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Hyperkalaemia is an electrolyte imbalance that impairs muscle function and myocardial excitability, and can potentially lead to fatal arrhythmias and sudden cardiac death. The prevalence of hyperkalaemia is estimated to be 6%-7% worldwide and 7%-10% in Asia. Hyperkalaemia frequently affects patients with chronic kidney disease, heart failure, and diabetes mellitus, particularly those receiving treatment with renin-angiotensin-aldosterone system (RAAS) inhibitors. Both hyperkalaemia and interruption of RAAS inhibitor therapy are associated with increased risks for cardiovascular events, hospitalisations, and death, highlighting a clinical dilemma in high-risk patients. Conventional potassium-binding resins are widely used for the treatment of hyperkalaemia; however, caveats such as the unpalatable taste and the risk of gastrointestinal side effects limit their chronic use. Recent evidence suggests that, with a rapid onset of action and improved gastrointestinal tolerability, novel oral potassium binders (e.g., patiromer and sodium zirconium cyclosilicate) are alternative treatment options for both acute and chronic hyperkalaemia. To optimise the care for patients with hyperkalaemia in the Asia-Pacific region, a multidisciplinary expert panel was convened to review published literature, share clinical experiences, and ultimately formulate 25 consensus statements, covering three clinical areas: (i) risk factors of hyperkalaemia and risk stratification in susceptible patients; (ii) prevention of hyperkalaemia for at-risk individuals; and (iii) correction of hyperkalaemia for at-risk individuals with cardiorenal disease. These statements were expected to serve as useful guidance in the management of hyperkalaemia for health care providers in the region.
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Consenso , Hiperpotassemia , Humanos , Hiperpotassemia/epidemiologia , Hiperpotassemia/terapia , Hiperpotassemia/diagnóstico , Ásia/epidemiologia , Fatores de Risco , Potássio/sangue , Silicatos/uso terapêutico , Silicatos/efeitos adversosRESUMO
WHAT IS THIS SUMMARY ABOUT?: This is a summary of a research survey called ACTION Teens. In our survey, 12,987 people from 10 countries answered questions about obesity. They were: 5275 teenagers with obesity, 5389 caregivers of teenagers with obesity, and 2323 doctors who provide medical care for teenagers with obesity. WHAT WERE THE MAIN RESULTS OF THE SURVEY?: Most teenagers with obesity were worried about their weight and thought that losing weight was their responsibility. Many teenagers had already tried to lose weight. For teenagers, wanting to be more fit or in better shape was the top reason for wanting to lose weight. Some caregivers did not realize how worried their teenager was about their own weight. There were also some caregivers who were not aware of their teenager's recent attempts to lose weight. As a group, the doctors did not know the main reasons why teenagers want to lose weight. They also did not know the main reasons preventing teenagers from losing weight. WHAT DO THE RESULTS OF THE SURVEY MEAN?: Teenagers with obesity will be better supported and understood if there is better communication between teenagers, caregivers, and doctors. Clinical Trial Registration: NCT05013359 (ClinicalTrials.gov).
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Cuidadores , Obesidade , Humanos , Adolescente , Obesidade/epidemiologia , Obesidade/terapia , Idioma , Redução de Peso , Atenção à SaúdeRESUMO
Geopolymer (GP) has been considered a potential material to replace ordinary Portland cement (OPC) because of its excellent mechanical properties and environmentally friendly process. However, the promotion of GP is limited due to the large shrinkage and the different operating procedures compared to cement. This study aims to reduce the shrinkage of ground granulated blast furnace slag (GGBFS) based GP by the hydration expansion properties of activated magnesium oxide (MgO). The slurry of GP was blended from GGBFS, MgO, and activator; and the compositions of the activator are sodium hydroxide (NaOH), sodium silicate (Na2SiO3), and alumina silicate(NaAlO2). Herein, the GGFBS and MgO were a binder and a shrinkage compensation agent of GP, respectively. After unmolding, the GP specimens were cured under four types of environments and the lengths of the specimens were measured at different time intervals to understand the length change ratio of GP. In this study, two groups of GP specimens were made by fixing the activator to binder (A/B) ratio and the fluidity. The test results show that adding MgO will reduce the shrinkage of GP as A/B ratio was fixed. However, fixing the fluidity exhibited the opposite results. The X-ray diffraction (XRD) was used to check the Mg(OH)2 that occurred due to the MgO hydration under four curing conditions. Three statistical and machine learning methods were used to analyze the length change of GP based on the test data. The testing and analysis results show that the influence of curing environments is more significant for improving the shrinkage of GP than additive MgO.
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BACKGROUND: There is limited evidence regarding the experiences, challenges, and needs of adolescents living with obesity (ALwO), their caregivers, and healthcare professionals (HCPs). OBJECTIVES: The cross-sectional, survey-based global ACTION Teens study aimed to identify perceptions, attitudes, behaviours, and barriers to effective obesity care among ALwO, caregivers of ALwO, and HCPs. METHODS: ALwO (aged 12 to <18 years; N = 5275), caregivers (N = 5389), and HCPs treating ALwO (N = 2323) from 10 countries completed an online survey (August-December 2021). RESULTS: Most ALwO perceived their weight as above normal (76% vs. 66% of caregivers), were worried about its impact on their health (85% vs. 80% of caregivers), and recently made a weight loss attempt (58%). While 45% of caregivers believed ALwO would slim down with age, only 24% of HCPs agreed. Most commonly reported weight loss motivators for ALwO were wanting to be more fit/in better shape according to ALwO (40%) and caregivers (32%), and improved confidence/social life according to HCPs (69%). ALwO weight loss barriers included lack of hunger control (most commonly reported by ALwO/caregivers), lack of motivation, unhealthy eating habits (most commonly agreed by HCPs), and lack of exercise. CONCLUSIONS: Misalignment between ALwO, caregivers, and HCPs-including caregivers' underestimation of the impact of obesity on ALwO and HCPs' misperception of key motivators/barriers for weight loss-suggests a need for improved communication and education.
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Cuidadores , Obesidade Infantil , Adolescente , Atitude do Pessoal de Saúde , Estudos Transversais , Humanos , Obesidade Infantil/epidemiologia , Obesidade Infantil/prevenção & controle , Redução de PesoRESUMO
The use of mean platelet diameter (MPD) to classify inherited thrombocytopenia (IT) has been demonstrated in several studies. Alternatively, the mean platelet volume (MPV) may be used, but in macrothrombocytopenia this may not be available. We hypothesized that platelet forward scatter (FSC) measurements using flow cytometry may be used for the size-based classification of IT. The study aimed to assess the ability of platelet FSC to measure platelet size and whether it could be used as an alternative to the MPD or MPV.Blood samples were obtained from individuals undergoing investigation for inherited platelet function disorders (IPFD, n = 40) or platelet number disorders (IPND, n = 46). A hematology analyzer was used to obtain MPV and platelet counts, flow cytometry to measure platelet FSC and ImageJ software to measure MPD from stained blood smears. The International Society of Thrombosis and Hemostasis (ISTH) Bleeding Assessment Tool (BAT) was used to calculate bleeding scores.Twenty-nine(63%) of IPND patients had an MPV that could not be reported. A significant correlation to platelet FSC was found to the MPD (p < .0001) and MPV (p < .0001) and an inverse correlation with platelet count (p < .0001). No significant correlation was found between FSC and bleeding history. In conclusion, platelet FSC is an alternative to MPV and may be used in macrothrombocytopenia where the MPV is not recorded.
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Transtornos Plaquetários , Trombocitopenia , Plaquetas , Citometria de Fluxo , Hemorragia , Humanos , Volume Plaquetário Médio , Contagem de Plaquetas , Trombocitopenia/diagnósticoRESUMO
Three studies used empirical equations to calculate the rainfall erosivity factor R, and all three equations appeared to be incorrect. All of the studies were published in the journal Science of the Total Environment, and none of them accurately cited the sources of the incorrect equations that were used in them. We were able to track down the original equation as well as the source of the equation. Additionally, it was discovered that the original equation contained an incorrect conversion factor, which needs to be corrected.
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Minas de Carvão , Água Subterrânea , China , Conservação dos Recursos Naturais , Ecossistema , Íons , Rios , SoloRESUMO
Colonic polyps are a common cause of persistent bloody stools in pediatric patients. Such polyps are easily diagnosed by a barium study of the lower gastrointestinal tract or by colonoscopy. Polypectomies utilizing electric ligators are generally performed on pediatric patients, and such patients can be easily operated on. However, giant colonic polyps have been reported in pediatric patients. In the past, a laparotomy or laparoscopy would have been performed in some pediatric patients diagnosed with a giant colonic polyp; however, the large size, location, or position of the polyp would sometimes be too large or the location or position of the polyp would make successful operation difficult. In general, larger stumps with large feeding arteries are associated with larger colonic polyps. Therefore, if such a polyp is removed via electric polypectomy alone, there may be a higher risk of post-polypectomy bleeding from its stump. We report a case of a 14-year-old male patient who presented with a 2-month history of bloody stools. A giant juvenile colonic polyp was detected by colonoscopy in the transverse colon. Finally, we successfully removed the giant polyp by using endoloop-assisted polypectomy.
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Variants of the Diaphanous-Related Formin 1 (DIAPH-1) gene have recently been reported causing inherited macrothrombocytopenia. The essential/"diagnostic" characteristics associated with the disorder are emerging; however, robust and complete criteria are not established. Here, we report the first cases of DIAPH1-related disorder in Australia caused by the autosomal dominant gain-of-function DIAPH1 R1213X variant formed by truncation of the protein within the diaphanous auto-regulatory domain (DAD) with loss of regulatory motifs responsible for autoinhibitory interactions within the DIAPH1 protein. We affirm phenotypic changes induced by the DIAPH1 R1213X variant to include macrothrombocytopenia, early-onset progressive sensorineural hearing loss, and mild asymptomatic neutropenia. High-resolution microscopy confirms perturbations of cytoskeletal dynamics caused by the DIAPH1 variant and we extend the repertoire of changes generated by this variant to include alteration of procoagulant platelet formation and possible dental anomalies.
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Plaquetas/metabolismo , Surdez/genética , Forminas/efeitos adversos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Surdez/patologia , Humanos , FenótipoRESUMO
Soil erosion can present a major threat to agriculture due to loss of soil, nutrients, and organic carbon. Therefore, soil erosion modelling is one of the steps used to plan suitable soil protection measures and detect erosion hotspots. A bibliometric analysis of this topic can reveal research patterns and soil erosion modelling characteristics that can help identify steps needed to enhance the research conducted in this field. Therefore, a detailed bibliometric analysis, including investigation of collaboration networks and citation patterns, should be conducted. The updated version of the Global Applications of Soil Erosion Modelling Tracker (GASEMT) database contains information about citation characteristics and publication type. Here, we investigated the impact of the number of authors, the publication type and the selected journal on the number of citations. Generalized boosted regression tree (BRT) modelling was used to evaluate the most relevant variables related to soil erosion modelling. Additionally, bibliometric networks were analysed and visualized. This study revealed that the selection of the soil erosion model has the largest impact on the number of publication citations, followed by the modelling scale and the publication's CiteScore. Some of the other GASEMT database attributes such as model calibration and validation have negligible influence on the number of citations according to the BRT model. Although it is true that studies that conduct calibration, on average, received around 30% more citations, than studies where calibration was not performed. Moreover, the bibliographic coupling and citation networks show a clear continental pattern, although the co-authorship network does not show the same characteristics. Therefore, soil erosion modellers should conduct even more comprehensive review of past studies and focus not just on the research conducted in the same country or continent. Moreover, when evaluating soil erosion models, an additional focus should be given to field measurements, model calibration, performance assessment and uncertainty of modelling results. The results of this study indicate that these GASEMT database attributes had smaller impact on the number of citations, according to the BRT model, than anticipated, which could suggest that these attributes should be given additional attention by the soil erosion modelling community. This study provides a kind of bibliographic benchmark for soil erosion modelling research papers as modellers can estimate the influence of their paper.
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Bibliometria , Erosão do Solo , Agricultura , Publicações , SoloRESUMO
To gain a better understanding of the global application of soil erosion prediction models, we comprehensively reviewed relevant peer-reviewed research literature on soil-erosion modelling published between 1994 and 2017. We aimed to identify (i) the processes and models most frequently addressed in the literature, (ii) the regions within which models are primarily applied, (iii) the regions which remain unaddressed and why, and (iv) how frequently studies are conducted to validate/evaluate model outcomes relative to measured data. To perform this task, we combined the collective knowledge of 67 soil-erosion scientists from 25 countries. The resulting database, named 'Global Applications of Soil Erosion Modelling Tracker (GASEMT)', includes 3030 individual modelling records from 126 countries, encompassing all continents (except Antarctica). Out of the 8471 articles identified as potentially relevant, we reviewed 1697 appropriate articles and systematically evaluated and transferred 42 relevant attributes into the database. This GASEMT database provides comprehensive insights into the state-of-the-art of soil- erosion models and model applications worldwide. This database intends to support the upcoming country-based United Nations global soil-erosion assessment in addition to helping to inform soil erosion research priorities by building a foundation for future targeted, in-depth analyses. GASEMT is an open-source database available to the entire user-community to develop research, rectify errors, and make future expansions.
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ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.
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Trifosfato de Adenosina/metabolismo , Heme/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Receptores Virais/metabolismo , Termogênese/fisiologia , Animais , Deficiência de Citocromo-c Oxidase , Complexo III da Cadeia de Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons , Metabolismo Energético/fisiologia , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Membranas Mitocondriais/metabolismo , Domínios Proteicos , Receptores Virais/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Transdução de Sinais , Células THP-1RESUMO
Peroxisomes are metabolic organelles that perform a diverse array of critical functions in human physiology. Traditional isolation methods for peroxisomes can take more than 1 h to complete and can be laborious to implement. To address this, we have now extended our prior work on rapid organellar isolation to peroxisomes via the development of a peroxisomally localized 3XHA epitope tag ("PEROXO-Tag") and associated immunoprecipitation ("PEROXO-IP") workflow. Our PEROXO-IP workflow has excellent reproducibility, is easy to implement, and achieves highly rapid (~10 min post homogenization) and specific isolation of human peroxisomes, which we characterize here via proteomic profiling. By offering speed, specificity, reproducibility, and ease of use, the PEROXO-IP workflow should facilitate studies on the biology of peroxisomes.
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Inherited disorders of platelet function (IPFD) and/or number (IPND) are heterogeneous conditions that result in variable mucocutaneous bleeding symptoms as a result of deranged primary haemostasis caused by platelet dysfunction or thrombocytopenia. Diagnosis is important to guide post-operative bleeding prophylactic strategies, to avoid treatment with inappropriate medications, and inform prognosis. Achieving an accurate diagnosis has traditionally been hampered by the requirement of multiple, often complex, laboratory tests that are not always available at single centres. To improve the diagnosis of these disorders a research collaborative was established, the Sydney Platelet Group, that explored an integrated approach combining traditional and contemporary platelet phenotypic and genetic diagnostic platforms available at four Sydney tertiary hospitals. Herein we report the outcomes of the first 50 patients evaluated using this approach. The cohort included 22 individuals with suspected IPFD and 28 with thrombocytopenia. Bleeding scores were higher in individuals with IPFD (mean 5.75; SD 4.83) than those with IPNDs (mean 2.14; SD 2.45). In cases with suspected IPFD, diagnosis to the level of the defective pathway was achieved in 71% and four individuals were found not to have a definitive platelet function defect. Dense granule secretion disorders were the most common platelet pathway abnormality detected (n=5). Mean bleeding scores in these individuals were not significantly different to individuals with defects in other commonly detected platelet pathways (dense granules, signal transduction and 'undetermined'). A molecular diagnosis was achieved in 52% of individuals with IPNDs and 5% with IPFD. Likely pathogenic and pathogenic variants detected included variants associated with extra-haematological complications (DIAPH1, MYH9) and potential for malignancy (ANKRD26 and RUNX1). The level of platelet investigation undertaken by this initiative is currently not available elsewhere in Australia and initial results confirm the utility of this integrated phenotypic-genetic approach.
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Transtornos Plaquetários/diagnóstico , Testes de Função Plaquetária/métodos , Adolescente , Adulto , Idoso , Austrália , Transtornos Plaquetários/genética , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
BACKGROUND: Recently, new evidence from large scale trials and updated guidelines have emerged on the risks and benefits of extended dual antiplatelet therapy (DAPT) for patients with acute coronary syndrome (ACS). AIMS: To discuss, clarify and advise on the application of the evidences and guidelines on individual patient selection for extended DAPT, with regard to balancing risk factors, particularly in Asian populations. METHODS: A total of 14 local cardiologists from Hong Kong with extensive experience in cardiology and cardiac interventions convened in a series of 3 advisory board meetings from October 2016 to September 2017, which included reviews of new evidence in the literature and discussions of the latest clinical trends, using an anonymous, electronic voting system for arriving at consensuses. RESULTS: Recommendations were produced for the following nine risk factors: old age (>65), chronic kidney disease (CKD), diabetes mellitus (DM), recurrent myocardial infarction (MI), multi-vessel disease (MVD), multiple stents, bioresorbable vascular scaffold (BVS) stent, left main stenting and peripheral artery disease (PAD). Strong ischaemic risk factors include DM, recurrent MI, MVD and/or >3 stents; less-strong ischaemic factors include CKD, left main stenting, BVS stent and PAD. Old age can be an unclear risk factor due to variations in physical fitness even among patients of identical age. DISCUSSION: The strengths and limitations of the results were acknowledged. CONCLUSION: ACS patients with ischaemic risk factors could be considered for extended DAPT beyond 12 months, while balancing the risk of bleeding.
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Síndrome Coronariana Aguda/terapia , Stents Farmacológicos/efeitos adversos , Inibidores da Agregação Plaquetária/administração & dosagem , Guias de Prática Clínica como Assunto , Síndrome Coronariana Aguda/diagnóstico , Comitês Consultivos , Esquema de Medicação , Quimioterapia Combinada , Hemorragia/induzido quimicamente , Hemorragia/prevenção & controle , Hong Kong , Humanos , Infarto do Miocárdio/etiologia , Inibidores da Agregação Plaquetária/efeitos adversos , Antagonistas do Receptor Purinérgico P2Y/administração & dosagem , Antagonistas do Receptor Purinérgico P2Y/efeitos adversos , Fatores de Risco , Trombose/etiologia , Trombose/prevenção & controleRESUMO
Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.
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Epitopos/imunologia , Mitocôndrias/imunologia , Animais , Hepatócitos/metabolismo , Immunoblotting , Lipídeos/fisiologia , Masculino , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/química , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Mitocôndrias Hepáticas/química , Mitocôndrias Hepáticas/imunologia , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/fisiologia , Proteômica/métodosRESUMO
The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth.
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Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos Essenciais/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sequência de Aminoácidos , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/genética , Animais , Arginina/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Alinhamento de SequênciaRESUMO
The genetic dependencies of human cancers widely vary. Here, we catalog this heterogeneity and use it to identify functional gene interactions and genotype-dependent liabilities in cancer. By using genome-wide CRISPR-based screens, we generate a gene essentiality dataset across 14 human acute myeloid leukemia (AML) cell lines. Sets of genes with correlated patterns of essentiality across the lines reveal new gene relationships, the essential substrates of enzymes, and the molecular functions of uncharacterized proteins. Comparisons of differentially essential genes between Ras-dependent and -independent lines uncover synthetic lethal partners of oncogenic Ras. Screens in both human AML and engineered mouse pro-B cells converge on a surprisingly small number of genes in the Ras processing and MAPK pathways and pinpoint PREX1 as an AML-specific activator of MAPK signaling. Our findings suggest general strategies for defining mammalian gene networks and synthetic lethal interactions by exploiting the natural genetic and epigenetic diversity of human cancer cells.
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Redes Reguladoras de Genes , Leucemia Mieloide Aguda/genética , Animais , Proteínas de Transporte , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Epigênese Genética , Genes Essenciais , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Proteínas Mitocondriais , Processamento de Proteína Pós-Traducional , Proteínas ras/genéticaRESUMO
Mitochondria carry out numerous metabolic reactions that are critical to cellular homeostasis. Here we present a protocol for interrogating mitochondrial metabolites and measuring their matrix concentrations. Our workflow uses high-affinity magnetic immunocapture to rapidly purify HA-tagged mitochondria from homogenized mammalian cells in â¼12 min. These mitochondria are extracted with methanol and water. Liquid chromatography and mass spectrometry (LC/MS) is used to determine the identities and mole quantities of mitochondrial metabolites using authentic metabolite standards and isotopically labeled internal standards, whereas the corresponding mitochondrial matrix volume is determined via immunoblotting, confocal microscopy of intact cells, and volumetric analysis. Once all values have been obtained, the matrix volume is combined with the aforementioned mole quantities to calculate the matrix concentrations of mitochondrial metabolites. With shortened isolation times and improved mitochondrial purity when compared with alternative methods, this LC/MS-compatible workflow allows for robust profiling of mitochondrial metabolites and serves as a strategy generalizable to the study of other mammalian organelles. Once all the necessary reagents have been prepared, quantifying the matrix concentrations of mitochondrial metabolites can be accomplished within a week.
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Separação Imunomagnética/métodos , Metaboloma/fisiologia , Metabolômica/métodos , Mitocôndrias/química , Mitocôndrias/metabolismo , Cromatografia Líquida , Técnicas Citológicas , Células HeLa , Humanos , Espectrometria de MassasRESUMO
Mitochondria house metabolic pathways that impact most aspects of cellular physiology. While metabolite profiling by mass spectrometry is widely applied at the whole-cell level, it is not routinely possible to measure the concentrations of small molecules in mammalian organelles. We describe a method for the rapid and specific isolation of mitochondria and use it in tandem with a database of predicted mitochondrial metabolites ("MITObolome") to measure the matrix concentrations of more than 100 metabolites across various states of respiratory chain (RC) function. Disruption of the RC reveals extensive compartmentalization of mitochondrial metabolism and signatures unique to the inhibition of each RC complex. Pyruvate enables the proliferation of RC-deficient cells but has surprisingly limited effects on matrix contents. Interestingly, despite failing to restore matrix NADH/NAD balance, pyruvate does increase aspartate, likely through the exchange of matrix glutamate for cytosolic aspartate. We demonstrate the value of mitochondrial metabolite profiling and describe a strategy applicable to other organelles.