RESUMO
Microglia maintain homeostasis in the brain, but whether aberrant microglial activation can cause neurodegeneration remains controversial. Here, we use transcriptome profiling to demonstrate that deficiency in frontotemporal dementia (FTD) gene progranulin (Grn) leads to an age-dependent, progressive upregulation of lysosomal and innate immunity genes, increased complement production, and enhanced synaptic pruning in microglia. During aging, Grn(-/-) mice show profound microglia infiltration and preferential elimination of inhibitory synapses in the ventral thalamus, which lead to hyperexcitability in the thalamocortical circuits and obsessive-compulsive disorder (OCD)-like grooming behaviors. Remarkably, deleting C1qa gene significantly reduces synaptic pruning by Grn(-/-) microglia and mitigates neurodegeneration, behavioral phenotypes, and premature mortality in Grn(-/-) mice. Together, our results uncover a previously unrecognized role of progranulin in suppressing aberrant microglia activation during aging. These results represent an important conceptual advance that complement activation and microglia-mediated synaptic pruning are major drivers, rather than consequences, of neurodegeneration caused by progranulin deficiency.
Assuntos
Envelhecimento/metabolismo , Encéfalo/metabolismo , Ativação do Complemento , Complemento C1q/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Microglia/metabolismo , Envelhecimento/imunologia , Animais , Líquido Cefalorraquidiano , Complemento C1q/genética , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Granulinas , Humanos , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/genética , Lisossomos/metabolismo , Redes e Vias Metabólicas , Camundongos , Transtorno Obsessivo-Compulsivo/genética , Transtorno Obsessivo-Compulsivo/metabolismo , Progranulinas , Sinapses/metabolismo , Tálamo/metabolismoRESUMO
Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids for energy or membrane synthesis and act as hubs for metabolic processes. Cells generate LDs de novo, converting cells to emulsions with LDs constituting the dispersed oil phase in the aqueous cytoplasm. Here we review our current view of LD biogenesis. We present a model of LD formation from the ER in distinct steps and highlight the biology of proteins that govern this biophysical process. Areas of incomplete knowledge are identified, as are connections with physiology and diseases linked to alterations in LD biology.
Assuntos
Gotículas Lipídicas/metabolismo , Animais , Fenômenos Biofísicos , Humanos , Modelos Biológicos , Proteínas/metabolismo , Triglicerídeos/metabolismoRESUMO
The amino acid cysteine and its oxidized dimeric form cystine are commonly believed to be synonymous in metabolic functions. Cyst(e)ine depletion not only induces amino acid response but also triggers ferroptosis, a non-apoptotic cell death. Here, we report that unlike general amino acid starvation, cyst(e)ine deprivation triggers ATF4 induction at the transcriptional level. Unexpectedly, it is the shortage of lysosomal cystine, but not the cytosolic cysteine, that elicits the adaptative ATF4 response. The lysosome-nucleus signaling pathway involves the aryl hydrocarbon receptor (AhR) that senses lysosomal cystine via the kynurenine pathway. A blockade of lysosomal cystine efflux attenuates ATF4 induction and sensitizes ferroptosis. To potentiate ferroptosis in cancer, we develop a synthetic mRNA reagent, CysRx, that converts cytosolic cysteine to lysosomal cystine. CysRx maximizes cancer cell ferroptosis and effectively suppresses tumor growth in vivo. Thus, intracellular nutrient reprogramming has the potential to induce selective ferroptosis in cancer without systematic starvation.
Assuntos
Cistos , Ferroptose , Humanos , Cisteína , Cistina , Ferroptose/genética , Aminoácidos , LisossomosRESUMO
Mitochondria are highly dynamic organelles that continuously grow, divide, and fuse. The division of mitochondria is crucial for human health. During mitochondrial division, the mechano-guanosine triphosphatase (GTPase) dynamin-related protein (Drp1) severs mitochondria at endoplasmic reticulum (ER)-mitochondria contact sites, where peripheral ER tubules interact with mitochondria. Here, we report that Drp1 directly shapes peripheral ER tubules in human and mouse cells. This ER-shaping activity is independent of GTP hydrolysis and located in a highly conserved peptide of 18 amino acids (termed D-octadecapeptide), which is predicted to form an amphipathic α helix. Synthetic D-octadecapeptide tubulates liposomes in vitro and the ER in cells. ER tubules formed by Drp1 promote mitochondrial division by facilitating ER-mitochondria interactions. Thus, Drp1 functions as a two-in-one protein during mitochondrial division, with ER tubulation and mechano-GTPase activities.
Assuntos
Dinaminas/metabolismo , Dinaminas/fisiologia , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Mitocôndrias/metabolismo , Animais , Dinaminas/genética , Retículo Endoplasmático/efeitos dos fármacos , GTP Fosfo-Hidrolases/genética , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial , Oligopeptídeos/farmacologiaRESUMO
Lipid droplets (LDs) store lipids for energy and are central to cellular lipid homeostasis. The mechanisms coordinating lipid storage in LDs with cellular metabolism are unclear but relevant to obesity-related diseases. Here we utilized genome-wide screening to identify genes that modulate lipid storage in macrophages, a cell type involved in metabolic diseases. Among â¼550 identified screen hits is MLX, a basic helix-loop-helix leucine-zipper transcription factor that regulates metabolic processes. We show that MLX and glucose-sensing family members MLXIP/MondoA and MLXIPL/ChREBP bind LDs via C-terminal amphipathic helices. When LDs accumulate in cells, these transcription factors bind to LDs, reducing their availability for transcriptional activity and attenuating the response to glucose. Conversely, the absence of LDs results in hyperactivation of MLX target genes. Our findings uncover a paradigm for a lipid storage response in which binding of MLX transcription factors to LD surfaces adjusts the expression of metabolic genes to lipid storage levels.
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Regulação da Expressão Gênica , Glucose/metabolismo , Gotículas Lipídicas/metabolismo , Proteoma/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/antagonistas & inibidores , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células Cultivadas , Testes Genéticos , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Ligação Proteica , Proteoma/análise , RNA Interferente Pequeno , Transcrição GênicaRESUMO
Among organelles, lipid droplets (LDs) uniquely constitute a hydrophobic phase in the aqueous environment of the cytosol. Their hydrophobic core of neutral lipids stores metabolic energy and membrane components, making LDs hubs for lipid metabolism. In addition, LDs are implicated in a number of other cellular functions, ranging from protein storage and degradation to viral replication. These processes are functionally linked to many physiological and pathological conditions, including obesity and related metabolic diseases. Despite their important functions and nearly ubiquitous presence in cells, many aspects of LD biology are unknown. In the past few years, the pace of LD investigation has increased, providing new insights. Here, we review the current knowledge of LD cell biology and its translation to physiology.
Assuntos
Células/metabolismo , Metabolismo dos Lipídeos , Lipídeos/química , Animais , Células/química , Humanos , Lipólise , Doenças Metabólicas/metabolismo , Obesidade/metabolismo , Organelas/metabolismoRESUMO
BACKGROUND: Studies evaluating surgical-site infection have had conflicting results with respect to the use of alcohol solutions containing iodine povacrylex or chlorhexidine gluconate as skin antisepsis before surgery to repair a fractured limb (i.e., an extremity fracture). METHODS: In a cluster-randomized, crossover trial at 25 hospitals in the United States and Canada, we randomly assigned hospitals to use a solution of 0.7% iodine povacrylex in 74% isopropyl alcohol (iodine group) or 2% chlorhexidine gluconate in 70% isopropyl alcohol (chlorhexidine group) as preoperative antisepsis for surgical procedures to repair extremity fractures. Every 2 months, the hospitals alternated interventions. Separate populations of patients with either open or closed fractures were enrolled and included in the analysis. The primary outcome was surgical-site infection, which included superficial incisional infection within 30 days or deep incisional or organ-space infection within 90 days. The secondary outcome was unplanned reoperation for fracture-healing complications. RESULTS: A total of 6785 patients with a closed fracture and 1700 patients with an open fracture were included in the trial. In the closed-fracture population, surgical-site infection occurred in 77 patients (2.4%) in the iodine group and in 108 patients (3.3%) in the chlorhexidine group (odds ratio, 0.74; 95% confidence interval [CI], 0.55 to 1.00; P = 0.049). In the open-fracture population, surgical-site infection occurred in 54 patients (6.5%) in the iodine group and in 60 patients (7.3%) in the chlorhexidine group (odd ratio, 0.86; 95% CI, 0.58 to 1.27; P = 0.45). The frequencies of unplanned reoperation, 1-year outcomes, and serious adverse events were similar in the two groups. CONCLUSIONS: Among patients with closed extremity fractures, skin antisepsis with iodine povacrylex in alcohol resulted in fewer surgical-site infections than antisepsis with chlorhexidine gluconate in alcohol. In patients with open fractures, the results were similar in the two groups. (Funded by the Patient-Centered Outcomes Research Institute and the Canadian Institutes of Health Research; PREPARE ClinicalTrials.gov number, NCT03523962.).
Assuntos
Anti-Infecciosos Locais , Clorexidina , Fixação de Fratura , Fraturas Ósseas , Iodo , Infecção da Ferida Cirúrgica , Humanos , 2-Propanol/administração & dosagem , 2-Propanol/efeitos adversos , 2-Propanol/uso terapêutico , Anti-Infecciosos Locais/administração & dosagem , Anti-Infecciosos Locais/efeitos adversos , Anti-Infecciosos Locais/uso terapêutico , Antissepsia/métodos , Canadá , Clorexidina/administração & dosagem , Clorexidina/efeitos adversos , Clorexidina/uso terapêutico , Etanol , Extremidades/lesões , Extremidades/microbiologia , Extremidades/cirurgia , Iodo/administração & dosagem , Iodo/efeitos adversos , Iodo/uso terapêutico , Cuidados Pré-Operatórios/efeitos adversos , Cuidados Pré-Operatórios/métodos , Pele/microbiologia , Infecção da Ferida Cirúrgica/etiologia , Infecção da Ferida Cirúrgica/prevenção & controle , Fraturas Ósseas/cirurgia , Estudos Cross-Over , Estados UnidosRESUMO
BACKGROUND: Phase 1-2 trials involving patients with resectable, macroscopic stage III melanoma have shown that neoadjuvant immunotherapy is more efficacious than adjuvant immunotherapy. METHODS: In this phase 3 trial, we randomly assigned patients with resectable, macroscopic stage III melanoma, in a 1:1 ratio, to receive two cycles of neoadjuvant ipilimumab plus nivolumab and then undergo surgery or to undergo surgery and then receive 12 cycles of adjuvant nivolumab. Only the patients in the neoadjuvant group who had a partial response or nonresponse received subsequent adjuvant treatment. The primary end point was event-free survival. RESULTS: A total of 423 patients underwent randomization. At a median follow-up of 9.9 months, the estimated 12-month event-free survival was 83.7% (99.9% confidence interval [CI], 73.8 to 94.8) in the neoadjuvant group and 57.2% (99.9% CI, 45.1 to 72.7) in the adjuvant group. The difference in restricted mean survival time was 8.00 months (99.9% CI, 4.94 to 11.05; P<0.001; hazard ratio for progression, recurrence, or death, 0.32; 99.9% CI, 0.15 to 0.66). In the neoadjuvant group, 59.0% of the patients had a major pathological response, 8.0% had a partial response, 26.4% had a nonresponse (>50% residual viable tumor), and 2.4% had progression; in 4.2%, surgery had not yet been performed or was omitted. The estimated 12-month recurrence-free survival was 95.1% among patients in the neoadjuvant group who had a major pathological response, 76.1% among those who had a partial response, and 57.0% among those who had a nonresponse. Adverse events of grade 3 or higher that were related to systemic treatment occurred in 29.7% of the patients in the neoadjuvant group and in 14.7% in the adjuvant group. CONCLUSIONS: Among patients with resectable, macroscopic stage III melanoma, neoadjuvant ipilimumab plus nivolumab followed by surgery and response-driven adjuvant therapy resulted in longer event-free survival than surgery followed by adjuvant nivolumab. (Funded by Bristol Myers Squibb and others; NADINA ClinicalTrials.gov number, NCT04949113.).
RESUMO
Hundreds of effector proteins of the human malaria parasite Plasmodium falciparum constitute a "secretome" carrying a host-targeting (HT) signal, which predicts their export from the intracellular pathogen into the surrounding erythrocyte. Cleavage of the HT signal by a parasite endoplasmic reticulum (ER) protease, plasmepsin V, is the proposed export mechanism. Here, we show that the HT signal facilitates export by recognition of the lipid phosphatidylinositol-3-phosphate (PI(3)P) in the ER, prior to and independent of protease action. Secretome HT signals, including those of major virulence determinants, bind PI(3)P with nanomolar affinity and amino acid specificities displayed by HT-mediated export. PI(3)P-enriched regions are detected within the parasite's ER and colocalize with endogenous HT signal on ER precursors, which also display high-affinity binding to PI(3)P. A related pathogenic oomycete's HT signal export is dependent on PI(3)P binding, without cleavage by plasmepsin V. Thus, PI(3)P in the ER functions in mechanisms of secretion and pathogenesis.
Assuntos
Eritrócitos/parasitologia , Malária Falciparum/parasitologia , Fosfatos de Fosfatidilinositol/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Antígenos de Protozoários/química , Antígenos de Protozoários/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Eritrócitos/metabolismo , Humanos , Malária Falciparum/patologia , Dados de Sequência Molecular , Plasmodium falciparum/citologia , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas de Protozoários/químicaRESUMO
Understanding cellular architecture is essential for understanding biology. Electron microscopy (EM) uniquely visualizes cellular structures with nanometre resolution. However, traditional methods, such as thin-section EM or EM tomography, have limitations in that they visualize only a single slice or a relatively small volume of the cell, respectively. Focused ion beam-scanning electron microscopy (FIB-SEM) has demonstrated the ability to image small volumes of cellular samples with 4-nm isotropic voxels1. Owing to advances in the precision and stability of FIB milling, together with enhanced signal detection and faster SEM scanning, we have increased the volume that can be imaged with 4-nm voxels by two orders of magnitude. Here we present a volume EM atlas at such resolution comprising ten three-dimensional datasets for whole cells and tissues, including cancer cells, immune cells, mouse pancreatic islets and Drosophila neural tissues. These open access data (via OpenOrganelle2) represent the foundation of a field of high-resolution whole-cell volume EM and subsequent analyses, and we invite researchers to explore this atlas and pose questions.
Assuntos
Conjuntos de Dados como Assunto , Disseminação de Informação , Microscopia Eletrônica de Varredura , Organelas/ultraestrutura , Animais , Linhagem Celular , Células Cultivadas , Drosophila melanogaster/citologia , Drosophila melanogaster/ultraestrutura , Feminino , Complexo de Golgi/ultraestrutura , Humanos , Interfase , Ilhotas Pancreáticas/citologia , Masculino , Camundongos , Microscopia Eletrônica de Varredura/métodos , Microscopia Eletrônica de Varredura/normas , Microtúbulos/ultraestrutura , Neuroglia/ultraestrutura , Neurônios/ultraestrutura , Publicação de Acesso Aberto , Neoplasias Ovarianas/imunologia , Neoplasias Ovarianas/ultraestrutura , Ribossomos/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/ultraestruturaRESUMO
Excessive levels of saturated fatty acids are toxic to cells, although the basis for this lipotoxicity remains incompletely understood. Here, we analyzed the transcriptome, lipidome, and genetic interactions of human leukemia cells exposed to palmitate. Palmitate treatment increased saturated glycerolipids, accompanied by a transcriptional stress response, including upregulation of the endoplasmic reticulum (ER) stress response. A comprehensive genome-wide short hairpin RNA (shRNA) screen identified >350 genes modulating lipotoxicity. Among previously unknown genetic modifiers of lipotoxicity, depletion of RNF213, a putative ubiquitin ligase mutated in Moyamoya vascular disease, protected cells from lipotoxicity. On a broader level, integration of our comprehensive datasets revealed that changes in di-saturated glycerolipids, but not other lipid classes, are central to lipotoxicity in this model. Consistent with this, inhibition of ER-localized glycerol-3-phosphate acyltransferase activity protected from all aspects of lipotoxicity. Identification of genes modulating the response to saturated fatty acids may reveal novel therapeutic strategies for treating metabolic diseases linked to lipotoxicity.
Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/efeitos dos fármacos , Glicerídeos/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Ácido Palmítico/toxicidade , Aciltransferases/genética , Aciltransferases/metabolismo , Adenosina Trifosfatases/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático/genética , Regulação Enzimológica da Expressão Gênica , Células HeLa , Células Hep G2 , Humanos , Células K562 , Metabolismo dos Lipídeos/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Transcriptoma , Ubiquitina-Proteína Ligases/metabolismoRESUMO
In Parkinson's disease (PD), α-synuclein (αS) pathologically impacts the brain, a highly lipid-rich organ. We investigated how alterations in αS or lipid/fatty acid homeostasis affect each other. Lipidomic profiling of human αS-expressing yeast revealed increases in oleic acid (OA, 18:1), diglycerides, and triglycerides. These findings were recapitulated in rodent and human neuronal models of αS dyshomeostasis (overexpression; patient-derived triplication or E46K mutation; E46K mice). Preventing lipid droplet formation or augmenting OA increased αS yeast toxicity; suppressing the OA-generating enzyme stearoyl-CoA-desaturase (SCD) was protective. Genetic or pharmacological SCD inhibition ameliorated toxicity in αS-overexpressing rat neurons. In a C. elegans model, SCD knockout prevented αS-induced dopaminergic degeneration. Conversely, we observed detrimental effects of OA on αS homeostasis: in human neural cells, excess OA caused αS inclusion formation, which was reversed by SCD inhibition. Thus, monounsaturated fatty acid metabolism is pivotal for αS-induced neurotoxicity, and inhibiting SCD represents a novel PD therapeutic approach.
Assuntos
Antiparkinsonianos/farmacologia , Descoberta de Drogas/métodos , Inibidores Enzimáticos/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolômica/métodos , Neurônios/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Estearoil-CoA Dessaturase/antagonistas & inibidores , alfa-Sinucleína/toxicidade , Animais , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/enzimologia , Caenorhabditis elegans/genética , Linhagem Celular , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/patologia , Diglicerídeos/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/enzimologia , Neurônios Dopaminérgicos/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/patologia , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/enzimologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Terapia de Alvo Molecular , Degeneração Neural , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/patologia , Neurônios/enzimologia , Neurônios/patologia , Ácido Oleico/metabolismo , Doença de Parkinson/enzimologia , Doença de Parkinson/genética , Doença de Parkinson/patologia , Ratos Sprague-Dawley , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Estearoil-CoA Dessaturase/metabolismo , Triglicerídeos/metabolismo , alfa-Sinucleína/genéticaRESUMO
The enzyme UDP-glucose: glycoprotein glucosyltransferase (UGGT) is the gatekeeper of protein folding within the endoplasmic reticulum (ER). One-third of the human proteome traverses the ER where folding and maturation are facilitated by a complex protein homeostasis network. Both glycan modifications and disulfide bonds are of key importance in the maturation of these ER proteins. The actions of UGGT are intimately linked to the glycan code for folding and maturation of secretory proteins in the ER. UGGT selectively glucosylates the N-linked glycan of misfolded proteins so that they can reenter the lectin-folding chaperone cycle and be retained within the ER for further attempts at folding. An intriguing aspect of UGGT function is its interaction with its poorly understood cochaperone, the 15 kDa selenoprotein known as SELENOF or SEP15. This small protein contains a rare selenocysteine residue proposed to act as an oxidoreductase toward UGGT substrates. AlphaFold2 predictions of the UGGT1/SEP15 complex provide insight into this complex at a structural level. The predicted UGGT1/SEP15 interaction interface was validated by mutagenesis and coimmunoprecipitation experiments. These results serve as a springboard for models of the integrated action of UGGT1 and SEP15.
Assuntos
Retículo Endoplasmático , Glucosiltransferases , Dobramento de Proteína , Selenoproteínas , Selenoproteínas/metabolismo , Selenoproteínas/genética , Retículo Endoplasmático/metabolismo , Humanos , Glucosiltransferases/metabolismo , Glucosiltransferases/genética , Ligação ProteicaRESUMO
Lipid droplets (LDs) are the main organelles for lipid storage, and their surfaces contain unique proteins with diverse functions, including those that facilitate the deposition and mobilization of LD lipids. Among organelles, LDs have an unusual structure with an organic, hydrophobic oil phase covered by a phospholipid monolayer. The unique properties of LD monolayer surfaces require proteins to localize to LDs by distinct mechanisms. Here we review the two pathways known to mediate direct LD protein localization: the CYTOLD pathway mediates protein targeting from the cytosol toLDs, and the ERTOLD pathway functions in protein targeting from the endoplasmic reticulum toLDs. We describe the emerging principles for each targeting pathway in animal cells and highlight open questions in the field.
Assuntos
Retículo Endoplasmático , Gotículas Lipídicas , Animais , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Transporte Proteico , Proteínas/metabolismoRESUMO
Maturation of membrane proteins is complicated by the need to fold in three distinct environments. While much is known about folding in the two aqueous milieus constituted by cytoplasm and ER lumen, our knowledge of the folding, arrangement, and quality control of transmembrane regions within the lipid bilayer, and its facilitation by molecular chaperones, is limited. New work by Bloemeke et al now reveals an expanded role of the ER chaperone calnexin acting within the lipid bilayer in a carbohydrate-independent manner.
Assuntos
Bicamadas Lipídicas , Paladar , Calnexina/metabolismo , Dobramento de Proteína , Chaperonas Moleculares/metabolismo , Carboidratos , Proteínas de Ligação ao Cálcio/metabolismoRESUMO
BACKGROUND: Clinical guidelines recommend low-molecular-weight heparin for thromboprophylaxis in patients with fractures, but trials of its effectiveness as compared with aspirin are lacking. METHODS: In this pragmatic, multicenter, randomized, noninferiority trial, we enrolled patients 18 years of age or older who had a fracture of an extremity (anywhere from hip to midfoot or shoulder to wrist) that had been treated operatively or who had any pelvic or acetabular fracture. Patients were randomly assigned to receive low-molecular-weight heparin (enoxaparin) at a dose of 30 mg twice daily or aspirin at a dose of 81 mg twice daily while they were in the hospital. After hospital discharge, the patients continued to receive thromboprophylaxis according to the clinical protocols of each hospital. The primary outcome was death from any cause at 90 days. Secondary outcomes were nonfatal pulmonary embolism, deep-vein thrombosis, and bleeding complications. RESULTS: A total of 12,211 patients were randomly assigned to receive aspirin (6101 patients) or low-molecular-weight heparin (6110 patients). Patients had a mean (±SD) age of 44.6±17.8 years, 0.7% had a history of venous thromboembolism, and 2.5% had a history of cancer. Patients received a mean of 8.8±10.6 in-hospital thromboprophylaxis doses and were prescribed a median 21-day supply of thromboprophylaxis at discharge. Death occurred in 47 patients (0.78%) in the aspirin group and in 45 patients (0.73%) in the low-molecular-weight-heparin group (difference, 0.05 percentage points; 96.2% confidence interval, -0.27 to 0.38; P<0.001 for a noninferiority margin of 0.75 percentage points). Deep-vein thrombosis occurred in 2.51% of patients in the aspirin group and 1.71% in the low-molecular-weight-heparin group (difference, 0.80 percentage points; 95% CI, 0.28 to 1.31). The incidence of pulmonary embolism (1.49% in each group), bleeding complications, and other serious adverse events were similar in the two groups. CONCLUSIONS: In patients with extremity fractures that had been treated operatively or with any pelvic or acetabular fracture, thromboprophylaxis with aspirin was noninferior to low-molecular-weight heparin in preventing death and was associated with low incidences of deep-vein thrombosis and pulmonary embolism and low 90-day mortality. (Funded by the Patient-Centered Outcomes Research Institute; PREVENT CLOT ClinicalTrials.gov number, NCT02984384.).
Assuntos
Anticoagulantes , Aspirina , Quimioprevenção , Fraturas Ósseas , Heparina de Baixo Peso Molecular , Adulto , Humanos , Pessoa de Meia-Idade , Anticoagulantes/efeitos adversos , Anticoagulantes/uso terapêutico , Aspirina/efeitos adversos , Aspirina/uso terapêutico , Quimioprevenção/métodos , Extremidades/lesões , Fraturas Ósseas/complicações , Fraturas Ósseas/mortalidade , Hemorragia/etiologia , Heparina de Baixo Peso Molecular/efeitos adversos , Heparina de Baixo Peso Molecular/uso terapêutico , Fraturas do Quadril/complicações , Fraturas do Quadril/mortalidade , Ossos Pélvicos/lesões , Ensaios Clínicos Pragmáticos como Assunto , Embolia Pulmonar/etiologia , Embolia Pulmonar/prevenção & controle , Fraturas da Coluna Vertebral/complicações , Fraturas da Coluna Vertebral/mortalidade , Tromboembolia Venosa/etiologia , Tromboembolia Venosa/prevenção & controle , Tromboembolia Venosa/tratamento farmacológico , Trombose Venosa/etiologia , Trombose Venosa/prevenção & controleRESUMO
Triacylglycerols store metabolic energy in organisms and have industrial uses as foods and fuels. Excessive accumulation of triacylglycerols in humans causes obesity and is associated with metabolic diseases1. Triacylglycerol synthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes2-4, the structures and catalytic mechanisms of which remain unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) family, by cryo-electron microscopy at approximately 3.0 Å resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at approximately 3.2 Å shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction centre is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density-possibly representing an acyl-acceptor molecule-is located within the reaction centre, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, provide the basis for a model of the catalysis of triacylglycerol synthesis by DGAT.
Assuntos
Biocatálise , Microscopia Crioeletrônica , Diacilglicerol O-Aciltransferase/metabolismo , Diacilglicerol O-Aciltransferase/ultraestrutura , Triglicerídeos/biossíntese , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Acil Coenzima A/ultraestrutura , Aciltransferases/química , Aciltransferases/metabolismo , Domínio Catalítico , Membrana Celular/química , Membrana Celular/metabolismo , Diacilglicerol O-Aciltransferase/química , Histidina/química , Histidina/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Multimerização Proteica , Especificidade por SubstratoRESUMO
Aberrant aggregation of the RNA-binding protein TDP-43 in neurons is a hallmark of frontotemporal lobar degeneration caused by haploinsufficiency in the gene encoding progranulin1,2. However, the mechanism leading to TDP-43 proteinopathy remains unclear. Here we use single-nucleus RNA sequencing to show that progranulin deficiency promotes microglial transition from a homeostatic to a disease-specific state that causes endolysosomal dysfunction and neurodegeneration in mice. These defects persist even when Grn-/- microglia are cultured ex vivo. In addition, single-nucleus RNA sequencing reveals selective loss of excitatory neurons at disease end-stage, which is characterized by prominent nuclear and cytoplasmic TDP-43 granules and nuclear pore defects. Remarkably, conditioned media from Grn-/- microglia are sufficient to promote TDP-43 granule formation, nuclear pore defects and cell death in excitatory neurons via the complement activation pathway. Consistent with these results, deletion of the genes encoding C1qa and C3 mitigates microglial toxicity and rescues TDP-43 proteinopathy and neurodegeneration. These results uncover previously unappreciated contributions of chronic microglial toxicity to TDP-43 proteinopathy during neurodegeneration.
Assuntos
Microglia/metabolismo , Microglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Progranulinas/deficiência , Proteinopatias TDP-43/metabolismo , Proteinopatias TDP-43/patologia , Envelhecimento/genética , Envelhecimento/patologia , Animais , Núcleo Celular/genética , Núcleo Celular/patologia , Ativação do Complemento/efeitos dos fármacos , Ativação do Complemento/imunologia , Complemento C1q/antagonistas & inibidores , Complemento C1q/imunologia , Complemento C3b/antagonistas & inibidores , Complemento C3b/imunologia , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Poro Nuclear/metabolismo , Poro Nuclear/patologia , Progranulinas/genética , RNA-Seq , Análise de Célula Única , Proteinopatias TDP-43/tratamento farmacológico , Proteinopatias TDP-43/genética , Tálamo/metabolismo , Tálamo/patologia , TranscriptomaRESUMO
Artificial cells are biomimetic structures formed from molecular building blocks that replicate biological processes, behaviors, and architectures. Of these building blocks, hydrogels have emerged as ideal, yet underutilized candidates to provide a gel-like chassis in which to incorporate both biological and nonbiological componentry which enables the replication of cellular functionality. Here, we demonstrate a microfluidic strategy to assemble biocompatible cell-sized hydrogel-based artificial cells with a variety of different embedded functional subcompartments, which act as engineered synthetic organelles. The organelles enable the recreation of increasingly biomimetic behaviors, including stimulus-induced motility, content release through activation of membrane-associated proteins, and enzymatic communication with surrounding bioinspired compartments. In this way, we showcase a foundational strategy for the bottom-up construction of hydrogel-based artificial cell microsystems which replicate fundamental cellular behaviors, paving the way for the construction of next-generation biotechnological devices.
Assuntos
Células Artificiais , Biomimética , Hidrogéis , Comunicação , OrganelasRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a lipid-enveloped virus that acquires its lipid bilayer from the host cell it infects. SARS-CoV-2 can spread from cell to cell or from patient to patient by undergoing assembly and budding to form new virions. The assembly and budding of SARS-CoV-2 is mediated by several structural proteins known as envelope (E), membrane (M), nucleoprotein (N), and spike (S), which can form virus-like particles (VLPs) when co-expressed in mammalian cells. Assembly and budding of SARS-CoV-2 from the host ER-Golgi intermediate compartment is a critical step in the virus acquiring its lipid bilayer. To date, little information is available on how SARS-CoV-2 assembles and forms new viral particles from host membranes. In this study, we used several lipid binding assays and found the N protein can strongly associate with anionic lipids including phosphoinositides and phosphatidylserine. Moreover, we show lipid binding occurs in the N protein C-terminal domain, which is supported by extensive in silico analysis. We demonstrate anionic lipid binding occurs for both the free and the N oligomeric forms, suggesting N can associate with membranes in the nucleocapsid form. Based on these results, we present a lipid-dependent model based on in vitro, cellular, and in silico data for the recruitment of N to assembly sites in the lifecycle of SARS-CoV-2.