase center is formed, indicating a possible role for DbpA promoting this conformational change.
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ARN Helicasas DEAD-box/fisiología , Proteínas de Escherichia coli/fisiología , Escherichia coli/genética , Subunidades Ribosómicas Grandes Bacterianas/metabolismo , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutación , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/químicaRESUMEN
The ribosome is a complex macromolecular machine responsible for protein synthesis in the cell. It consists of two subunits, each of which contains both RNA and protein components. Ribosome assembly is subject to intricate regulatory control and is aided by a multitude of assembly factors in vivo, but can also be carried out in vitro. The details of the assembly process remain unknown even in the face of atomic structures of the entire ribosome and after more than three decades of research. Some of the earliest research on ribosome assembly produced the Nomura assembly map of the small subunit, revealing a hierarchy of protein binding dependencies for the 20 proteins involved and suggesting the possibility of a single intermediate. Recent work using a combination of RNA footprinting and pulse-chase quantitative mass spectrometry paints a picture of small subunit assembly as a dynamic and varied landscape, with sequential and hierarchical RNA folding and protein binding events finally converging on complete subunits. Proteins generally lock tightly into place in a 5' to 3' direction along the ribosomal RNA, stabilizing transient RNA conformations, while RNA folding and the early stages of protein binding are initiated from multiple locations along the length of the RNA.
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Modelos Químicos , Modelos Moleculares , Proteínas Ribosómicas/química , Proteínas Ribosómicas/ultraestructura , Subunidades Ribosómicas Pequeñas/química , Subunidades Ribosómicas Pequeñas/ultraestructura , Sitios de Unión , Simulación por Computador , Unión Proteica , Conformación ProteicaRESUMEN
BACKGROUND: An important aspect of proteomic mass spectrometry involves quantifying and interpreting the isotope distributions arising from mixtures of macromolecules with different isotope labeling patterns. These patterns can be quite complex, in particular with in vivo metabolic labeling experiments producing fractional atomic labeling or fractional residue labeling of peptides or other macromolecules. In general, it can be difficult to distinguish the contributions of species with different labeling patterns to an experimental spectrum and difficult to calculate a theoretical isotope distribution to fit such data. There is a need for interactive and user-friendly software that can calculate and fit the entire isotope distribution of a complex mixture while comparing these calculations with experimental data and extracting the contributions from the differently labeled species. RESULTS: Envelope has been developed to be user-friendly while still being as flexible and powerful as possible. Envelope can simultaneously calculate the isotope distributions for any number of different labeling patterns for a given peptide or oligonucleotide, while automatically summing these into a single overall isotope distribution. Envelope can handle fractional or complete atom or residue-based labeling, and the contribution from each different user-defined labeling pattern is clearly illustrated in the interactive display and is individually adjustable. At present, Envelope supports labeling with 2H, 13C, and 15N, and supports adjustments for baseline correction, an instrument accuracy offset in the m/z domain, and peak width. Furthermore, Envelope can display experimental data superimposed on calculated isotope distributions, and calculate a least-squares goodness of fit between the two. All of this information is displayed on the screen in a single graphical user interface. Envelope supports high-quality output of experimental and calculated distributions in PNG or PDF format. Beyond simply comparing calculated distributions to experimental data, Envelope is useful for planning or designing metabolic labeling experiments, by visualizing hypothetical isotope distributions in order to evaluate the feasibility of a labeling strategy. Envelope is also useful as a teaching tool, with its real-time display capabilities providing a straightforward way to illustrate the key variable factors that contribute to an observed isotope distribution. CONCLUSION: Envelope is a powerful tool for the interactive calculation and visualization of complex isotope distributions for comparison to experimental data. It is available under the GNU General Public License from http://williamson.scripps.edu/envelope/.
Asunto(s)
Biología Computacional/métodos , Isótopos/análisis , Modelos Químicos , Programas Informáticos , Algoritmos , Aminoácidos/análisis , Almacenamiento y Recuperación de la Información/métodos , Marcaje Isotópico/métodos , Isótopos/química , Espectrometría de Masas/métodos , Oligonucleótidos/análisis , Oligonucleótidos/química , Péptidos/análisis , Péptidos/química , Interfaz Usuario-ComputadorRESUMEN
Quantitative proteomic mass spectrometry involves comparison of the amplitudes of peaks resulting from different isotope labeling patterns, including fractional atomic labeling and fractional residue labeling. We have developed a general and flexible analytical treatment of the complex isotope distributions that arise in these experiments, using Fourier transform convolution to calculate labeled isotope distributions and least-squares for quantitative comparison with experimental peaks. The degree of fractional atomic and fractional residue labeling can be determined from experimental peaks at the same time as the integrated intensity of all of the isotopomers in the isotope distribution. The approach is illustrated using data with fractional (15)N-labeling and fractional (13)C-isoleucine labeling. The least-squares Fourier transform convolution approach can be applied to many types of quantitative proteomic data, including data from stable isotope labeling by amino acids in cell culture and pulse labeling experiments.