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1.
Viruses ; 13(2)2021 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-33567490

RESUMO

Recent research indicates that most tissue and cell types can secrete and release membrane-enclosed small vesicles, known as exosomes, whose content reflects the physiological/pathological state of the cells from which they originate. These exosomes participate in the communication and cell-to-cell transfer of biologically active proteins, lipids, and nucleic acids. Studies of RNA viruses have demonstrated that exosomes release regulatory factors from infected cells and deliver other functional host genetic elements to neighboring cells, and these functions are involved in the infection process and modulate the cellular responses. This review provides an overview of the biogenesis, composition, and some of the most striking functions of exosome secretion and identifies physiological/pathological areas in need of further research. While initial indications suggest that exosome-mediated pathways operate in vivo, the exosome mechanisms involved in the related effects still need to be clarified. The current review focuses on the role of exosomes in RNA virus infections, with an emphasis on the potential contributions of exosomes to pathogenesis.


Assuntos
Exossomos/metabolismo , Infecções por Vírus de RNA/patologia , Vírus de RNA/fisiologia , Exossomos/química , Biogênese de Organelas , Infecções por Vírus de RNA/metabolismo , Infecções por Vírus de RNA/transmissão , Infecções por Vírus de RNA/virologia , Vírus de RNA/classificação , Replicação Viral
2.
Anal Chem ; 93(3): 1709-1716, 2021 01 26.
Artigo em Inglês | MEDLINE | ID: mdl-33369394

RESUMO

Tumor exosomes are promising biomarkers for early cancer diagnosis in a noninvasive manner. However, precise capture and direct analysis of tumor-specific exosomes in complex biological samples are still challenging. Herein, we present a highly efficient dual-aptamer recognition system for precisely isolating and quantifying tumor exosomes from the complex biological environment based on hyperbranched DNA superstructure-facilitated signal amplification and ratiometric dual-signal strategies. When tumor exosomes were captured by the dual-aptamer recognition system, the cholesterol-modified DNA probe was anchored on the surface of the exosomes, activating DNA tetrahedron-based hyperbranched hybridization chain reaction to generate a sandwich complex. Then, the sandwich complex could bind a large number of Ru(NH3)63+ (Ru(III)), leading to a small amount of unbound Ru(III) left in the supernatant after magnetic separation. Hence, the redox reaction between Ru(II) and [Fe(CN)6]3- (Fe(III)) was significantly prevented, causing an obviously enhanced IFe(III)/IRu(III) value. Consequently, highly sensitive detection of tumor exosomes was achieved. The developed approach successfully realized direct isolation and analysis of tumor exosomes in complex sample media and human serum samples as well. More significantly, this ratiometric dual-signal mode and immobilization-free strategy effectively circumvented the systematic errors caused by external factors and the tedious probe immobilization processes, thus displaying the excellent performances of high reliability, improved accuracy, and easy manipulation. Overall, this approach is expected to offer novel ways for nondestructive early cancer diagnosis.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Técnicas Eletroquímicas , Exossomos/química , Compostos Férricos/química , Humanos , Células MCF-7 , Nanopartículas/química , Dióxido de Silício/química , Células Tumorais Cultivadas
3.
Anal Chem ; 93(2): 709-714, 2021 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-33315384

RESUMO

Exosomes are considered promising indicators for early cancer diagnosis. The multiple protein biomarkers carried by exosomes are associated with diverse significant biological processes and are important biomarkers of cancer subtypes. However, it is challenging to sensitively and accurately quantify protein biomarkers from a few exosomes. Herein, we propose an ultrasensitive method for quantitatively profiling protein biomarkers on the surface of exosomes by integrating mass spectrometry imaging and gold nanoparticle (AuNP)-based signal amplification. Organic oligomers as mass tags and specific antibodies are modified on AuNPs to form biomarker probes. Exosomes captured by the antibody-coated gold chip are recognized by the AuNPs probes, forming a sandwich immunoassay. By mass spectrometry imaging the mass tags, multiple protein biomarkers can be quantitatively detected from the exosomes, with a limit-of-detection (LOD) down to 50 exosome particles. As a proof of concept, exosomes secreted by different breast-cancer cell subtypes, i.e. MCF-7 and MDA-MB231, were distinguished by the level of surface protein biomarkers of CD9, CD44, and epithelial cell adhesion molecule (EpCAM) acquired by the method, demonstrating that exosomes could be used for the diagnosis of cancer at subtype level. In consideration of the advantages of the ultrasensitivity, accuracy, and simplicity, the strategy has potential prospects in biomarker discovery, cellular phenotype characterization, and cancer diagnosis.


Assuntos
Exossomos/química , Imunoensaio/métodos , Espectrometria de Massas/métodos , Biomarcadores/química , Neoplasias da Mama/classificação , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial , Feminino , Humanos , Receptores de Hialuronatos , Limite de Detecção , Análise Serial de Proteínas , Tetraspanina 29
4.
Int J Nanomedicine ; 15: 6917-6934, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33061359

RESUMO

Exosomes are nano-sized small extracellular vesicles secreted by cells, carrying nucleic acids, proteins, lipids and other bioactive substances to play a role in the body's physiological and pathological processes. Compared to synthetic carriers such as liposomes and nanoparticles, the endogeneity and heterogeneity of exosomes give them extensive and unique advantages in the field of disease diagnosis and treatment. However, the storage stability, low yield, low purity, and weak targeting of exosomes limit its clinical application. For this reason, further exploration is needed to optimize the above problems and facilitate future functional studies of exosomes. In this paper, the origin, classification, preparation and characterization, storage stability and applications of exosome delivery system are summarized and discussed by searching a large number of literatures.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Exossomos , Biologia Molecular/métodos , Terapia de Alvo Molecular/métodos , Criopreservação , Exossomos/química , Exossomos/metabolismo , Exossomos/transplante , Alimentos , Liofilização , Humanos , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Proteínas/química , Proteínas/metabolismo
5.
PLoS One ; 15(9): e0232442, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32956358

RESUMO

Exosomes are vesicles involved in intercellular communication. Their membrane structure and core content is largely dependent on the cell of origin. Exosomes have been investigated both for their biological roles and their possible use as disease biomarkers and drug carriers. These potential technological applications require the rigorous characterization of exosomal blood brain barrier permeability and a description of their lipid bilayer composition. To achieve these goals, we have established a 3D static blood brain barrier system based on existing systems for liposomes and a complementary LC-MS/MS and 31P nuclear magnetic resonance methodology for the analysis of purified human plasma-derived exosome-like vesicles. Results show that the isolated vesicles pass the blood brain barrier and are taken up in endothelial cells. The compositional analysis revealed that the isolated vesicles are enriched in lyso phospholipids and do not contain phosphatidylserine. These findings deviate significantly from the composition of exosomes originating from cell culture, and may reflect active removal by macrophages that respond to exposed phosphahtidylserine.


Assuntos
Barreira Hematoencefálica/metabolismo , Exossomos/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Animais , Astrócitos/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , Modelos Biológicos , Ratos , Suínos
6.
Int J Nanomedicine ; 15: 5911-5926, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32848396

RESUMO

Purpose: Chronic refractory wounds are a multifactorial comorbidity of diabetes mellitus with the characteristic of impaired vascular networks. Currently, there is a lack of effective treatments for such wounds. Various types of mesenchymal stem cell-derived exosomes (MSC-exos) have been shown to exert multiple therapeutic effects on skin regeneration. We aimed to determine whether a constructed combination of human umbilical cord MSC (hUCMSC)-derived exosomes (hUCMSC-exos) and Pluronic F-127 (PF-127) hydrogel could improve wound healing. Materials and Methods: We topically applied human umbilical cord-derived MSC (hUCMSC)-derived exosomes (hUCMSC-exos) encapsulated in a thermosensitive PF-127 hydrogel to a full-thickness cutaneous wound in a streptozotocin-induced diabetic rat model. The material properties and wound healing ability of the hydrogel and cellular responses were analyzed. Results: Compared with hUCMSC-exos, PF-127-only or control treatment, the combination of PF-127 and hUCMSC-exos resulted in a significantly accelerated wound closure rate, increased expression of CD31 and Ki67, enhanced regeneration of granulation tissue and upregulated expression of vascular endothelial growth factor (VEGF) and factor transforming growth factor beta-1 (TGFß-1). Conclusion: The efficient delivery of hUCMSC-exos in PF-127 gel and improved exosome ability could promote diabetic wound healing. Thus, this biomaterial-based exosome therapy may represent a new therapeutic approach for cutaneous regeneration of chronic wounds.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Exossomos/química , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/citologia , Poloxâmero/química , Cicatrização/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/complicações , Pé Diabético/tratamento farmacológico , Pé Diabético/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Hidrogéis/química , Masculino , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Ratos Sprague-Dawley , Regeneração , Pele/efeitos dos fármacos , Pele/lesões , Fenômenos Fisiológicos da Pele , Estreptozocina , Cordão Umbilical/citologia , Fator A de Crescimento do Endotélio Vascular/genética , Cicatrização/fisiologia
7.
Int J Nanomedicine ; 15: 4257-4273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606676

RESUMO

In recent years, it has been found that exosomes can be used as nanocarriers, which can be used in the treatment of tumors by carrying contents. The exosomes are derived from the secretion of the organism's own cells and are characterized by a phospholipid bilayer structure and a small particle size. These characteristics guarantee that the exosomes can carry a wide range of tumor drugs, deliver the drug to the cancer, and reduce or eliminate the tumor drug band. The toxic side effects were significantly eliminated; meanwhile, the therapeutic effects of the drug on the tumor were remarkably improved. This paper reviewed the strategies and drugs presented by different scholars for the treatment of tumors based on the drugs carried by exosomes.


Assuntos
Portadores de Fármacos/química , Exossomos/química , Nanopartículas/química , Neoplasias/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Humanos , Tamanho da Partícula
8.
Adv Exp Med Biol ; 1195: 149, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32468469

RESUMO

Alzheimer's disease and other neurodegenerative diseases have long preclinical phases with active and progressively irreversible pathology. Therefore, biomarkers are essential for identifying patients early in the course of these diseases, when they may benefit the most from disease-modifying interventions. A limitation of biomarkers measured in the soluble phase of blood is their tenuous link to brain pathology. A new approach to biomarker discovery that addresses this limitation is deriving extracellular vesicles (EVs) enriched for neuronal and astrocytic origin from peripheral blood. EVs are membranous particles (subdivided into smaller exosomes and larger microvesicles) that are shed by all cells and found in all biofluids. Neuronal and astrocytic EVs have been implicated in the pathogenesis of several neurodegenerative diseases. Given their origin, neuronal and astrocytic enriched EVs harvested from blood can be used to interrogate brain pathologic processes previously inaccessible in vivo. In a long series of case control studies based on these EV subpopulations, we have identified candidate protein biomarkers for Alzheimer's disease and other neurodegenerative diseases. In GeNeDis 2018, an update of these studies and results from a validation study of these biomarkers in preclinical Alzheimer's disease will be presented. In addition, we will present results from studies demonstrating EV biomarker responses to experimental interventions. EV-based biomarkers are a valuable new tool that will enable researchers to test hypotheses in proof of concept studies with carefully selected participants at the preclinical phase, spearheading therapeutic discovery in neurodegenerative disease.


Assuntos
Biomarcadores/análise , Ensaios Clínicos como Assunto/métodos , Exossomos/química , Doenças Neurodegenerativas/diagnóstico , Doenças Neurodegenerativas/terapia , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Doença de Alzheimer/terapia , Humanos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Resultado do Tratamento
9.
Neurology ; 94(23): e2412-e2423, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32461282

RESUMO

OBJECTIVE: To measure exosomal and plasma levels of candidate blood biomarkers in veterans with history of mild traumatic brain injury (mTBI) and test their relationship with chronic symptoms. METHODS: Exosomal and plasma levels of neurofilament light (NfL) chain, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-10, and vascular endothelial growth factor (VEGF) were measured using an ultrasensitive assay in a cohort of 195 veterans, enrolled in the Chronic Effects of Neurotrauma Consortium Longitudinal Study. We examined relationships between candidate biomarkers and symptoms of postconcussive syndrome (PCS), posttraumatic stress disorder (PTSD), and depression. Biomarker levels were compared among those with no traumatic brain injury (TBI) (controls), 1-2 mTBIs, and repetitive (3 or more) mTBIs. RESULTS: Elevated exosomal and plasma levels of NfL were associated with repetitive mTBIs and with chronic PCS, PTSD, and depression symptoms. Plasma TNF-α levels correlated with PCS and PTSD symptoms. The total number of mTBIs correlated with exosomal and plasma NfL levels and plasma IL-6. Increased number of years since the most recent TBI correlated with higher exosomal NfL and lower plasma IL-6 levels, while increased number of years since first TBI correlated with higher levels of exosomal and plasma NfL, as well as plasma TNF-α and VEGF. CONCLUSION: Repetitive mTBIs are associated with elevated exosomal and plasma levels of NfL, even years following these injuries, with the greatest elevations in those with chronic PCS, PTSD, and depression symptoms. Our results suggest a possible neuroinflammatory and axonal disruptive basis for symptoms that persist years after mTBI, especially repetitive.


Assuntos
Concussão Encefálica/sangue , Exossomos/química , Proteínas de Neurofilamentos/sangue , Saúde dos Veteranos , Veteranos , Adulto , Biomarcadores , Traumatismos por Explosões/sangue , Traumatismos por Explosões/complicações , Concussão Encefálica/complicações , Estudos Transversais , Depressão/sangue , Depressão/etiologia , Feminino , Seguimentos , Humanos , Interleucina-6/sangue , Masculino , Pessoa de Meia-Idade , Síndrome Pós-Concussão/sangue , Síndrome Pós-Concussão/etiologia , Prognóstico , Degeneração Retrógrada , Índice de Gravidade de Doença , Transtornos de Estresse Pós-Traumáticos/sangue , Transtornos de Estresse Pós-Traumáticos/etiologia , Fator de Necrose Tumoral alfa/análise , Fator A de Crescimento do Endotélio Vascular/sangue , Guerra
10.
Sci Rep ; 10(1): 8686, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457479

RESUMO

Exosomes are cell-secreted microvesicles that play important roles in epithelial ovarian cancer (EOC) progression, as they are constantly secreted into ascites fluids. While cells spontaneously release exosomes, alterations in intracellular calcium or extracellular pH can release additional exosomes. Yet, little is known about how these exosomes compare to those that are continuously released without stimulation and how they mediate cellular activities important in cancer progression. Here, we demonstrate that chelation of extracellular calcium leads to release of chelation-induced exosomes (CI-exosomes) from OVCAR-3 EOC cells. CI-exosomes display a unique miRNA profile compared to naturally secreted exosomes (SEC-exosomes). Furthermore, treatment with CI- and SEC-exosomes leads to differential biophysical and functional changes including, adhesion and migration in EOC-derived fibroblasts that suggest the development of a malignant tumor microenvironment. This result highlights how tumor environmental factors contribute to heterogeneity in exosome populations and how different exosome populations mediate diversity in stromal cell behavior.


Assuntos
Fibroblastos Associados a Câncer/metabolismo , Exossomos/metabolismo , Actinas/metabolismo , Cálcio/química , Fibroblastos Associados a Câncer/citologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Quelantes/química , Quelantes/farmacologia , Progressão da Doença , Exossomos/química , Feminino , Humanos , MicroRNAs/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Análise de Componente Principal , Vinculina/metabolismo
11.
Toxicology ; 440: 152490, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32418910

RESUMO

Testicular injury is often observed in drug development. Serum hormones are usually used as noninvasive biomarkers for testicular injury; however, their sensitivities are low. Therefore, it is difficult to monitor testicular injury in drug development. In recent years, molecules in body fluid exosomes have attracted attention as biomarkers for diseases. In this study, small RNAs in serum exosomes were analyzed to identify noninvasive biomarkers of testicular injury in rats, which are often used in preclinical drug development. The rat models of testicular injury were prepared by a single oral administration of 2000 mg/kg ethylene glycol monomethyl ether, in which spermatocyte degeneration and Sertoli cell vacuolation were observed, or 400 mg/kg carbendazim, in which Sertoli cell vacuolation and seminiferous tubule dilation were observed. Serum exosomal small RNA-seq analysis of these models was performed. The analysis identified 3 small RNAs that fluctuated in common between the models, and miR-423-5p and miR-128-3p were selected as candidate markers. For evaluating these candidate markers in other testicular injury models, the models were prepared by a single oral administration of 60 mg/kg 1,3-dinitrobenzene or 500 mg/kg nitrofurazone, and spermatocyte degeneration and Sertoli cell vacuolation were observed. In qPCR analysis, these exosomal miRNAs were upregulated in all models except for the 1,3-dinitrobenzene model, in which severe hemolysis was observed. By contrast, these miRNAs in whole serum extracts did not significantly change in any of the models. In conclusion, we identified miR-423-5p and miR-128-3p in serum exosomes as noninvasive biomarkers for testicular injury in rats.


Assuntos
Biomarcadores/análise , Exossomos/química , RNA Citoplasmático Pequeno/análise , Doenças Testiculares/diagnóstico , Animais , Benzimidazóis/toxicidade , Carbamatos/toxicidade , Dinitrobenzenos/toxicidade , Masculino , MicroRNAs/efeitos dos fármacos , Nitrofurazona/toxicidade , Ratos , Ratos Sprague-Dawley , Células de Sertoli/química , Células de Sertoli/patologia , Espermatócitos/química , Espermatócitos/efeitos dos fármacos , Vacúolos/efeitos dos fármacos , Vacúolos/patologia
12.
Mikrochim Acta ; 187(5): 282, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32318822

RESUMO

A sandwich-type fluorescent biosensor for the determination of tumor-related exosome was designed. It is based on magnetic nanoparticle (MNP) capture and horseradish peroxidase (HRP) catalysis. MNPs were used as the substrate to capture exosomes by modifying the CD63 antibody on MNPs surface. After that, the biotinylated epithelial cell adhesion molecule (EpCAM) antibody was used to capture the tumor-related exosomes, which specifically express EpCAM. A novel method for the fluorescence measurement of tumor-associated exosome was achieved, with a detection limit as low as 200 (± 9) particles mL-1. The analytical range of this method is from 576 (± 15) particles mL-1 to 5.76 × 107 (± 5.1 × 105) particles mL-1. For the fluorescence measurement, the excitation wavelength was set to 320 nm. Fluorescent spectra were collected at emission wavelength in the range 370 to 550 nm; the data shown in the calibration plot were studied by using the fluorescence intensity at 406 nm. This sensor was also able to successfully detect the exosomes from the plasma of patients with hepatocellular carcinoma (HCC) and healthy humans. Graphical abstract Schematic representation of the sensing process of immunoassay-type biosensor based on magnetic nanoparticle capture and the fluorescence signal formed by the horseradish peroxidase (HRP) catalysis for tumor-related exosome determination.


Assuntos
Técnicas Biossensoriais , Carcinoma Hepatocelular/sangue , Exossomos/química , Fluorescência , Peroxidase do Rábano Silvestre/metabolismo , Imunoensaio , Neoplasias Hepáticas/sangue , Biocatálise , Calibragem , Carcinoma Hepatocelular/diagnóstico , Exossomos/metabolismo , Células Hep G2 , Peroxidase do Rábano Silvestre/química , Humanos , Neoplasias Hepáticas/diagnóstico , Nanopartículas de Magnetita/química , Tamanho da Partícula , Espectrometria de Fluorescência , Propriedades de Superfície , Células Tumorais Cultivadas
13.
Talanta ; 214: 120851, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32278412

RESUMO

As non-invasive biomarkers, exosomes are of great significance to diseases diagnosis. However, sensitive and accurate detection of exosomes still remains technical challenges. Herein, inspired by nature's "one-to-many" concept, we design a biosensor mimicking the cactus with numerous thorns to detect exosomes. The biosensor is composed of CD63 antibodies, resembling the roots of cactus, to capture exosomes, and the exosomes resemble the stems. Cholesterol-labeled DNA (DNA anchor) binding to streptavidin modified horseradish peroxidase (HRP) can insert into exosomes membrane, which seems the thorns. The readout signal is produced through HRP-catalyzed hydrogen peroxide (H2O2) mediated oxidation of 1,4-phenylenediamine (PPD) to form 2,5-diamino-NN'-bis-(p-aminophenyl)-1,4-benzoquinone di-imine (PPDox). The PPDox can quench fluorescence of fluorescein through inner filter effect (IFE), which provides fluorescent signal for exosomes detection. Based on this principle, the obtained exosomes solution is qualitatively and quantitatively analyzed by our biosensor, with the comparison to current standard methods by nanoparticle tracking analysis (NTA) and commercial enzyme-linked immunosorbent assay (ELISA) kit. The linear range is from 1.0 × 104 to 5.0 × 105 particles µL-1 with the limit of detection 3.40 × 103 particles µL-1 and 3.12 × 103 particles µL-1 for colorimetric and fluorescent assays, respectively. Meanwhile, our biosensor exhibits good selectivity, and can eliminate the interference from proteins. This dual-modal biosensor shows favorable performance towards analytical application in clinic samples, pushing one step further towards practical clinical use.


Assuntos
Técnicas Biossensoriais , Colorimetria , Ensaio de Imunoadsorção Enzimática , Exossomos/química , Fluorescência , Biocatálise , Biomarcadores/análise , Biomarcadores/metabolismo , Exossomos/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Tamanho da Partícula , Fenilenodiaminas/química , Fenilenodiaminas/metabolismo , Propriedades de Superfície
14.
Life Sci ; 248: 117473, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32114007

RESUMO

MicroRNAs (miRNAs) are a group of tiny molecules of 18-22 nucleotide long noncoding RNA that regulate the post-transcriptional gene expression through translational inhibition and/or mRNA destabilization. Because of their involvement in important developmental processes, it is highly likely that the altered expression of miRNAs could be associated with abnormal conditions like suboptimal growth or diseases. Thus, the expression of miRNAs can be used as biomarkers in pathophysiological conditions. Recently, a handful of miRNAs are detected in cell-free conditions including biofluids and cell culture media and they exhibit specific expression patterns that are associated with altered physiological conditions. Extracellular miRNAs are not only extremely stable outside cells in a variety of biofluids but also they are easy to acquire. These characteristics led to the idea of using extracellular miRNAs as a potential biomarker for the onset and prognosis of cancer. Although miRNAs have been proposed as a potential diagnostic tool for cancer detection, their application in the routine clinical investigation is yet to come. First, this review will provide an insight into the extracellular miRNAs, particularly, their release mechanisms and characteristics, and the potential of extracellular miRNAs as a biomarker in cancer detection. Finally, it will discuss the potential of using extracellular miRNAs in different cancer diagnoses and challenges associated with the clinical application of extracellular miRNAs as noninvasive biomarkers.


Assuntos
Biomarcadores Tumorais/genética , MicroRNA Circulante/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/diagnóstico , Neoplasias/genética , RNA Neoplásico/genética , Biomarcadores Tumorais/sangue , MicroRNA Circulante/sangue , Detecção Precoce de Câncer/métodos , Exossomos/química , Feminino , Humanos , Masculino , Neoplasias/sangue , Neoplasias/patologia , Especificidade de Órgãos , Prognóstico , RNA Neoplásico/sangue
15.
PLoS One ; 15(3): e0228871, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32119684

RESUMO

Exosomes are extracellular vesicles (EVs) of ~20-200 nm diameter that shuttle DNAs, RNAs, proteins and other biomolecules between cells. The large number of biomolecules present in exosomes demands the frequent use of high-throughput analysis. This, in turn, requires technical replicates (TRs), and biological replicates (BRs) to produce accurate results. As the number and abundance of identified biomolecules varies between replicates (Rs), establishing the replicate variability predicted for the event under study is essential in determining the number of Rs required. Although there have been few reports of replicate variability in high throughput biological data, none of them focused on exosomes. Herein, we determined the replicate variability in protein profiles found in exosomes released from 3 lung adenocarcinoma cell lines, H1993, A549 and H1975. Since exosome isolates are invariably contaminated by a small percentage of ~200-300 nm microvesicles, we refer to our samples as exosome-enriched EVs (EE-EVs). We generated BRs of EE-EVs from each cell line, and divided each group into 3 TRs. All Rs were analyzed by liquid chromatography/mass spectrometry (LC/MS/MS) and customized bioinformatics and biostatistical workflows (raw data available via ProteomeXchange: PXD012798). We found that the variability among TRs as well as BRs, was largely qualitative (protein present or absent) and higher among BRs. By contrast, the quantitative (protein abundance) variability was low, save for the H1975 cell line where the quantitative variability was significant. Importantly, our replicate strategy identified 90% of the most abundant proteins, thereby establishing the utility of our approach.


Assuntos
Exossomos/química , Vesículas Extracelulares/química , Proteínas/análise , Proteômica/métodos , Células A549 , Biologia Computacional/métodos , Humanos
16.
Nucleic Acids Res ; 48(5): 2220-2231, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32020194

RESUMO

Hybridization chain reaction (HCR) was a significant discovery for the development of nanoscale materials and devices. One key challenge for HCR is the vulnerability to background leakage in the absence of the initiator. Here, we systematically analyze the sources of leakage and refine leak-resistant rule by using molecular thermodynamics and dynamics, biochemical and biophysical methods. Transient melting of DNA hairpin is revealed to be the underlying cause of leakage and that this can be mitigated through careful consideration of the sequence thermodynamics. The transition threshold of the energy barrier is proposed as a testing benchmark of leak-resistance DNA hairpins. The universal design of DNA hairpins is illustrated by the analysis of hsa-miR-21-5p as biomarker when used in conjunction with surface-enhanced Raman spectroscopy. We further extend the strategy for specific signal amplification of miRNA homologs. Significantly, it possibly provides a practical route to improve the accuracy of DNA self-assembly for signal amplification, and that could facilitate the development of sensors for the sensitive detection of interest molecules in biotechnology and clinical medicine.


Assuntos
DNA/química , Sequências Repetidas Invertidas , MicroRNAs/química , Hibridização de Ácido Nucleico/métodos , Pareamento de Bases , Benchmarking , DNA/genética , DNA/metabolismo , Exossomos/química , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Análise Espectral Raman , Termodinâmica , Neoplasias da Bexiga Urinária/química , Neoplasias da Bexiga Urinária/urina
17.
Science ; 367(6478)2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32029601

RESUMO

The study of extracellular vesicles (EVs) has the potential to identify unknown cellular and molecular mechanisms in intercellular communication and in organ homeostasis and disease. Exosomes, with an average diameter of ~100 nanometers, are a subset of EVs. The biogenesis of exosomes involves their origin in endosomes, and subsequent interactions with other intracellular vesicles and organelles generate the final content of the exosomes. Their diverse constituents include nucleic acids, proteins, lipids, amino acids, and metabolites, which can reflect their cell of origin. In various diseases, exosomes offer a window into altered cellular or tissue states, and their detection in biological fluids potentially offers a multicomponent diagnostic readout. The efficient exchange of cellular components through exosomes can inform their applied use in designing exosome-based therapeutics.


Assuntos
Exossomos , Animais , Doenças Cardiovasculares/metabolismo , Comunicação Celular , Exossomos/química , Exossomos/metabolismo , Exossomos/fisiologia , Humanos , Infecções/imunologia , Doenças Metabólicas/metabolismo , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Reprodução
18.
Sci Rep ; 10(1): 1500, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001768

RESUMO

The polycystin-1 (PC1), polycystin-2 (PC2) and fibrocystin proteins, the respective products of the PKD1, PKD2 and PKHD1 genes, are abundant in urinary exosome-like vesicles (ELVs) where they form the polycystin complex (PCC). ELVs are 100 nm diameter membrane vesicles shed into the urine by the cells lining the nephron. Using MS/MS analysis of ELVs from individuals with PKD1 mutations and controls, we show that in addition to the well-described GPS/GAIN cleavage event in PC1 at 3048 aa and the proprotein convertase cleavage (PPC) event in fibrocystin at 3616 aa, there are multiple other cleavage events in these proteins. The C-terminal 11 transmembrane portion of PC1 undergoes three cleavage events in vivo. The absence of peptides from the C-terminal cytoplasmic tail of fibrocystin implies a cleavage event close to its single TM domain prior to loading onto the ELVs. There is also evidence that the C-terminal tail of PC2 is also cleaved in ELVs. Native gel analysis of the PCC shows that the entire complex is  > 2 MDa in size and that N-terminal GPS/GAIN cleaved PC1 and PPC cleaved fibrocystin ectodomains can be released under non-reducing conditions and resolve at 300 kDa. This paper shows that the three major human cystogene proteins are detectable in human urinary ELVs and that all three undergo post-translational proteolytic processing. Human urinary ELVs may be a useful source of material in the search for proteins that interact with the PCC.


Assuntos
Receptores de Superfície Celular/análise , Canais de Cátion TRPP/urina , Sequência de Aminoácidos , Exossomos/química , Glicosilação , Humanos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/urina , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/urina , Proteólise , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Canais de Cátion TRPP/química , Canais de Cátion TRPP/genética
19.
Biosens Bioelectron ; 154: 112066, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32056961

RESUMO

Non-small cell lung cancer (NSCLC) have been reported to secret a high concentration of exosomes into blood circulatory system, which is one of sensitive and non-invasive biomarkers for NSCLC's early-stage diagnosis. But it is still lack of feasible and accurate methods to analyze the different NSCLC cells-derived exosomes. Herein, we built a SPRi biosensing assay for high-sensitive and multiplex characterizations of NSCLC-derived exosomes by bioaffinity interactions of antibodies and different recognition sites. By this way, the exosomes derived from normal lung and NSCLC cells can be effectively distinguished through precise identification of the exosomal protein pattern. And the multiplex characterizations of NSCLC-related exosomes are also achieved by anti-CD63, anti-EGFR and anti-EpCAM modified SPRi array. The limit of detection (LOD) of this SPRi-based biosensor approaches to the level of 104 particles/µL with the help of functionalized gold nanoparticles. Besides, the developed biosensing assay was successfully applied in the determination of exosomes purified from clinical plasma samples. This SPRi biosensing strategy might offer a potential alternative for massive high-throughput screening for NSCLC in clinical specimens.


Assuntos
Técnicas Biossensoriais , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Exossomos/química , Nanopartículas Metálicas/química , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Exossomos/patologia , Ouro/química , Humanos , Pulmão/química , Pulmão/patologia
20.
Fertil Steril ; 113(2): 364-373.e2, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32106990

RESUMO

OBJECTIVE: To demonstrate the feasibility of studying exosomes directly from peritoneal fluid, we isolated exosomes from endometriosis patient samples and from controls, and characterized their cargo. DESIGN: Case-control experimental study. SETTING: Academic clinical center. PATIENT (S): Women with and without endometriosis who underwent laparoscopic surgery (n = 28 in total). INTERVENTION (S): None. MAIN OUTCOME MEASURE (S): Concentration of exosomes within peritoneal fluid and protein content of the isolated exosomes. RESULT (S): Peritoneal fluid samples were pooled according to the cycle phase and disease stage to form six experimental groups, from which the exosomes were isolated. Exosomes were successfully isolated from peritoneal fluid in all the study groups. The concentration varied with cycle phase and disease stage. Proteomic analysis showed specific proteins in the exosomes derived from endometriosis patients that were absent in the controls. Five proteins were found exclusively in the endometriosis groups: PRDX1, H2A type 2-C, ANXA2, ITIH4, and the tubulin α-chain. CONCLUSION (S): Exosomes are present in peritoneal fluid. The characterization of endometriosis-specific exosomes opens up new avenues for the diagnosis and investigation of endometriosis.


Assuntos
Líquido Ascítico/química , Endometriose/metabolismo , Exossomos/química , Proteínas/análise , Adulto , Anexina A2/análise , Líquido Ascítico/patologia , Estudos de Casos e Controles , Endometriose/patologia , Exossomos/ultraestrutura , Estudos de Viabilidade , Feminino , Histonas/análise , Humanos , Pessoa de Meia-Idade , Peroxirredoxinas/análise , Proteínas Secretadas Inibidoras de Proteinases/análise , Proteômica , Tubulina (Proteína)/análise , Adulto Jovem
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