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1.
Nature ; 584(7820): 291-297, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32728216

RESUMO

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.


Assuntos
Espaço Extracelular/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteólise , Proteínas Recombinantes de Fusão/metabolismo , Animais , Anticorpos/química , Anticorpos/metabolismo , Antígenos CD/metabolismo , Apolipoproteína E4/metabolismo , Antígeno B7-H1/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Receptores ErbB/metabolismo , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Humanos , Ligantes , Proteínas de Membrana/química , Camundongos , Domínios Proteicos , Transporte Proteico , Receptor IGF Tipo 2/metabolismo , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/química , Solubilidade , Especificidade por Substrato
2.
PLoS One ; 15(7): e0235546, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32609743

RESUMO

Resistin and resistin-like molecules are pleiotropic cytokines that are involved in inflammatory diseases. Our previous work suggested that resistin has the potential to be used as a biomarker and therapeutic target for human pulmonary arterial hypertension. However, data are limited on the distribution of resistin in healthy human organs. In this study, we used our newly developed anti-human resistin (hResistin) antibody to immunohistochemically detect the expression, localization, and intracellular/extracellular compartmentalization of hResistin in a full human tissue panel from healthy individuals. The potential cross reactivity of this monoclonal anti-hResistin IgG1 with normal human tissues also was verified. Results showed that hResistin is broadly distributed and principally localized in the cytoplasmic granules of macrophages scattered in the interstitium of most human tissues. Bone marrow hematopoietic precursor cells also exhibited hResistin signals in their cytoplasmic granules. Additionally, hResistin labeling was observed in the cytoplasm of nervous system cells. Notably, the cytokine activity of hResistin was illustrated by positively stained extracellular material in most human tissues. These data indicate that our generated antibody binds to the secreted hResistin and support its potential use for immunotherapy to reduce circulating hResistin levels in human disease. Our findings comprehensively document the basal expression patterns of hResistin protein in normal human tissues, suggest a critical role of this cytokine in normal and pathophysiologic inflammatory processes, and offer key insights for using our antibody in future pharmacokinetic studies and immunotherapeutic strategies.


Assuntos
Anticorpos Monoclonais/imunologia , Regulação da Expressão Gênica , Resistina/imunologia , Resistina/metabolismo , Espaço Extracelular/metabolismo , Células HEK293 , Humanos , Imuno-Histoquímica , Espaço Intracelular/metabolismo , Especificidade de Órgãos , Transporte Proteico
3.
Nature ; 584(7821): 410-414, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641833

RESUMO

In metazoans, the secreted proteome participates in intercellular signalling and innate immunity, and builds the extracellular matrix scaffold around cells. Compared with the relatively constant intracellular environment, conditions for proteins in the extracellular space are harsher, and low concentrations of ATP prevent the activity of intracellular components of the protein quality-control machinery. Until now, only a few bona fide extracellular chaperones and proteases have been shown to limit the aggregation of extracellular proteins1-5. Here we performed a systematic analysis of the extracellular proteostasis network in Caenorhabditis elegans with an RNA interference screen that targets genes that encode the secreted proteome. We discovered 57 regulators of extracellular protein aggregation, including several proteins related to innate immunity. Because intracellular proteostasis is upregulated in response to pathogens6-9, we investigated whether pathogens also stimulate extracellular proteostasis. Using a pore-forming toxin to mimic a pathogenic attack, we found that C. elegans responded by increasing the expression of components of extracellular proteostasis and by limiting aggregation of extracellular proteins. The activation of extracellular proteostasis was dependent on stress-activated MAP kinase signalling. Notably, the overexpression of components of extracellular proteostasis delayed ageing and rendered worms resistant to intoxication. We propose that enhanced extracellular proteostasis contributes to systemic host defence by maintaining a functional secreted proteome and avoiding proteotoxicity.


Assuntos
Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/microbiologia , Espaço Extracelular/metabolismo , Agregados Proteicos , Proteostase , Envelhecimento/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sistema de Sinalização das MAP Quinases , Agregação Patológica de Proteínas/prevenção & controle , Proteoma/genética , Proteoma/metabolismo , Interferência de RNA
4.
Nat Commun ; 11(1): 3440, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-32651387

RESUMO

In recent years, exploration of the brain extracellular space (ECS) has made remarkable progress, including nanoscopic characterizations. However, whether ECS precise conformation is altered during brain pathology remains unknown. Here we study the nanoscale organization of pathological ECS in adult mice under degenerative conditions. Using electron microscopy in cryofixed tissue and single nanotube tracking in live brain slices combined with super-resolution imaging analysis, we find enlarged ECS dimensions and increased nanoscale diffusion after α-synuclein-induced neurodegeneration. These animals display a degraded hyaluronan matrix in areas close to reactive microglia. Furthermore, experimental hyaluronan depletion in vivo reduces dopaminergic cell loss and α-synuclein load, induces microgliosis and increases ECS diffusivity, highlighting hyaluronan as diffusional barrier and local tissue organizer. These findings demonstrate the interplay of ECS, extracellular matrix and glia in pathology, unraveling ECS features relevant for the α-synuclein propagation hypothesis and suggesting matrix manipulation as a disease-modifying strategy.


Assuntos
Encéfalo/metabolismo , Espaço Extracelular/metabolismo , Ácido Hialurônico/metabolismo , Sinucleinopatias/metabolismo , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/ultraestrutura , Microscopia Eletrônica , Doença de Parkinson/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho
5.
Anticancer Res ; 40(7): 3931-3937, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32620634

RESUMO

BACKGROUND/AIM: Extracellular water-to-total body water ratio (ECW/TBW) measured by bioelectrical impedance analysis (BIA) reportedly predicts clinical outcomes of various diseases. The aim of this retrospective study was to examine the association between ECW/TBW and therapeutic durability of chemotherapy and/or immune checkpoint inhibitors in advanced lung cancer. PATIENTS AND METHODS: Patients with advanced lung cancer underwent BIA before chemotherapy and/or treatment with immune checkpoint inhibitors at our hospital between June 2018 and November 2019. RESULTS: Of 75 patients, 18 with ECW/TBW ≥0.4 were assigned to the overhydrated group (OH-G) and 57 patients ECW/TBW <0.4 were assigned to the non-overhydrated group (NOH-G). The median time-to-treatment failure was significantly shorter in the OH-G than in the NOH-G (p=0.003). Multivariate analysis revealed that ECW/TBW ≥0.4 predicted treatment failure [hazard ratio (HR)=2.508, 95% confidence interval (CI)=1.19-5.27; p=0.01]. CONCLUSION: The ECW/TBW may be an objective parameter for predicting therapeutic durability in advanced lung cancer.


Assuntos
Antineoplásicos/uso terapêutico , Água Corporal/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Água/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/uso terapêutico , Composição Corporal , Impedância Elétrica , Espaço Extracelular/metabolismo , Humanos , Neoplasias Pulmonares/patologia , Pessoa de Meia-Idade , Estudos Retrospectivos , Taxa de Sobrevida
6.
PLoS Biol ; 18(6): e3000722, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32569301

RESUMO

Inflammation and infection can trigger local tissue Na+ accumulation. This Na+-rich environment boosts proinflammatory activation of monocyte/macrophage-like cells (MΦs) and their antimicrobial activity. Enhanced Na+-driven MΦ function requires the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5), which augments nitric oxide (NO) production and contributes to increased autophagy. However, the mechanism of Na+ sensing in MΦs remained unclear. High extracellular Na+ levels (high salt [HS]) trigger a substantial Na+ influx and Ca2+ loss. Here, we show that the Na+/Ca2+ exchanger 1 (NCX1, also known as solute carrier family 8 member A1 [SLC8A1]) plays a critical role in HS-triggered Na+ influx, concomitant Ca2+ efflux, and subsequent augmented NFAT5 accumulation. Moreover, interfering with NCX1 activity impairs HS-boosted inflammatory signaling, infection-triggered autolysosome formation, and subsequent antibacterial activity. Taken together, this demonstrates that NCX1 is able to sense Na+ and is required for amplifying inflammatory and antimicrobial MΦ responses upon HS exposure. Manipulating NCX1 offers a new strategy to regulate MΦ function.


Assuntos
Macrófagos/metabolismo , Trocador de Sódio e Cálcio/metabolismo , Sódio/metabolismo , Processamento Alternativo/genética , Animais , Cálcio/metabolismo , Espaço Extracelular/metabolismo , Inativação Gênica/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Íons , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Camundongos , Óxido Nítrico/biossíntese , Células RAW 264.7 , Cloreto de Sódio/farmacologia
7.
Nat Commun ; 11(1): 2092, 2020 04 29.
Artigo em Inglês | MEDLINE | ID: mdl-32350252

RESUMO

Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. Our previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, dim fluorescence and the inability to make stably-expressing cell lines limit its use. We incorporated a stabilizing mutation in the pHluorin moiety, M153R, which now exhibits higher, stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader-follower behavior in 2D and 3D migration. Incorporating an additional non-pH-sensitive red fluorescent tag allows visualization of the exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes.


Assuntos
Movimento Celular , Exossomos/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Sobrevivência Celular , Exossomos/ultraestrutura , Espaço Extracelular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Corpos Multivesiculares/metabolismo , Corpos Multivesiculares/ultraestrutura , Nanopartículas/química , Nanopartículas/ultraestrutura , Tetraspanina 30/química , Tetraspanina 30/metabolismo , Fatores de Tempo
8.
PLoS One ; 15(5): e0234009, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470038

RESUMO

One of the potential contributing factors for iron overload-induced osteoporosis is the iron toxicity on bone forming cells, osteoblasts. In this study, the comparative effects of Fe3+ and Fe2+ on osteoblast differentiation and mineralization were studied in UMR-106 osteoblast cells by using ferric ammonium citrate and ferrous ammonium sulfate as Fe3+ and Fe2+ donors, respectively. Effects of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and iron chelator deferiprone on iron uptake ability of osteoblasts were examined, and the potential protective ability of 1,25(OH)2D3, deferiprone and extracellular calcium treatment in osteoblast cell survival under iron overload was also elucidated. The differential effects of Fe3+ and Fe2+ on reactive oxygen species (ROS) production in osteoblasts were also compared. Our results showed that both iron species suppressed alkaline phosphatase gene expression and mineralization with the stronger effects from Fe3+ than Fe2+. 1,25(OH)2D3 significantly increased the intracellular iron but minimally affected osteoblast cell survival under iron overload. Deferiprone markedly decreased intracellular iron in osteoblasts, but it could not recover iron-induced osteoblast cell death. Interestingly, extracellular calcium was able to rescue osteoblasts from iron-induced osteoblast cell death. Additionally, both iron species could induce ROS production and G0/G1 cell cycle arrest in osteoblasts with the stronger effects from Fe3+. In conclusions, Fe3+ and Fe2+ differentially compromised the osteoblast functions and viability, which can be alleviated by an increase in extracellular ionized calcium, but not 1,25(OH)2D3 or iron chelator deferiprone. This study has provided the invaluable information for therapeutic design targeting specific iron specie(s) in iron overload-induced osteoporosis. Moreover, an increase in extracellular calcium could be beneficial for this group of patients.


Assuntos
Calcitriol/farmacologia , Deferiprona/farmacologia , Espaço Extracelular/química , Sobrecarga de Ferro/metabolismo , Ferro/farmacologia , Osteoblastos/citologia , Animais , Biomarcadores/metabolismo , Calcificação Fisiológica/efeitos dos fármacos , Cálcio/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo
9.
Nat Commun ; 11(1): 2058, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32345973

RESUMO

Anaerobic ammonium oxidation (anammox) bacteria contribute significantly to the global nitrogen cycle and play a major role in sustainable wastewater treatment. Anammox bacteria convert ammonium (NH4+) to dinitrogen gas (N2) using intracellular electron acceptors such as nitrite (NO2-) or nitric oxide (NO). However, it is still unknown whether anammox bacteria have extracellular electron transfer (EET) capability with transfer of electrons to insoluble extracellular electron acceptors. Here we show that freshwater and marine anammox bacteria couple the oxidation of NH4+ with transfer of electrons to insoluble extracellular electron acceptors such as graphene oxide or electrodes in microbial electrolysis cells. 15N-labeling experiments revealed that NH4+ was oxidized to N2 via hydroxylamine (NH2OH) as intermediate, and comparative transcriptomics analysis revealed an alternative pathway for NH4+ oxidation with electrode as electron acceptor. Complete NH4+ oxidation to N2 without accumulation of NO2- and NO3- was achieved in EET-dependent anammox. These findings are promising in the context of implementing EET-dependent anammox process for energy-efficient treatment of nitrogen.


Assuntos
Compostos de Amônio/metabolismo , Bactérias/metabolismo , Espaço Extracelular/metabolismo , Anaerobiose , Eletroquímica , Eletrólise , Transporte de Elétrons , Oxirredução , Fatores de Tempo
10.
J Cancer Res Clin Oncol ; 146(7): 1647-1658, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32335720

RESUMO

BACKGROUND: Additional prognostic factors and personalized therapeutic alternatives for vulvar squamous cell carcinoma (VSCC), especially for advanced stages with poor prognosis, are urgently needed. OBJECTIVES: To review and assess literature regarding underlying molecular mechanisms of VSCC target therapeutic and prognostic approaches. METHODS: We performed a narrative literature review from the inception of the database up to January 2020 limited to English language, organizing knowledge in five main fields: extracellular and intracellular cell cycle deregulation, tumor immune microenvironment, tumor angiogenesis and hormones. RESULTS: EGFR immunohistochemical overexpression/gene amplification, representing early events in VSCC carcinogenesis, have been correlated with a worse prognosis and led to inclusion of erlotinib in cancer guidelines. p16 expression and HPV positivity are linked to a better prognosis, while p53 overexpression is linked to a worse prognosis; thus, biomarkers could help tailoring conventional treatment and follow-up. The implications of PD-L1 positivity in reference to HPV status and prognosis are still not clear, even though pembrolizumab is part of available systemic therapies. The role of tumor angiogenesis emerges through data on microvessel density, immunohistochemical VEGF staining and evaluation of serum VEGF concentrations. Few data exist on hormonal receptor expression, even though hormonal therapy showed great manageability. CONCLUSIONS: We suggest adding p16, p53 and HPV status to routine hystopathological examination of vulvar biopsies or surgical specimens. Predictive biomarkers for anti-EGFR and anti-PD-1/PD-L1 drugs are needed. Enough preclinical data supporting anti-angiogenic target therapies in clinical trials are existing. Hormonal receptor expression deserves further investigation.


Assuntos
Biomarcadores Tumorais , Carcinoma de Células Escamosas/etiologia , Carcinoma de Células Escamosas/metabolismo , Regulação Neoplásica da Expressão Gênica , Transdução de Sinais , Neoplasias Vulvares/etiologia , Neoplasias Vulvares/metabolismo , Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/patologia , Ciclo Celular/genética , Tomada de Decisão Clínica , Suscetibilidade a Doenças , Espaço Extracelular/metabolismo , Feminino , Humanos , Espaço Intracelular/metabolismo , Terapia de Alvo Molecular , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Neoplasias Vulvares/tratamento farmacológico , Neoplasias Vulvares/patologia
11.
Beijing Da Xue Xue Bao Yi Xue Ban ; 52(2): 362-367, 2020 Apr 18.
Artigo em Chinês | MEDLINE | ID: mdl-32306023

RESUMO

OBJECTIVE: To compare the changes of extracellular space (ECS) structure and local drug distribution in adult brain and aged brain at different drug delivery rates in minimally invasive treatment of encephalopathy by convection enhanced delivery (CED) via ECS pathway. METHODS: Thirty-six SD male rats were divided into adult rats group (2-8 months, n=18) and aged rats group (18-24 months, n=18) according to the age of the month. According to the drug rates (0.1 µL/min, 0.2 µL/min, and 0.3 µL/min), they were randomly divided into 3 subgroups, 6 in each subgroup. Gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) with a concentration of 10 mmol/L were introduced into the caudate nucleus of each group of rats by stereotactic injection. Tracer-based magnetic resonance imaging (MRI) was used to dynamically monitor the diffusion and distribution images of the Gd-DTPA in the brain interstitial system (ISS). Using the self-developed MRI image measurement and analysis system software to process and analyze the obtained images, the diffusion coefficient, clearance rate, volume fraction, and half-life of each group of rats in the caudate nucleus ECS could be acquired. The effects and differences of drug clearance and ECS structural function in the brain of aged rats and adult rats were compared and analyzed at different drug delivery rates. Magnetic tracer DECS-mapping technique was used to observe the distribution and drainage of tracer in caudate nucleus. RESULTS: At the injection rate of 0.1 µL/min, the volume fraction in the aged rats was increased compared with that in the adult rats (18.20%±0.04% vs. 17.20%±0.03%, t=3.752, P=0.004), and the degree of tortuosity was decreased (1.63±0.04 vs. 1.78±0.09, t=-3.680, P=0.004), the drug clearance rate was decreased [(1.94±0.68) mm2/s vs. (3.25±0.43) mm2/s, t=-3.971, P=0.003], and the molecular diffusion in ECS was increased [(3.99±0.21)×10-4 mm2/s vs. (3.36±0.37)×10-4 mm2/s, t=3.663, P=0.004]. When the rate of injection increased to 0.2 µL/min, the drug clearance in ECS of the aged rats was slowed down [(2.53±0.45) mmol/L vs. (3.37±0.72) mmol/L, t=-1.828, P=0.021]. However, there were no significant differences in volume fraction, molecular diffusion in ECS and macroscopic drug metabolism parameters. When the rate of injection increased to 0.3 µL/min, the volume fraction in the aged rats was decreased (17.20%±0.03% vs. 18.20%±0.05%, t=-0.869, P=0.045), and the drug clearance rate in ECS was significantly accelerated [(4.04±0.76) mmol/L vs. (3.26±0.55) mmol/L, t=1.786, P=0.014], and there was no significant difference in tortuosity and the rate of molecular diffusion in the ECS. CONCLUSION: The drug clearance and ECS structural parameters of brain ECS in aged brain with CED administration were changed at different rates, and it has the least effect on ECS in the aged brain at the injection rate of 0.2 µL/min. For the application of CED for the treatment of encephalopathy, we should consider the influence of factors such as age and injection rate, and provide reference for the development of individualized clinical treatment plan for minimally invasive treatment of encephalopathy via ECS pathway.


Assuntos
Convecção , Espaço Extracelular , Animais , Encéfalo , Gadolínio DTPA , Imagem por Ressonância Magnética , Masculino , Ratos , Ratos Sprague-Dawley
12.
PLoS Comput Biol ; 16(4): e1007780, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32298259

RESUMO

Metabolism plays an essential role in cell fate decisions. However, the methods used for metabolic characterization and for finding potential metabolic regulators are still based on characterizing cellular metabolic steady-state which is dependent on the extracellular environment. In this work, we hypothesized that the response dynamics of intracellular metabolic pools to extracellular stimuli is controlled in a cell type-specific manner. We applied principles of process dynamics and control to human induced pluripotent stem cells (hiPSC) and human neural stem cells (hNSC) subjected to a sudden extracellular glutamine step. The fold-changes of steady-states and the transient profiles of metabolic pools revealed that dynamic responses were reproducible and cell type-specific. Importantly, many amino acids had conserved dynamics and readjusted their steady state concentration in response to the increased glutamine influx. Overall, we propose a novel methodology for systematic metabolic characterization and identification of potential metabolic regulators.


Assuntos
Células-Tronco Pluripotentes Induzidas , Redes e Vias Metabólicas/fisiologia , Células-Tronco Neurais , Reatores Biológicos , Células Cultivadas , Biologia Computacional , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Glutamina/metabolismo , Glutamina/farmacologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo
13.
PLoS One ; 15(4): e0230903, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32267858

RESUMO

Magnetic resonance electrical properties tomography (MREPT) uses the B1 mapping technique to provide the high-frequency conductivity distribution at Larmor frequency that simultaneously reflects the intracellular and extracellular effects. In biological tissues, the electrical conductivity can be described as the concentration and mobility of charge carriers. For the water molecule diffusivity, diffusion weighted imaging (DWI) measures the random Brownian motion of water molecules within biological tissues. The DWI data can quantitatively access the mobility of microscopic water molecules within biological tissues. By measuring multi-b-value DWI data and the recovered high-frequency conductivity at Larmor frequency, we propose a new method to decompose the conductivity into the total ion concentration and mobility in the extracellular space (ECS) within a routinely applicable MR scan time. Using the measured multi-b-value DWI data, a constrained compartment model is designed to estimate the extracellular volume fraction and extracellular mean diffusivity. With the extracted extracellular volume fraction and water molecule diffusivity, we directly reconstruct the low-frequency electrical properties including the extracellular mean conductivity and extracellular conductivity tensor. To demonstrate the proposed method by comparing the ion concentration and the ion mobility, we conducted human experiments for the proposed low-frequency conductivity imaging. Human experiments verify that the proposed method can recover the low-frequency electrical properties using a conventional MRI scanner.


Assuntos
Imagem de Difusão por Ressonância Magnética , Condutividade Elétrica , Espaço Extracelular/diagnóstico por imagem , Espaço Extracelular/metabolismo , Humanos , Processamento de Imagem Assistida por Computador
14.
Nature ; 580(7803): 402-408, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32296183

RESUMO

Global insights into cellular organization and genome function require comprehensive understanding of the interactome networks that mediate genotype-phenotype relationships1,2. Here we present a human 'all-by-all' reference interactome map of human binary protein interactions, or 'HuRI'. With approximately 53,000 protein-protein interactions, HuRI has approximately four times as many such interactions as there are high-quality curated interactions from small-scale studies. The integration of HuRI with genome3, transcriptome4 and proteome5 data enables cellular function to be studied within most physiological or pathological cellular contexts. We demonstrate the utility of HuRI in identifying the specific subcellular roles of protein-protein interactions. Inferred tissue-specific networks reveal general principles for the formation of cellular context-specific functions and elucidate potential molecular mechanisms that might underlie tissue-specific phenotypes of Mendelian diseases. HuRI is a systematic proteome-wide reference that links genomic variation to phenotypic outcomes.


Assuntos
Proteoma/metabolismo , Espaço Extracelular/metabolismo , Humanos , Especificidade de Órgãos , Mapeamento de Interação de Proteínas
15.
Mol Immunol ; 120: 136-145, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32120181

RESUMO

Evasion of the immune system is often associated with malignant tumors. The cancer cell microenvironment plays an important role in tumor progression, but its mechanism is largely unknown. Here we show that an extracellular compound derived from gastric cancer (GC-EC) selectively suppresses CD161+CD3- natural killer (NK) cells. Splenocytes treated with GC-EC showed considerable proliferation and the CD161+CD3- NK cell population was time-dependently suppressed. Intracellular staining of IFN-γ was shown to be down-regulated in concert with granzyme B and perforin. A cytotoxicity assay of splenocytes treated with GC-EC against K-562 cells showed a significant reduction in cytolytic activity. Further, the immune-suppressive effect of GC-EC was more evident in a syngeneic tumor model in C57BL/6 mice. Animals treated with B16 F10 and GC-EC exhibited more aggravated tumor formation than animals treated with B16 F10 only. We demonstrated that inhibition of apoptosis while increasing PI3 K/AKT levels may provoke tumor formation by GC-EC. A cytokine array revealed the presence of several cytokines in GC-EC that negatively regulate immune cytolytic activity and could be potential candidates for immune-suppressive effects.


Assuntos
Células Matadoras Naturais/imunologia , Neoplasias Gástricas/imunologia , Animais , Apoptose/imunologia , Complexo CD3/imunologia , Proliferação de Células , Citocinas/imunologia , Citotoxicidade Imunológica , Espaço Extracelular/imunologia , Humanos , Tolerância Imunológica , Células K562 , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Ratos , Ratos Sprague-Dawley , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Microambiente Tumoral/imunologia
16.
Chimia (Aarau) ; 74(3): 122-128, 2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32197669

RESUMO

Harmful cyanobacterial blooms in freshwater ecosystems produce bioactive secondary metabolites including cyanopeptides that pose ecological and human health risks. Only adverse effects of one class of cyanopeptides, microcystins, have been studied extensively and have consequently been included in water quality assessments. Inhibition is a commonly observed effect for enzymes exposed to cyanopeptides and has mostly been investigated for human biologically relevant model enzymes. Here, we investigated the inhibition of ubiquitous aquatic enzymes by cyanobacterial metabolites. Hydrolytic enzymes are utilized in the metabolism of aquatic organisms and extracellularly by heterotrophic bacteria to obtain assimilable substrates. The ubiquitous occurrence of hydrolytic enzymes leads to the co-occurrence with cyanopeptides especially during cyanobacterial blooms. Bacterial leucine aminopeptidase and alkaline phosphatase were exposed to cyanopeptide extracts of different cyanobacterial strains ( Microcystis aeruginosa wild type and microcystin-free mutant, Planktothrix rubescens) and purified cyanopeptides. We observed inhibition of aminopeptidase and phosphatase upon exposure, especially to the apolar fractions of the cyanobacterial extracts. Exposure to the dominant cyanopeptides in these extracts confirmed that purified microcystins, aerucyclamide A and cyanopeptolin A inhibit the aminopeptidase in the low mg L-1 range while the phosphatase was less affected. Inhibition of aquatic enzymes can reduce the turnover of nutrients and carbon substrates and may also impair metabolic functions of grazing organisms.


Assuntos
Cianobactérias , Ecossistema , Espaço Extracelular , Água Doce , Humanos , Microcystis , Peptídeos
17.
J Vis Exp ; (155)2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32065130

RESUMO

Cross-kingdom biofilms consisting of both fungal and bacterial cells are involved in a variety of oral diseases, such as endodontic infections, periodontitis, mucosal infections and, most notably, early childhood caries. In all of these conditions, the pH in the biofilm matrix impacts microbe-host interactions and thus the disease progression. The present protocol describes a confocal microscopy-based method to monitor pH dynamics inside cross-kingdom biofilms comprising Candida albicans and Streptococcus mutans. The pH-dependent dual-emission spectrum and the staining properties of the ratiometric probe C-SNARF-4 are exploited to determine drops in pH in extracellular areas of the biofilms. Use of pH ratiometry with the probe requires a meticulous choice of imaging parameters, a thorough calibration of the dye, and careful, threshold-based post-processing of the image data. When used correctly, the technique allows for the rapid assessment of extracellular pH in different areas of a biofilm and thus the monitoring of both horizontal and vertical pH gradients over time. While the use of confocal microscopy limits Z-profiling to thin biofilms of 75 µm or less, the use of pH ratiometry is ideally suited for the noninvasive study of an important virulence factor in cross-kingdom biofilms.


Assuntos
Biofilmes , Candida albicans/fisiologia , Espaço Extracelular/química , Microscopia Confocal/métodos , Streptococcus mutans/fisiologia , Biofilmes/efeitos dos fármacos , Calibragem , Candida albicans/efeitos dos fármacos , Glucose/farmacologia , Concentração de Íons de Hidrogênio , Processamento de Imagem Assistida por Computador , Streptococcus mutans/efeitos dos fármacos
18.
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi ; 34(2): 226-233, 2020 Feb 15.
Artigo em Chinês | MEDLINE | ID: mdl-32030956

RESUMO

Objective: To explore the possibility of constructing tissue engineered adipose by adipose tissue derived extracellular vesicles (hAT-EV) combined with decellularized adipose tissue (DAT) scaffolds, and to provide a new therapy for soft tissue defects. Methods: The adipose tissue voluntarily donated by the liposuction patient was divided into two parts, one of them was decellularized and observed by HE and Masson staining and scanning electron microscope (SEM). Immunohistochemical staining and Western blot detection for collagen type Ⅰ and Ⅳ and laminin were also employed. Another one was incubated with exosome-removed complete medium for 48 hours, then centrifuged to collect the medium and to obtain hAT-EV via ultracentrifugation. The morphology of hAT-EV was observed by transmission electron microscopy; the nanoparticle tracking analyzer (NanoSight) was used to analyze the size distribution; Western blot was used to analyse membrane surface protein of hAT-EV. Adipose derived stem cells (ADSCs) were co-cultured with PKH26 fluorescently labeled hAT-EV, confocal fluorescence microscopy was used to observe the uptake of hAT-EV by ADSCs. Oil red O staining was used to evaluate adipogenic differentiation after hAT-EV and ADSCs co-cultured for 15 days. The DAT was scissored and then injected into the bilateral backs of 8 C57 mice (6-week-old). In experimental group, 0.2 mL hAT-EV was injected weekly, and 0.2 mL PBS was injected weekly in control group. After 12 weeks, the mice were sacrificed, and the new fat organisms on both sides were weighed. The amount of new fat was evaluated by HE and peri-lipoprotein immunofluorescence staining to evaluate the ability of hAT-EV to induce adipogenesis in vivo. Results: After acellularization of adipose tissue, HE and Masson staining showed that DAT was mainly composed of loosely arranged collagen with no nucleus; SEM showed that no cells and cell fragments were found in DAT, and thick fibrous collagen bundles could be seen; immunohistochemical staining and Western blot detection showed that collagen type Ⅰ and Ⅳ and laminin were retained in DAT. It was found that hAT-EV exhibited a spherical shape of double-layer envelope, with high expressions of CD63, apoptosis-inducible factor 6 interacting protein antibody, tumor susceptibility gene 101, and the particle size of 97.9% hAT-EV ranged from 32.67 nmto 220.20 nm with a peak at 91.28 nm. Confocal fluorescence microscopy and oil red O staining showed that hAT-EV was absorbed by ADSCs and induced adipogenic differentiation. In vivo experiments showed that the wet weight of fat new organisms in the experimental group was significantly higher than that in the control group ( t=2.278, P=0.048). HE staining showed that the structure of lipid droplets in the experimental group was more than that in the control group, and the collagen content in the control group was higher than that in the experimental group. The proportion of new fat in the experimental group was significantly higher than that in the control group ( t=4.648, P=0.017). Conclusion: DAT carrying hAT-EV can be used as a new method to induce adipose tissue regeneration and has a potential application prospect in the repair of soft tissue defects.


Assuntos
Tecido Adiposo , Adipócitos , Animais , Diferenciação Celular , Células Cultivadas , Espaço Extracelular , Humanos , Camundongos , Células-Tronco , Engenharia Tecidual , Tecidos Suporte
19.
Mutat Res ; 849: 503128, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32087849

RESUMO

A physiological decrease in extracellular pH (pHe) alters the efficiency of DNA repair and increases formation of DNA double-strand breaks (DSBs). Whether this could translate into genetic instability and variations, was investigated using the TK6 cell model, in which positive selection of the TK1 gene loss-of-function mutations can be achieved from resistance to trifluorothymidine. Cell exposure to suboptimal pH (down to 6.9) for 3 weeks resulted in the 100 % frequency of a stronger frameshift mutation that has spread to both TK1 alleles, whereas weaker frameshift mutations within the 3'exon were eliminated during the selection. Suboptimal pHe values were also found to alter the proportion of the TK1 splicing variant expressed as percent spliced in index values and promote selection of truncated exons as well as intron retention. Although recovery at pH 7.4 did not reverse the selected frameshift mutation, reversal of splice variants and exon truncation towards control values were observed. Hence, suboptimal pHe can induce a combination of mutational events and splicing alterations within the same gene in the resistant clones. This model of positive selection for loss-of-function clearly demonstrates that suboptimal pHe may confer a similar growth advantage when such instability occurs within tumor suppressor genes.


Assuntos
Processamento Alternativo , Mutação da Fase de Leitura , Linfócitos/metabolismo , Modelos Genéticos , Mutagênese , Timidina Quinase/genética , Antimetabólitos/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos dos fármacos , Éxons , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Íntrons , Linfócitos/citologia , Linfócitos/efeitos dos fármacos , Timidina Quinase/metabolismo , Trifluridina/farmacologia
20.
Nat Commun ; 11(1): 758, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32029728

RESUMO

We test the hypothesis that the frequency and cost of extracellular proteins produced by bacteria, which often depend on cooperative processes, vary with habitat structure and community diversity. The integration of the environmental distribution of bacteria (using 16S datasets) and their genomes shows that bacteria living in more structured habitats encode more extracellular proteins. In contrast, the effect of community diversity depends on protein function: it's positive for proteins implicated in antagonistic interactions and negative for those involved in nutrient acquisition. Extracellular proteins are costly and endure stronger selective pressure for low cost and for low diffusivity in less structured habitats and in more diverse communities. Finally, Bacteria found in multiple types of habitats, including host-associated generalists, encode more extracellular proteins than niche-restricted bacteria. These results show that ecological variables, notably habitat structure and community diversity, shape the evolution of the repertoires of genes encoding extracellular proteins and thus affect the ability of bacteria to manipulate their environment.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Microbiota/fisiologia , Bactérias/genética , Proteínas de Bactérias/genética , Biodiversidade , Ecossistema , Espaço Extracelular/metabolismo , Genes Bacterianos , Microbiota/genética , Modelos Biológicos , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética
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