RESUMO
BACKGROUND: Coronavirus disease 2019 (Covid-19) disproportionately results in hospitalization or death in older patients and those with underlying conditions. Sotrovimab is a pan-sarbecovirus monoclonal antibody that was designed to prevent progression of Covid-19 in high-risk patients early in the course of disease. METHODS: In this ongoing, multicenter, double-blind, phase 3 trial, we randomly assigned, in a 1:1 ratio, nonhospitalized patients with symptomatic Covid-19 (≤5 days after the onset of symptoms) and at least one risk factor for disease progression to receive a single infusion of sotrovimab at a dose of 500 mg or placebo. The primary efficacy outcome was hospitalization (for >24 hours) for any cause or death within 29 days after randomization. RESULTS: In this prespecified interim analysis, which included an intention-to-treat population of 583 patients (291 in the sotrovimab group and 292 in the placebo group), 3 patients (1%) in the sotrovimab group, as compared with 21 patients (7%) in the placebo group, had disease progression leading to hospitalization or death (relative risk reduction, 85%; 97.24% confidence interval, 44 to 96; P = 0.002). In the placebo group, 5 patients were admitted to the intensive care unit, including 1 who died by day 29. Safety was assessed in 868 patients (430 in the sotrovimab group and 438 in the placebo group). Adverse events were reported by 17% of the patients in the sotrovimab group and 19% of those in the placebo group; serious adverse events were less common with sotrovimab than with placebo (in 2% and 6% of the patients, respectively). CONCLUSIONS: Among high-risk patients with mild-to-moderate Covid-19, sotrovimab reduced the risk of disease progression. No safety signals were identified. (Funded by Vir Biotechnology and GlaxoSmithKline; COMET-ICE ClinicalTrials.gov number, NCT04545060.).
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Tratamento Farmacológico da COVID-19 , Progressão da Doença , SARS-CoV-2/imunologia , Adulto , Idoso , Assistência Ambulatorial , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/efeitos adversos , Anticorpos Neutralizantes/efeitos adversos , Método Duplo-Cego , Feminino , Hospitalização/estatística & dados numéricos , Humanos , Incidência , Infusões Intravenosas , Análise de Intenção de Tratamento , Tempo de Internação , Masculino , Pessoa de Meia-IdadeRESUMO
Osteocrin (OSTN) is an endogenous protein sharing structural similarities with the natriuretic peptides [NPs; atrial (ANP), B-type (BNP) and C-type (CNP) NP], which are hormones known for their crucial role in maintaining pressure/volume homeostasis. Osteocrin competes with the NPs for binding to the receptor involved in their clearance (NPR-C). In the present study, having identified, for the first time, the major circulating form of OSTN in human and ovine plasma, we examined the integrated haemodynamic, endocrine and renal effects of vehicle-controlled incremental infusions of ovine proOSTN (83-133) and its metabolism in eight conscious normal sheep. Incremental i.v. doses of OSTN produced stepwise increases in circulating concentrations of the peptide, and its metabolic clearance rate was inversely proportional to the dose. Osteocrin increased plasma levels of ANP, BNP and CNP in a dose-dependent manner, together with concentrations of their intracellular second messenger, cGMP. Increases in plasma cGMP were associated with progressive reductions in arterial pressure and central venous pressure. Plasma cAMP, renin and aldosterone were unchanged. Despite significant increases in urinary cGMP levels, OSTN administration was not associated with natriuresis or diuresis in normal sheep. These results support OSTN as an endogenous ligand for NPR-C in regulating plasma concentrations of NPs and associated cGMP-mediated bioactivity. Collectively, our findings support a role for OSTN in maintaining cardiovascular homeostasis.
Assuntos
GMP Cíclico , Hemodinâmica , Rim , Animais , Ovinos , Rim/metabolismo , GMP Cíclico/metabolismo , Hemodinâmica/efeitos dos fármacos , Hemodinâmica/fisiologia , Fator Natriurético Atrial/metabolismo , Fator Natriurético Atrial/sangue , Feminino , Peptídeo Natriurético Encefálico/metabolismo , Renina/metabolismo , Renina/sangue , AMP Cíclico/metabolismo , Peptídeo Natriurético Tipo C/metabolismo , Aldosterona/sangue , Aldosterona/metabolismo , Pressão Sanguínea/fisiologia , Pressão Sanguínea/efeitos dos fármacos , Peptídeos Natriuréticos/metabolismo , NatriureseRESUMO
This open-label, extension study assessed long-term safety, tolerability, and efficacy of ambrisentan in a pediatric population (age 8- < 18 years) with pulmonary arterial hypertension (PAH). Following completion of a 6-month, randomized study, participants entered the long-term extension at individualized ambrisentan dosages (2.5/5/7.5 or 10 mg/day). Safety assessments included adverse events (AEs), AEs of special interest, and serious AEs (SAEs); efficacy outcomes included 6-min walking distance (6MWD) and World Health Organization functional class (WHO FC). Thirty-eight of 41 (93%) randomized study participants entered the extension; 21 (55%) completed (reaching age 18 years). Most participants received concomitant phosphodiesterase-5 inhibitors (n = 25/38, 66%). Median ambrisentan exposure was 3.5 years. Most participants experienced ≥ 1 AE (n = 34/38, 89%), and 21 (55%) experienced SAEs, most commonly worsening PAH (n = 3/38, 8%), acute cardiac failure, pneumonia, or anemia (n = 2/38; 5% each); none considered ambrisentan-related. Seven participants (18%) died, with recorded reasons (MedDRA preferred term): cardiac failure (n = 2), PAH (n = 2), COVID-19 (n = 1), acute right ventricular failure (n = 1), and failure to thrive (n = 1); median time to death: 5.2 years. Anemia and hepatotoxicity AEs were generally mild to moderate and did not require ambrisentan dose adjustment. Assessed at study end in 29 participants (76%), mean 6MWD improved by 17% (standard deviation: 34.3%), and all (29/29, 100%) had improved or unchanged WHO FC. Conclusion: Long-term weight-based ambrisentan dosing, alone or combined with other PAH therapies in children with PAH aged 8- < 18 years, exhibited tolerability and clinical improvements consistent with prior randomized study results. Trial registration: NCT01342952, April 27, 2011. What is Known: ⢠The endothelin receptor antagonist, ambrisentan, is indicated for treatment of pulmonary arterial hypertension (PAH). Previous studies have shown similar efficacy and tolerability in pediatric patients as in adults. What is New: ⢠This open-label extension study assessed the long-term use of ambrisentan in pediatric patients (8-<18 years) with PAH, most of whom were also receiving recommended background PAH treatment. ⢠Weight-based dosing of ambrisentan, given alone or in combination with other PAH therapies, was well tolerated with clinical improvements consistent with prior randomized study results.
Assuntos
Fenilpropionatos , Hipertensão Arterial Pulmonar , Piridazinas , Humanos , Piridazinas/efeitos adversos , Piridazinas/uso terapêutico , Piridazinas/administração & dosagem , Fenilpropionatos/administração & dosagem , Fenilpropionatos/efeitos adversos , Fenilpropionatos/uso terapêutico , Masculino , Criança , Feminino , Adolescente , Resultado do Tratamento , Hipertensão Arterial Pulmonar/tratamento farmacológico , Anti-Hipertensivos/efeitos adversos , Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/uso terapêutico , Relação Dose-Resposta a Droga , Teste de Caminhada , Hipertensão Pulmonar/tratamento farmacológicoRESUMO
District nursing vacancies are recognised as high in comparison to many other nursing specialities. The role is often considered as an option for nurses after a period of registration as a nurse, which may be informed by multiple factors. The objectives of the study were to explore barriers to district nurse employment from the perspective of undergraduate nurses. A descriptive qualitative design was employed using focus groups as the method of data collection in accordance with study approval reference no: 21/NAH/006, 30/03/23. Participants were recruited from the current undergraduate nursing programme (n=60). Thematic analysis was chosen as a flexible method of qualitative data analysis. The study produced themes relating to role ambiguity, personal preferences, educational opportunities and recruitment and employer engagement. The experiences provided an insight into the barriers to recruitment of student nurses into district nursing on qualification. Increased engagement from community nurse providers is required to inform the role and incentivise entering this career option. Educational strategies that support community career pathways are required to ensure a balanced view of nursing roles among undergraduates.
Assuntos
Bacharelado em Enfermagem , Estudantes de Enfermagem , Humanos , Pesquisa Qualitativa , Grupos Focais , EscolaridadeRESUMO
Cholesterol biosynthesis is a highly regulated pathway, with over 20 enzymes controlled at the transcriptional and posttranslational levels. While some enzymes remain stable, increased sterol levels can trigger degradation of several synthesis enzymes via the ubiquitin-proteasome system. Of note, we previously identified four cholesterol synthesis enzymes as substrates for one E3 ubiquitin ligase, membrane-associated RING-CH-type finger 6 (MARCHF6). Whether MARCHF6 targets the cholesterol synthesis pathway at other points is unknown. In addition, the posttranslational regulation of many cholesterol synthesis enzymes, including the C4-demethylation complex (sterol-C4-methyl oxidase-like, SC4MOL; NAD(P)-dependent steroid dehydrogenase-like, NSDHL; hydroxysteroid 17-beta dehydrogenase, HSD17B7), is largely uncharacterized. Using cultured mammalian cell lines (human-derived and Chinese hamster ovary cells), we show SC4MOL, the first acting enzyme of C4-demethylation, is a MARCHF6 substrate and is rapidly turned over and sensitive to sterols. Sterol depletion stabilizes SC4MOL protein levels, while sterol excess downregulates both transcript and protein levels. Furthermore, we found SC4MOL depletion by siRNA results in a significant decrease in total cell cholesterol. Thus, our work indicates SC4MOL is the most regulated enzyme in the C4-demethylation complex. Our results further implicate MARCHF6 as a crucial posttranslational regulator of cholesterol synthesis, with this E3 ubiquitin ligase controlling levels of at least five enzymes of the pathway.
Assuntos
Fitosteróis , Esteróis , Cricetinae , Animais , Humanos , Esteróis/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Células CHO , Cricetulus , Colesterol/metabolismo , Oxirredutases , 3-Hidroxiesteroide DesidrogenasesRESUMO
Mass spectrometry is a powerful technique for investigating renal pathologies and identifying biomarkers, and efficient protein extraction from kidney tissue is essential for bottom-up proteomic analyses. Detergent-based strategies aid cell lysis and protein solubilization but are poorly compatible with downstream protein digestion and liquid chromatography-coupled mass spectrometry, requiring additional purification and buffer-exchange steps. This study compares two well-established detergent-based methods for protein extraction (in-solution sodium deoxycholate (SDC); suspension trapping (S-Trap)) with the recently developed sample preparation by easy extraction and digestion (SPEED) method, which uses strong acid for denaturation. We compared the quantitative performance of each method using label-free mass spectrometry in both sheep kidney cortical tissue and plasma. In kidney tissue, SPEED quantified the most unique proteins (SPEED 1250; S-Trap 1202; SDC 1197). In plasma, S-Trap produced the most unique protein quantifications (S-Trap 150; SDC 148; SPEED 137). Protein quantifications were reproducible across biological replicates in both tissue (R2 = 0.85-0.90) and plasma (SPEED R2 = 0.84; SDC R2 = 0.76, S-Trap R2 = 0.65). Our data suggest SPEED as the optimal method for proteomic preparation in kidney tissue and S-Trap or SPEED as the optimal method for plasma, depending on whether a higher number of protein quantifications or greater reproducibility is desired.
Assuntos
Detergentes , Espectrometria de Massas em Tandem , Animais , Ovinos , Detergentes/química , Espectrometria de Massas em Tandem/métodos , Proteômica/métodos , Reprodutibilidade dos Testes , ProteínasRESUMO
ISSUE ADDRESSED: Australia has smoking prevalence of less than 15% among adults, but there are concerns that the rates of decline have stabilised. Sustained mass media campaigns are central to decreasing prevalence, and the emotions evoked by campaigns contribute to their impact. This study investigates the association between potential exposure to campaigns that evoke different emotions on quitting salience (thinking about quitting), quitting intentions and quitting attempts. METHODS: Data on quitting outcomes were obtained from weekly cross-sectional telephone surveys with adult smokers and recent quitters between 2013 and 2018. Campaign activity data were collated, and population-level potential campaign exposure was measured by time and dose. RESULTS: Using multivariate analyses, a positive association between potential exposure to 'hope' campaigns and thinking about quitting and intending to quit was noted, but no association was seen with quit attempts. Potential exposure to 'sadness' evoking campaigns was positively associated with quitting salience and negatively associated with quit attempts, whereas those potentially exposed to campaigns evoking multiple negative emotions (fear, guilt and sadness) were approximately 30% more likely to make a quit attempt. CONCLUSIONS: This study suggests a relationship between the emotional content of campaigns, quitting behaviours. Campaign planners should consider campaigns that evoke negative emotions for population-wide efforts to bring about quitting activity alongside hopeful campaigns that promote quitting salience and quitting intentions. The emotional content of campaigns provides an additional consideration for campaigns targeting smokers and influencing quitting activity. SO WHAT?: This study demonstrates the importance of balancing the emotional content of campaigns to ensure that campaign advertising is given the greatest chance to achieve its objectives. Utilising campaigns that evoke negative emotions appear to be needed to encourage quitting attempts but maintaining hopeful campaigns to promote thinking about quitting and intending to quit is also an important component of the mix of tobacco control campaigns.
Assuntos
Fumantes , Abandono do Hábito de Fumar , Adulto , Humanos , Fumantes/psicologia , Meios de Comunicação de Massa , Tristeza , Controle do Tabagismo , Estudos Transversais , Promoção da Saúde , Prevenção do Hábito de Fumar , EmoçõesRESUMO
This research study was undertaken to elicit a group of final-year student nurses' perceptions of their motivations and approaches to learning, and the implications of their views. It is important to explore this subject because students' motivations and approaches to learning can potentially impact patient care. This study was part of a larger research project. The sample consisted of 18 final-year student nurses at a large UK university. Students completed semi-structured interviews that used a qualitative constructivist approach to explore their educational experience. Students described what motivated them to learn, and how they approached their learning because of their understanding of which subjects they believed were and were not important. Students felt that clinical skills were the most important subjects, and topics such as health promotion, law and ethics, were less important and therefore they approached these subjects in a superficial way, learning just enough to pass their course. Clinical skills were perceived as more useful because they would be used directly in clinical practice. The findings of this study are significant to inform nurse educators as they plan curricula and provide an insight into what may potentially adversely affect patient care when students become registered nurses.
Assuntos
Bacharelado em Enfermagem , Enfermeiras e Enfermeiros , Estudantes de Enfermagem , Humanos , Motivação , Pesquisa QualitativaRESUMO
BACKGROUND: Over half the population (51%) of England and Wales is female, most of whom will experience menopause, either as a result of endocrine ageing or medical treatment. AIM: The project aimed to undertake a review of the literature to determine the level of knowledge about menopause that healthcare students are exposed to, and to highlight why it is important for them to have an understanding of this subject both for their own clinical practice and for supporting colleagues in the workplace. METHOD: A literature review was conducted by the project team. FINDINGS: There is a lack of education for healthcare students, who will go on to care for those affected by menopause, and will also work with colleagues experiencing menopause. CONCLUSION: Educational programmes should include menopause as a component, which will allow for a breaking down of barriers on a subject that is still generally considered taboo. RECOMMENDATIONS: A national audit should be conducted on menopause coverage in UK pre-registration nursing. The addition of menopause to the Liverpool John Moores University pre-registration nursing curriculum is also recommended based on agreed competencies.
Assuntos
Educação em Enfermagem , Ocupações em Saúde , Menopausa , Feminino , Humanos , Currículo , Educação em Enfermagem/organização & administração , Ocupações em Saúde/educação , Reino Unido , Pesquisa em Educação em EnfermagemRESUMO
Importance: Older patients and those with comorbidities who are infected with SARS-CoV-2 may be at increased risk of hospitalization and death. Sotrovimab is a neutralizing antibody for the treatment of high-risk patients to prevent COVID-19 progression. Objective: To evaluate the efficacy and adverse events of sotrovimab in preventing progression of mild to moderate COVID-19 to severe disease. Design, Setting, and Participants: Randomized clinical trial including 1057 nonhospitalized patients with symptomatic, mild to moderate COVID-19 and at least 1 risk factor for progression conducted at 57 sites in Brazil, Canada, Peru, Spain, and the US from August 27, 2020, through March 11, 2021; follow-up data were collected through April 8, 2021. Interventions: Patients were randomized (1:1) to an intravenous infusion with 500 mg of sotrovimab (n = 528) or placebo (n = 529). Main Outcomes and Measures: The primary outcome was the proportion of patients with COVID-19 progression through day 29 (all-cause hospitalization lasting >24 hours for acute illness management or death); 5 secondary outcomes were tested in hierarchal order, including a composite of all-cause emergency department (ED) visit, hospitalization of any duration for acute illness management, or death through day 29 and progression to severe or critical respiratory COVID-19 requiring supplemental oxygen or mechanical ventilation. Results: Enrollment was stopped early for efficacy at the prespecified interim analysis. Among 1057 patients randomized (median age, 53 years [IQR, 42-62], 20% were ≥65 years of age, and 65% Latinx), the median duration of follow-up was 103 days for sotrovimab and 102 days for placebo. All-cause hospitalization lasting longer than 24 hours or death was significantly reduced with sotrovimab (6/528 [1%]) vs placebo (30/529 [6%]) (adjusted relative risk [RR], 0.21 [95% CI, 0.09 to 0.50]; absolute difference, -4.53% [95% CI, -6.70% to -2.37%]; P < .001). Four of the 5 secondary outcomes were statistically significant in favor of sotrovimab, including reduced ED visit, hospitalization, or death (13/528 [2%] for sotrovimab vs 39/529 [7%] for placebo; adjusted RR, 0.34 [95% CI, 0.19 to 0.63]; absolute difference, -4.91% [95% CI, -7.50% to -2.32%]; P < .001) and progression to severe or critical respiratory COVID-19 (7/528 [1%] for sotrovimab vs 28/529 [5%] for placebo; adjusted RR, 0.26 [95% CI, 0.12 to 0.59]; absolute difference, -3.97% [95% CI, -6.11% to -1.82%]; P = .002). Adverse events were infrequent and similar between treatment groups (22% for sotrovimab vs 23% for placebo); the most common events were diarrhea with sotrovimab (n = 8; 2%) and COVID-19 pneumonia with placebo (n = 22; 4%). Conclusions and Relevance: Among nonhospitalized patients with mild to moderate COVID-19 and at risk of disease progression, a single intravenous dose of sotrovimab, compared with placebo, significantly reduced the risk of a composite end point of all-cause hospitalization or death through day 29. The findings support sotrovimab as a treatment option for nonhospitalized, high-risk patients with mild to moderate COVID-19, although efficacy against SARS-CoV-2 variants that have emerged since the study was completed is unknown. Trial Registration: ClinicalTrials.gov Identifier: NCT04545060.
Assuntos
Antivirais , Tratamento Farmacológico da COVID-19 , COVID-19 , SARS-CoV-2 , Doença Aguda , Adulto , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais Humanizados/uso terapêutico , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/uso terapêutico , Antivirais/administração & dosagem , Antivirais/uso terapêutico , COVID-19/mortalidade , Progressão da Doença , Hospitalização , Humanos , Infusões Intravenosas , Pessoa de Meia-Idade , Respiração Artificial , Resultado do TratamentoRESUMO
One-quarter of patients with acute decompensated heart failure (ADHF) experience acute kidney injury (AKI)-an abrupt reduction or loss of kidney function associated with increased long-term mortality. There is a critical need to identify early and real-time markers of AKI in ADHF; however, to date, no protein biomarkers have exhibited sufficient diagnostic or prognostic performance for widespread clinical uptake. We aimed to identify novel protein biomarkers of AKI associated with ADHF by quantifying changes in protein abundance in the kidneys that occur during ADHF development and recovery in an ovine model. Relative quantitative protein profiling was performed using sequential window acquisition of all theoretical fragment ion spectra-mass spectrometry (SWATH-MS) in kidney cortices from control sheep (n = 5), sheep with established rapid-pacing-induced ADHF (n = 8), and sheep after ~4 weeks recovery from ADHF (n = 7). Of the 790 proteins quantified, we identified 17 candidate kidney injury markers in ADHF, 1 potential kidney marker of ADHF recovery, and 2 potential markers of long-term renal impairment (differential abundance between groups of 1.2-2.6-fold, adjusted p < 0.05). Among these 20 candidate protein markers of kidney injury were 6 candidates supported by existing evidence and 14 novel candidates not previously implicated in AKI. Proteins of differential abundance were enriched in pro-inflammatory signalling pathways: glycoprotein VI (activated during ADHF development; adjusted p < 0.01) and acute phase response (repressed during recovery from ADHF; adjusted p < 0.01). New biomarkers for the early detection of AKI in ADHF may help us to evaluate effective treatment strategies to prevent mortality and improve outcomes for patients.
Assuntos
Injúria Renal Aguda/diagnóstico , Biomarcadores/metabolismo , Insuficiência Cardíaca/metabolismo , Proteômica/métodos , Injúria Renal Aguda/sangue , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/urina , Animais , Biomarcadores/sangue , Biomarcadores/urina , Modelos Animais de Doenças , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/complicações , Insuficiência Cardíaca/urina , Humanos , Glicoproteínas da Membrana de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/urina , Prognóstico , OvinosRESUMO
Ensuring that pre-registration student nurses gain the richness of clinical practice to allow them to feel confident and knowledgeable to care for patients of the 21st century is often a challenge. Clinical practice challenges often mean that students will be strategic learners. This article discusses the development of a virtual case-based learning site to provide a rich experience for students to learn.
Assuntos
Bacharelado em Enfermagem , Educação em Enfermagem , Estudantes de Enfermagem , Competência Clínica , Humanos , Relações Interprofissionais , AprendizagemRESUMO
Cholesterol synthesis is a tightly controlled pathway, with over 20 enzymes involved. Each of these enzymes can be distinctly regulated, helping to fine-tune the production of cholesterol and its functional intermediates. Several enzymes are degraded in response to increased sterol levels, whilst others remain stable. We hypothesised that an enzyme at a key branch point in the pathway, lanosterol 14α-demethylase (LDM) may be post-translationally regulated. Here, we show that the preceding enzyme, lanosterol synthase is stable, whilst LDM is rapidly degraded. Surprisingly, this degradation is not triggered by sterols. However, the E3 ubiquitin ligase membrane-associated ring-CH-type finger 6 (MARCH6), known to control earlier rate-limiting steps in cholesterol synthesis, also control levels of LDM and the terminal cholesterol synthesis enzyme, 24-dehydrocholesterol reductase. Our work highlights MARCH6 as the first example of an E3 ubiquitin ligase that targets multiple steps in a biochemical pathway and indicates new facets in the control of cholesterol synthesis.
Assuntos
Colesterol/biossíntese , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Esterol 14-Desmetilase/genética , Ubiquitina-Proteína Ligases/genética , Animais , Células CHO , Colesterol/genética , Cricetulus , Células HeLa , Humanos , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Lipogênese/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Processamento de Proteína Pós-Traducional/genética , Proteólise , Esterol 14-Desmetilase/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The E3 ligase membrane-associated ring-CH-type finger 6 (MARCH6) is a polytopic enzyme bound to the membranes of the endoplasmic reticulum. It controls levels of several known protein substrates, including a key enzyme in cholesterol synthesis, squalene monooxygenase. However, beyond its own autodegradation, little is known about how MARCH6 itself is regulated. Using CRISPR/Cas9 gene-editing, MARCH6 overexpression, and immunoblotting, we found here that cholesterol stabilizes MARCH6 protein endogenously and in HEK293 cells that stably express MARCH6. Conversely, MARCH6-deficient HEK293 and HeLa cells lost their ability to degrade squalene monooxygenase in a cholesterol-dependent manner. The ability of cholesterol to boost MARCH6 did not seem to involve a putative sterol-sensing domain in this E3 ligase, but was abolished when either membrane extraction by valosin-containing protein (VCP/p97) or proteasomal degradation was inhibited. Furthermore, cholesterol-mediated stabilization was absent in two MARCH6 mutants that are unable to degrade themselves, indicating that cholesterol stabilizes MARCH6 protein by preventing its autodegradation. Experiments with chemical chaperones suggested that this likely occurs through a conformational change in MARCH6 upon cholesterol addition. Moreover, cholesterol reduced the levels of at least three known MARCH6 substrates, indicating that cholesterol-mediated MARCH6 stabilization increases its activity. Our findings highlight an important new role for cholesterol in controlling levels of proteins, extending the known repertoire of cholesterol homeostasis players.
Assuntos
Colesterol/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismo , Sistemas CRISPR-Cas , Colesterol/genética , Ativação Enzimática/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Ubiquitina-Proteína Ligases/genética , Proteína com Valosina/genética , Proteína com Valosina/metabolismoRESUMO
Squalene monooxygenase (SM) is an essential rate-limiting enzyme in cholesterol synthesis. SM degradation is accelerated by excess cholesterol, and this requires the first 100 amino acids of SM (SM N100). This process is part of a protein quality control pathway called endoplasmic reticulum-associated degradation (ERAD). In ERAD, SM is ubiquitinated by MARCH6, an E3 ubiquitin ligase located in the endoplasmic reticulum (ER). However, several details of the ERAD process for SM remain elusive, such as the extraction mechanism from the ER membrane. Here, we used SM N100 fused to GFP (SM N100-GFP) as a model degron to investigate the extraction process of SM in ERAD. We showed that valosin-containing protein (VCP) is important for the cholesterol-accelerated degradation of SM N100-GFP and SM. In addition, we revealed that VCP acts following ubiquitination of SM N100-GFP by MARCH6. We demonstrated that the amphipathic helix (Gln62-Leu73) of SM N100-GFP is critical for regulation by VCP and MARCH6. Replacing this amphipathic helix with hydrophobic re-entrant loops promoted degradation in a VCP-dependent manner. Finally, we showed that inhibiting VCP increases cellular squalene and cholesterol levels, indicating a functional consequence for VCP in regulating the cholesterol synthesis pathway. Collectively, we established VCP plays a key role in ERAD that contributes to the cholesterol-mediated regulation of SM.
Assuntos
Colesterol/metabolismo , Degradação Associada com o Retículo Endoplasmático , Esqualeno Mono-Oxigenase/metabolismo , Proteína com Valosina/metabolismo , Colesterol/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Conformação Proteica em alfa-Hélice , Proteólise , Esqualeno/metabolismo , Esqualeno Mono-Oxigenase/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína com Valosina/genéticaRESUMO
Some 5-10% of all breast cancers are associated with a pathogenic variant in a breast cancer-associated gene (BRCA1/BRCA2). Historically, with referral to the Nottingham University Hospitals NHS Trust's clinical genetics department for genetic testing, waiting times were on average 12-14 weeks for an initial appointment and 4-6 months to obtain results from the date of testing. A specialist, nurse-led mainstreaming cancer genetics (MCG) service was set up in the trust's Nottingham Breast Institute (NBI) to: reduce waiting times for the initial consultation, counselling, consent and obtaining results for BRCA1/BRCA2 gene testing; and to ensure appropriate patients with breast cancer were offered genetic testing. Two breast clinical nurse specialists were trained so they could counsel, consent and give results for the BRCA1/BRCA2 gene testing directly to patients. Average waiting times for results from the time of testing were reduced to 35.8 days under the nurse-led service, which enabled oncologists and patients to consider individual treatment options at an earlier time. The MCG service reduced waiting times, resulting in an improved, more streamlined service for patients undergoing genetic testing. The MCG service extended the scope of practice of the breast nurse clinical specialists, embedded an expert advanced nursing role in the breast multidisciplinary team and developed nurse mentoring opportunities.
Assuntos
Genes BRCA1 , Genes BRCA2 , Aconselhamento Genético/métodos , Testes Genéticos/métodos , Síndrome Hereditária de Câncer de Mama e Ovário/diagnóstico , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Padrões de Prática em Enfermagem/organização & administração , Adulto , Inglaterra , Feminino , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND:: clinical audits highlight areas where care may not be of the desired quality; they are essential to ensure care is safe and effective. Effective assessment and management of pain have been shown to improve patient wellbeing and clinical outcomes. AIM:: this audit aimed to identify compliance with pain assessment tools and documentation within intensive care and make recommendations to improve practice. DISCUSSION:: compliance with documenting pain assessments was poor, a finding that is consistent with the literature. Although a wealth of evidence has shown pain assessments are not being completed effectively, this continues to be a problem. Intensive care has significant areas for improvement in this area, which would improve patients' experiences and outcomes. Nurses should be educated in the use of pain assessment tools and compliance. CONCLUSION:: providing patients in intensive care with appropriate analgesia benefits their physical and psychological health. Areas for improvement identified in this audit include that pain assessments need to be carried out and documented regularly. The audit has implications for practice in that it shows a need for reinforced education for staff, better communication and updates to promote pain assessment and the implementation of guidelines.
Assuntos
Benchmarking , Documentação/normas , Unidades de Terapia Intensiva/normas , Auditoria Médica/normas , Avaliação em Enfermagem/normas , Medição da Dor/enfermagem , Humanos , Medição da Dor/normas , Medicina Estatal , Reino UnidoRESUMO
BACKGROUND: Collecting data in phenomenological research can be difficult and an often under-explored area of data collection is the use of diaries. AIM: To enable the reader to understand how diaries can be used to collect data in phenomenological research. DISCUSSION: The author discusses how diaries can provide unadulterated insight into a phenomenon. CONCLUSION: Diaries can be useful to gain a personal insight into a phenomenon. IMPLICATIONS FOR PRACTICE: Diaries can be a challenge for those maintaining them, but offer a personal insight which can be useful as a data collection method and also cathartic for the participant.
Assuntos
Pesquisa Metodológica em Enfermagem , Pesquisa em Enfermagem , Coleta de Dados/métodos , Pesquisa Qualitativa , Projetos de PesquisaRESUMO
A qualitative phenomenological research study sought to determine the perceptions of final-year nursing students about the value of simulation during their nurse education. The study sample consisted of 18 final-year students, and semi-structured interviews were undertaken. Data analysis using interpretative phenomenological analysis was carried out. The findings suggest that nursing students perceive simulation as a useful educational tool, which can increase their confidence and encourage deeper learning. The relevance of this research also relates to the potential for more simulated time within the new educational standards.