RESUMO
Acylglycerol kinase (AGK) is a mitochondrial lipid kinase that catalyzes the phosphorylation of monoacylglycerol and diacylglycerol to lysophosphatidic acid and phosphatidic acid, respectively. Mutations in AGK cause Sengers syndrome, which is characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy, exercise intolerance, and lactic acidosis. Here we identified AGK as a subunit of the mitochondrial TIM22 protein import complex. We show that AGK functions in a kinase-independent manner to maintain the integrity of the TIM22 complex, where it facilitates the import and assembly of mitochondrial carrier proteins. Mitochondria isolated from Sengers syndrome patient cells and tissues show a destabilized TIM22 complex and defects in the biogenesis of carrier substrates. Consistent with this phenotype, we observe perturbations in the tricarboxylic acid (TCA) cycle in cells lacking AGK. Our identification of AGK as a bona fide subunit of TIM22 provides an exciting and unexpected link between mitochondrial protein import and Sengers syndrome.
Assuntos
Cardiomiopatias/enzimologia , Catarata/enzimologia , Mitocôndrias/enzimologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Cardiomiopatias/genética , Catarata/genética , Ciclo do Ácido Cítrico , Predisposição Genética para Doença , Células HEK293 , Células HeLa , Humanos , Proteínas de Transporte da Membrana Mitocondrial/genética , Complexos Multiproteicos , Mutação , Fenótipo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Estabilidade Proteica , Transporte Proteico , TransfecçãoRESUMO
Epidemiological studies suggest that diabetes and obesity increases the risk of colorectal cancer (CRC) and lowers the patient survival rate. An important attribute in diabetes and obesity is the presence of high levels of growth factors including insulin in blood which can activate the PI3K/Akt signalling pathway. Dysregulation of PI3K/Akt signalling pathway leads to sustained proliferative signals thereby allowing the cells susceptible to cancer. Extracellular vesicles (EVs), secreted nanovesicles of endocytic origin, are implicated in mediating the transfer of oncogenic cargo in the tumour microenvironment. In this study, CRC cells were treated with insulin to activate PI3K/Akt signaling pathway. Insulin treatment significantly increased the number of EVs secreted by CRC cells. Furthermore, pAkt was exclusively packaged in EVs secreted by PI3K/Akt activated cells. Quantitative proteomics analysis confirmed that the protein cargo of EVs are modified upon activation of PI3K/Akt signaling pathway. Bioinformatics analysis highlighted the enrichment of proteins implicated in cell proliferation in EVs secreted by PI3K/Akt activated cells. Furthermore, incubation of EVs secreted by PI3K/Akt activated cells induced proliferation in recipient CRC cells. These findings suggest that EVs can amplify the signal provided by the growth factors in the tumor microenvironment and hence aid in cancer progression.
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Neoplasias Colorretais/metabolismo , Vesículas Extracelulares/metabolismo , Insulina/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteômica/métodos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Biologia Computacional/métodos , Vesículas Extracelulares/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Células Tumorais Cultivadas , Microambiente Tumoral/efeitos dos fármacosRESUMO
Cancer cells actively release extracellular vesicles, including exosomes, into the surrounding microenvironment. Exosomes play pleiotropic roles in cancer progression and metastasis, including invasion, angiogenesis, and immune modulation. However, the proteome profile of exosomes isolated from cells with different metastatic potential and the role of these exosomes in driving metastasis remains unclear. Here, we conduct a comparative proteomic analysis of exosomes isolated from several genetically related mouse breast tumor lines with different metastatic propensity. The amount of exosomes produced and the extent of cancer-associated protein cargo vary significantly between nonmetastatic and metastatic cell-derived exosomes. Metastatic cell-derived exosomes contain proteins that promote migration, proliferation, invasion, and angiogenesis while the nonmetastatic cell-derived exosomes contain proteins involved in cell-cell/cell-matrix adhesion and polarity maintenance. The metastatic exosomes contain a distinct set of membrane proteins including Ceruloplasmin and Metadherin which could presumably aid in targeting the primary cancer cells to specific metastatic sites. Hence, it can be concluded that the exosomes contain different protein cargo based on the host cells metastatic properties and can facilitate in the dissemination of the primary tumors to distant sites.
Assuntos
Exossomos/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Mamárias Animais/metabolismo , Proteoma/metabolismo , Proteômica/métodos , Animais , Neoplasias Ósseas/secundário , Adesão Celular , Linhagem da Célula , Movimento Celular , Proliferação de Células , Ceruloplasmina/metabolismo , Progressão da Doença , Feminino , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Animais/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Ligação a RNA , Células Tumorais CultivadasRESUMO
A genetic diagnosis of autosomal-dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short-read next-generation sequencing approaches, such as whole-genome sequencing and whole-exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long-read single-molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long-read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long-read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance.
Assuntos
Doenças Renais Policísticas/genética , Canais de Cátion TRPP/genética , Alelos , Estudos de Coortes , Biblioteca Gênica , Testes Genéticos , Genótipo , Humanos , Perda de Heterozigosidade , Rim Policístico Autossômico Dominante/genética , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Pseudogenes , Análise de Sequência de DNARESUMO
Extracellular vesicles (EVs) are signaling organelles that are released by many cell types and is highly conserved in both prokaryotes and eukaryotes. Based on the mechanism of biogenesis, these membranous vesicles can be classified as exosomes, shedding microvesicles, and apoptotic blebs. It is becoming clearer that these EVs mediate signal transduction in both autocrine and paracrine fashion by the transfer of proteins and RNA. While the role of EVs including exosomes in pathogenesis is well established, very little is known about their function in normal physiological conditions. Recent evidences allude that EVs can mediate both protective and pathogenic effects depending on the precise state. In this review, we discuss the involvement of EVs as mediators of signal transduction in neurodegenerative diseases and cancer. In addition, the role of EVs in mediating Wnt and PI3K signaling pathways is also discussed. Additional findings on the involvement of EVs in homeostasis and disease progression will promote a better biological understanding, advance future therapeutic, and diagnostic applications.
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Exossomos/metabolismo , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Transdução de Sinais , Animais , Exossomos/patologia , Humanos , Neoplasias/patologia , Doenças Neurodegenerativas/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Wnt/metabolismoRESUMO
A proteogenomic analysis is presented for Venturia pirina, a fungus that causes scab disease on European pear (Pyrus communis). V. pirina is host-specific, and the infection is thought to be mediated by secreted effector proteins. Currently, only 36 V. pirina proteins are catalogued in GenBank, and the genome sequence is not publicly available. To identify putative effectors, V. pirina was grown in vitro on and in cellophane sheets mimicking its growth in infected leaves. Secreted extracts were analyzed by tandem mass spectrometry, and the data (ProteomeXchange identifier PXD000710) was queried against a protein database generated by combining in silico predicted transcripts with six frame translations of a whole genome sequence of V. pirina (GenBank Accession JEMP00000000 ). We identified 1088 distinct V. pirina protein groups (FDR 1%) including 1085 detected for the first time. Thirty novel (not in silico predicted) proteins were found, of which 14 were identified as potential effectors based on characteristic features of fungal effector protein sequences. We also used evidence from semitryptic peptides at the protein N-terminus to corroborate in silico signal peptide predictions for 22 proteins, including several potential effectors. The analysis highlights the utility of proteogenomics in the study of secreted effectors.
Assuntos
Ascomicetos/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteoma/genética , Pyrus/microbiologia , Ascomicetos/metabolismo , Cromatografia Líquida , Bases de Dados de Proteínas , Genômica/métodos , Folhas de Planta/genética , Folhas de Planta/microbiologia , Proteômica/métodos , Espectrometria de Massas em TandemRESUMO
BACKGROUND AND PURPOSE: White matter hyperintensities (WMH) on MRI are a quantitative marker for sporadic cerebral small vessel disease and are highly heritable. To date, large-scale genetic studies have identified only a single locus influencing WMH burden. This might in part relate to biological heterogeneity of sporadic WMH. The current study searched for genetic modifiers of WMH volume in cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a monogenic small vessel disease. METHODS: We performed a genome-wide association study to identify quantitative trait loci for WMH volume by combining data from 517 CADASIL patients collected through 7 centers across Europe. WMH volumes were centrally analyzed and quantified on fluid attenuated inversion recovery images. Genotyping was performed using the Affymetrix 6.0 platform. Individuals were assigned to 2 distinct genetic clusters (cluster 1 and cluster 2) based on their genetic background. RESULTS: Four hundred sixty-six patients entered the final genome-wide association study analysis. The phenotypic variance of WMH burden in CADASIL explained by all single nucleotide polymorphisms in cluster 1 was 0.85 (SE=0.21), suggesting a substantial genetic contribution. Using cluster 1 as derivation and cluster 2 as a validation sample, a polygenic score was significantly associated with WMH burden (P=0.001) after correction for age, sex, and vascular risk factors. No single nucleotide polymorphism reached genome-wide significance. CONCLUSIONS: We found a polygenic score to be associated with WMH volume in CADASIL subjects. Our findings suggest that multiple variants with small effects influence WMH burden in CADASIL. The identification of these variants and the biological pathways involved will provide insights into the pathophysiology of white matter disease in CADASIL and possibly small vessel disease in general.
Assuntos
CADASIL/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla , Leucoencefalopatias/genética , Modelos Genéticos , Adulto , Idoso , CADASIL/epidemiologia , CADASIL/patologia , Feminino , Predisposição Genética para Doença/epidemiologia , Humanos , Hipertensão/epidemiologia , Hipertensão/genética , Hipertensão/patologia , Leucoencefalopatias/epidemiologia , Leucoencefalopatias/patologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Locos de Características Quantitativas , Fatores de RiscoRESUMO
BACKGROUND: Postmortem studies have indicated the potential of high-field magnetic resonance imaging (MRI) to visualize amyloid depositions in the cerebral cortex. The aim of this study is to test this hypothesis in patients with Alzheimer's disease (AD). METHODS: T2*-weighted MRI was performed in 16 AD patients and 15 control subjects. All magnetic resonance images were scored qualitatively by visual assessment, and quantitatively by measuring phase shifts in the cortical gray matter and hippocampus. Statistical analysis was performed to assess differences between groups. RESULTS: Patients with AD demonstrated an increased phase shift in the cortex in the temporoparietal, frontal, and parietal regions (P < .005), and this was associated with individual Mini-Mental State Examination scores (r = -0.54, P < .05). CONCLUSION: Increased cortical phase shift in AD patients demonstrated on 7-tesla T2*-weighted MRI is a potential new biomarker for AD, which may reflect amyloid pathology in the early stages.
Assuntos
Doença de Alzheimer/patologia , Córtex Cerebral/patologia , Imageamento por Ressonância Magnética , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Masculino , Estatísticas não ParamétricasRESUMO
Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active.
Assuntos
Exossomos/química , Plasma/química , Plasma/citologia , Proteoma/análise , Proteômica/métodos , Biomarcadores , Western Blotting , Humanos , Espectrometria de Massas , Proteoma/química , ViscosidadeRESUMO
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by stereotyped missense mutations in NOTCH3. Whether these mutations lead to the CADASIL phenotype via a neomorphic effect, or rather by a hypomorphic effect, is subject of debate. Here, we report two novel NOTCH3 mutations, both leading to a premature stop codon with predicted loss of NOTCH3 function. The first mutation, c.307C>T, p.Arg103*, was detected in two brothers aged 50 and 55 years, with a brain MRI and skin biopsy incompatible with CADASIL. The other mutation was found in a 40-year-old CADASIL patient compound heterozygous for a pathogenic NOTCH3 mutation (c.2129A>G, p.Tyr710Cys) and an intragenic frameshift deletion. The deletion was inherited from his father, who did not have the skin biopsy abnormalities seen in CADASIL patients. These individuals with rare NOTCH3 mutations indicate that hypomorphic NOTCH3 alleles do not cause CADASIL.
Assuntos
Alelos , CADASIL/diagnóstico , CADASIL/genética , Fenótipo , Receptores Notch/genética , Adulto , Encéfalo/patologia , Análise Mutacional de DNA , Heterozigoto , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Linhagem , Receptor Notch3 , Deleção de SequênciaRESUMO
OBJECTIVE: Diffuse iron deposition in the brain is commonly found in older people. One of the possible mechanisms that contribute to this iron deposition is cerebral small vessel disease. The aim of this study is to quantify diffuse iron deposition in patients with the hereditary small vessel disease cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). METHODS: 25 NOTCH3 mutation carriers and 18 healthy controls were examined using high-resolution T2*-weighted imaging on a 7 T whole body MRI scanner. Susceptibility-weighted MRI scans were analysed for areas of signal loss and increased phase shift. Phase shift measurements in deep grey nuclei, cortex and subcortical white matter were compared between mutation carriers and controls. For confirmation, ex vivo brain specimens from another three patients with CADASIL were analysed for iron deposition using ex vivo MRI combined with iron histochemistry. RESULTS: In vivo MRI showed areas of decreased signal intensity and increased phase shift in mutation carriers. Compared with healthy controls, mutation carriers had significantly higher phase shift in the putamen (p=0.0002) and caudate nucleus (p=0.006). Ex vivo MRI showed decreased signal intensity in the putamen and caudate nucleus in all specimens. Histochemistry confirmed the presence of iron deposition in these nuclei. CONCLUSIONS: This study demonstrates increased diffuse iron accumulation in the putamen and caudate nucleus in patients with the small vessel disease CADASIL. This supports the hypothesis that small vessel disease contributes to the process of increased iron accumulation in the general population.
Assuntos
CADASIL/metabolismo , Núcleo Caudado/metabolismo , Distúrbios do Metabolismo do Ferro/metabolismo , Ferro/metabolismo , Putamen/metabolismo , Adulto , Autopsia , CADASIL/patologia , Núcleo Caudado/patologia , Hemorragia Cerebral/patologia , Imagem Ecoplanar , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação , Putamen/patologia , Receptor Notch3 , Receptores Notch/genética , Imagem Corporal TotalRESUMO
BACKGROUND AND PURPOSE: Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary small vessel disease. Although postmortem studies have demonstrated mural thickening in leptomeningeal arteries and lenticulostriate perforating arteries, it is unclear whether this also leads to luminal narrowing. High-field MRI scanners enable in vivo imaging of the lumen of the lenticulostriate arteries. The aim of this study is to examine the luminal diameters of lenticulostriate arteries in living patients with CADASIL and to investigate whether luminal narrowing is correlated with the number of lacunar infarcts in the basal ganglia. METHODS: Twenty-two NOTCH3 mutation carriers and 11 healthy control subjects were examined using high-resolution 3-dimensional time-of-flight MR angiography imaging on a 7-T MRI scanner. Scans were analyzed for the presence of focal stenotic segments. The total number, length, and total cross-sectional area of lenticulostriate arteries were measured and compared between mutation carriers and control subjects. These measurements were correlated with age, disease duration, and number of lacunar infarcts in the basal ganglia. RESULTS: No stenotic segments were observed. No differences between mutation carriers and control subjects were found in total number of end branches (mutation carriers: mean, 14.6; control subjects: mean, 12.8), length of the lenticulostriate system, or total cross-sectional area of lenticulostriate artery lumina. Measurements of lenticulostriate artery lumina were not associated with lacunar infarct load in the basal ganglia area or with basal ganglia hyperintensities. CONCLUSIONS: Three-dimensional time-of-flight MR angiographic on 7 T showed no differences in luminal diameters of lenticulostriate arteries between patients with CADASIL and control subjects.
Assuntos
Doença Cerebrovascular dos Gânglios da Base/patologia , CADASIL/patologia , Adulto , Fatores Etários , Idoso , Anatomia Transversal , Gânglios da Base/patologia , CADASIL/epidemiologia , Artérias Cerebrais/patologia , Constrição Patológica , Feminino , Heterozigoto , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Mutação , Receptor Notch3 , Receptores Notch/genéticaRESUMO
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is caused by mutations in the NOTCH3 gene and is clinically characterized by recurrent stroke, cognitive decline, psychiatric disturbances and migraine. The prevalence of migraine in CADASIL is slightly higher than in the general population, and the proportion of migraine with aura is much higher. The pathophysiological mechanism that leads to increased aura prevalence in CADASIL is unknown. Possible mechanisms of the excess of migraine with aura are an increased susceptibility to cortical spreading depression (CSD) or a different expression of CSD. It is also possible that the brainstem migraine area is involved in CADASIL. Last, it is possible that the NOTCH3 mutation acts as a migraine aura susceptibility gene by itself. In this narrative review we summarize the literature about migraine in CADASIL, with a special focus on what CADASIL might teach us about the pathophysiology of migraine.
Assuntos
CADASIL/complicações , CADASIL/fisiopatologia , Transtornos de Enxaqueca/complicações , CADASIL/epidemiologia , Suscetibilidade a Doenças , Humanos , Transtornos de Enxaqueca/epidemiologia , Receptor Notch3 , Receptores Notch/genéticaRESUMO
One of the hallmark behaviors of social groups is division of labor, where different group members become specialized to carry out complementary tasks. By dividing labor, cooperative groups increase efficiency, thereby raising group fitness even if these behaviors reduce individual fitness. We find that antibiotic production in colonies of Streptomyces coelicolor is coordinated by a division of labor. We show that S. coelicolor colonies are genetically heterogeneous because of amplifications and deletions to the chromosome. Cells with chromosomal changes produce diversified secondary metabolites and secrete more antibiotics; however, these changes reduced individual fitness, providing evidence for a trade-off between antibiotic production and fitness. Last, we show that colonies containing mixtures of mutants and their parents produce significantly more antibiotics, while colony-wide spore production remains unchanged. By generating specialized mutants that hyper-produce antibiotics, streptomycetes reduce the fitness costs of secreted secondary metabolites while maximizing the yield and diversity of these products.
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Antibacterianos/biossíntese , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Streptomyces/genética , Streptomyces/metabolismo , Heterogeneidade Genética , Genoma Bacteriano , Genômica/métodos , Mutação , Fenótipo , Proteoma , Metabolismo SecundárioRESUMO
Neuroblastoma accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is a well-established poor prognostic marker for neuroblastoma. Whilst N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressiveness of the disease is poorly understood. Exosomes are released by many cell types including cancer cells and are implicated as key mediators in cell-cell communication via the transfer of molecular cargo. Hence, characterising the exosomal protein components from N-Myc amplified and non-amplified neuroblastoma cells will improve our understanding on their role in the progression of neuroblastoma. In this study, a comparative proteomic analysis of exosomes isolated from cells with varying N-Myc amplification status was performed. Label-free quantitative proteomic profiling revealed 968 proteins that are differentially abundant in exosomes released by the neuroblastoma cells. Gene ontology-based analysis highlighted the enrichment of proteins involved in cell communication and signal transduction in N-Myc amplified exosomes. Treatment of SH-SY5Y cells with N-Myc amplified SK-N-BE2 cell-derived exosomes increased the migratory potential, colony forming abilities and conferred resistance to doxorubicin induced apoptosis. Incubation of exosomes from N-Myc knocked down SK-N-BE2 cells abolished the transfer of resistance to doxorubicin induced apoptosis. These findings suggest that exosomes could play a pivotal role in N-Myc-driven aggressive neuroblastoma and transfer of chemoresistance between cells. Abbreviations: RNA = ribonucleic acid; DNA = deoxyribonucleic acid; FCS = foetal calf serum; NTA = nanoparticle tracking analysis; LC-MS = liquid chromatography-mass spectrometry; KD = knockdown; LTQ = linear trap quadropole; TEM = transmission electron microscopy.
RESUMO
Extracellular vesicles (EVs) are an important class of membrane-bound structures that have been widely investigated for their roles in intercellular communication in the contexts of tumor progression, vascular function, immunity and regenerative medicine. Much of the current knowledge on the functions of EVs pertains to those derived from viable cells (e.g. exosomes and microvesicles) or apoptotic cells (e.g. apoptotic bodies) whilst the generation of EVs from dying cells under non-apoptotic conditions remains poorly characterized. Herein, the release of EVs from THP-1 monocytes under conditions of primary necrosis, secondary necrosis and pyroptosis, was investigated. A comprehensive analysis of THP-1-derived EVs revealed that cells undergoing lytic forms of cell death generated a high number of EVs compared with viable or apoptotic cells in vitro. Differential centrifugation via 16,000 g and 100,000 g revealed that dying THP-1 cells release both medium and small EVs, respectively, consistent with the known characteristics of microvesicles and/or exosomes. In addition, large EVs isolated via 2000 g centrifugation were also present in all samples. These findings suggest that lytic cell death under both sterile and non-sterile inflammatory conditions induces monocytes to generate EVs, which could potentially act as mediators of cell-to-cell communication.
Assuntos
Morte Celular/fisiologia , Micropartículas Derivadas de Células/metabolismo , Exossomos/metabolismo , Vesículas Extracelulares/metabolismo , Monócitos/metabolismo , Comunicação Celular , Linhagem Celular Tumoral , Centrifugação , Humanos , Monócitos/citologia , Células THP-1RESUMO
Objective: To validate whether serum Neurofilament Light-chain (NfL) levels correlate with disease severity in CADASIL, and to determine whether serum NfL predicts disease progression and survival. Methods: Fourty-one (pre-) manifest individuals with CADASIL causing NOTCH3 mutations and 22 healthy controls were recruited from CADASIL families. At baseline, MRI-lesion load and clinical severity was determined and serum was stored. Disease progression was measured in 30/41 patients at 7-year follow-up, and survival of all individuals was determined at 17-year follow-up. Serum NfL levels were quantified using an ultra-sensitive molecule array. Generalized estimated equation regression (GEE) was used to analyze association between serum NfL, MRI-lesion load, disease severity, and disease progression. With GEE-based Cox regression, survival was analyzed. Results: At baseline, serum NfL levels correlated with MRI-lesion load [lacune count (s = 0.64, P = 0.002), brain atrophy (r = -0.50, P = 0.001), and microbleed count (s = 0.48, P = 0.044)], cognition [CAMCOG (s = -0.45, P = 0.010), MMSE (r = -0.61, P = 0.003), GIT (r = -0.61, P < 0.001), TMT-A (r = 0.70, P < 0.001)) and disability (mRS (r = 0.70, P = 0.002)]. Baseline serum NfL predicted 7-year changes in disability (B = 0.34, P < 0.001) and cognition (CAMCOG B = -4.94, P = 0.032), as well as 17-year survival. Higher NfL levels were associated with increased mortality (HR=1.8 per twofold increase in NfL levels, P = 0.006). Interpretation: Serum NfL levels correlate with disease severity, disease progression and 17-year survival in CADASIL patients. Serum NfL is a promising biomarker to monitor and predict disease course in CADASIL, as well as potentially assessing therapeutic response in future clinical trials.
Assuntos
CADASIL/sangue , CADASIL/diagnóstico , Proteínas de Neurofilamentos/sangue , Adulto , Biomarcadores/sangue , CADASIL/patologia , Estudos Transversais , Progressão da Doença , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Sensibilidade e Especificidade , Índice de Gravidade de DoençaRESUMO
Mutations in ß-catenin, especially at the residues critical for its degradation, render it constitutively active. Here, we show that mutant ß-catenin can be transported via extracellular vesicles (EVs) and activate Wnt signalling pathway in the recipient cells. An integrative proteogenomic analysis identified the presence of mutated ß-catenin in EVs secreted by colorectal cancer (CRC) cells. Follow-up experiments established that EVs released from LIM1215 CRC cells stimulated Wnt signalling pathway in the recipient cells with wild-type ß-catenin. SILAC-based quantitative proteomics analysis confirmed the transfer of mutant ß-catenin to the nucleus of the recipient cells. In vivo tracking of DiR-labelled EVs in mouse implanted with RKO CRC cells revealed its bio-distribution, confirmed the activation of Wnt signalling pathway in tumour cells and increased the tumour burden. Overall, for the first time, this study reveals that EVs can transfer mutant ß-catenin to the recipient cells and promote cancer progression.
RESUMO
Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1∆ or chs3∆ or the vps23∆chs3∆ double knockout strain were unable to rescue the yeast cells as compared to vps23∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling.
Assuntos
Parede Celular/metabolismo , Vesículas Extracelulares/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Antifúngicos/farmacologia , Caspofungina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Vesículas Extracelulares/efeitos dos fármacos , Mutação/genética , Proteômica , Saccharomyces cerevisiae/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacosRESUMO
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