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1.
J Extracell Vesicles ; 13(7): e12476, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38978287

RESUMO

The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.


Assuntos
Vesículas Extracelulares , Infecções por HIV , HIV-1 , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/virologia , Infecções por HIV/virologia , Infecções por HIV/metabolismo , Vírion/metabolismo , Ultracentrifugação/métodos , Linfócitos T/virologia , Linfócitos T/metabolismo , Tetraspanina 30/metabolismo , Tamanho da Partícula
2.
Mikrochim Acta ; 191(8): 448, 2024 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-38967796

RESUMO

Surface functionalization strategy is becoming a crucial bridge from magnetic nanoparticles (MNPs) to their broad bio-application. To realize the multiple functions of MNPs such as magnetic manipulation, target capture, and signal amplification in their use of electrochemical biosensing, co-crosslinking strategy was proposed here to construct dual-functionalized MNPs by combining ultra-sensitive redox moieties and specific biological probes. In this work, MNPs with a TEM size of 10 nm were synthesized by co-precipitation for amination and PEGylation to maintain colloid stability once dispersed in high-ionic-strength buffer (such as phosphate-buffered saline). Then, MNPs@IgG were prepared via the bis(sulfosuccinimidyl) suberate (BS3) cross-linker to conjugate these IgG onto the MNP surface, with a binding efficiency of 73%. To construct dual-functionalized MNPs, these redox probes of ferrocene-NHS (Fc) were co-crosslinked onto the MNP surface, together with IgG, by using BS3. The developed MNPs@Redox@IgG were characterized by SDS‒PAGE to identify IgG binding and by square wave voltammetry (SWV) to validate the redox signal. Additionally, the anti-CD63 antibodies were selected for the development of MNPs@anti-CD63 for use in the bio-testing of exosome sample capture. Therefore, co-crosslinking strategy paved a way to develop dual-functionalized MNPs that can be an aid of their potential utilization in diagnostic assay or electrochemical methods.


Assuntos
Reagentes de Ligações Cruzadas , Imunoglobulina G , Nanopartículas de Magnetita , Oxirredução , Nanopartículas de Magnetita/química , Imunoglobulina G/química , Humanos , Reagentes de Ligações Cruzadas/química , Compostos Ferrosos/química , Metalocenos/química , Técnicas Biossensoriais/métodos , Tetraspanina 30/imunologia , Técnicas Eletroquímicas/métodos
3.
Anal Chim Acta ; 1314: 342779, 2024 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-38876518

RESUMO

Alzheimer's disease (AD) is the most common neurodegenerative disease in the world and poses a huge challenge to global healthcare. Early and accurate detection of amyloid-ß (1-42) (Aß42), a key biomarker of AD, is crucial for effective diagnosis and intervention of AD. Specific or overexpressed proteins on extracellular vesicles (EVs) describe a close correlation with the occurrence and development of diseases. EVs are a very promising non-invasive biomarker for the diagnosis of AD and other diseases. As a sensitive, simple and rapid analytical method, fluorescence resonance energy transfer (FRET) has been widely applied in the detection of EVs. Herein, we developed a dual labelling strategy for simultaneously detecting EV membrane proteins of Aß42 and CD63 based on FRET pair consisting of Au nanoclusters (AuNCs) and polydopamine nanospheres (PDANSs). The constructed nanoprobe, termed EVMPFAP assay, could specifically measure the Aß42 and CD63 on EVs with excellent sensitivity, high specificity and satisfactory accuracy. The limit of detection of EVMPFAP assay was 1.4 × 103 particles mL-1 and the linear range was from 104 to 108 particles mL-1. EVMPFAP assay was successfully used to analyze plasma EVs to distinguish AD and healthy mice. We expect that EVMPFAP assay can be routinely applied for early diagnosis and development-monitoring of AD, thus facilitating the fight against AD.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Vesículas Extracelulares , Transferência Ressonante de Energia de Fluorescência , Ouro , Nanopartículas Metálicas , Tetraspanina 30 , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/metabolismo , Vesículas Extracelulares/química , Animais , Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/sangue , Camundongos , Humanos , Tetraspanina 30/metabolismo , Ouro/química , Nanopartículas Metálicas/química , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/química , Polímeros/química , Indóis/química , Limite de Detecção
4.
Mol Biol Rep ; 51(1): 749, 2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38874800

RESUMO

Background The incidence of various types of cancers, including leukemia, is on the rise and many challenges in both drug resistance and complications related to chemotherapy appeared. Recently, the development and application of extracellular vesicles (EV) such as exosomes in the management of cancers, especially leukemia, holds great significance. In this article, we extracted exosomes from NALM6 cells and assessed their regulatory effects on proliferation and apoptosis in mesenchymal stem cells (MSCs). Method and result We first verified the exosomes using various techniques, including flow cytometry, transient electron microscopy, dynamic light scattering (DLS), and BCA protein assay. Then MTT analysis and flowcytometry (apoptosis and cell cycle assay) besides gene expressions were employed to determine the state of MSC proliferations. The results indicated that exosome-specific pan markers like CD9, CD63, and CD81 were present. Through DLS, we found out that the mean size of the exosomes was 89.68 nm. The protein content was determined to be 956.292 µg/ml. Analysis of MTT, flow cytometry (cell cycle and apoptosis assay), and RT-qPCR showed that in the dose of 50 µg/ml the proliferation of MSCs was increased significantly (p-value < 0.05). Conclusion All these data showed that exosomes use several signaling pathways to increase the MSCs' proliferation and drug resistance, ultimately leading to high mortalities and morbidities of acute lymphoblastic leukemia.


Assuntos
Apoptose , Proliferação de Células , Exossomos , Células-Tronco Mesenquimais , Exossomos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Humanos , Proliferação de Células/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Tetraspanina 29/metabolismo , Tetraspanina 29/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Tetraspanina 30/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética
5.
Int J Mol Sci ; 25(11)2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38892363

RESUMO

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.


Assuntos
Autofagia , Vesículas Extracelulares , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestrutura , Linhagem Celular Tumoral , Microdomínios da Membrana/metabolismo , Exossomos/metabolismo , Exossomos/ultraestrutura , Tetraspanina 30/metabolismo , Fibrossarcoma/metabolismo , Fibrossarcoma/patologia , Proteínas Associadas aos Microtúbulos/metabolismo
6.
BMC Cancer ; 24(1): 698, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38849760

RESUMO

BACKGROUND: Tumor-associated macrophages (TAMs) constitute a substantial part of human hepatocellular carcinoma (HCC). The present study was devised to explore TAM diversity and their roles in HCC progression. METHODS: Through the integration of multiple 10 × single-cell transcriptomic data derived from HCC samples and the use of consensus nonnegative matrix factorization (an unsupervised clustering algorithm), TAM molecular subtypes and expression programs were evaluated in detail. The roles played by these TAM subtypes in HCC were further probed through pseudotime, enrichment, and intercellular communication analyses. Lastly, vitro experiments were performed to validate the relationship between CD63, which is an inflammatory TAM expression program marker, and tumor cell lines. RESULTS: We found that the inflammatory expression program in TAMs had a more obvious interaction with HCC cells, and CD63, as a marker gene of the inflammatory expression program, was associated with poor prognosis of HCC patients. Both bulk RNA-seq and vitro experiments confirmed that higher TAM CD63 expression was associated with the growth of HCC cells as well as their epithelial-mesenchymal transition, metastasis, invasion, and the reprogramming of lipid metabolism. CONCLUSIONS: These analyses revealed that the TAM inflammatory expression program in HCC is closely associated with malignant tumor cells, with the hub gene CD63 thus representing an ideal target for therapeutic intervention in this cancer type.


Assuntos
Carcinoma Hepatocelular , Progressão da Doença , Transição Epitelial-Mesenquimal , Neoplasias Hepáticas , Tetraspanina 30 , Macrófagos Associados a Tumor , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Transição Epitelial-Mesenquimal/genética , Macrófagos Associados a Tumor/metabolismo , Macrófagos Associados a Tumor/imunologia , Macrófagos Associados a Tumor/patologia , Tetraspanina 30/metabolismo , Tetraspanina 30/genética , Metabolismo dos Lipídeos/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Prognóstico , Reprogramação Celular/genética
7.
Biosens Bioelectron ; 261: 116492, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-38870828

RESUMO

Exosomes have been considered as promising biomarkers for cancer diagnosis due to their abundant information from originating cells. However, sensitive and reliable detection of exosomes is still facing technically challenges due to the lack of a sensing platform with high sensitivity and reproducibility. To address the challenges, here we propose a portable surface plasmon resonance (SPR) sensing of exosomes with a three-layer Au mirror/SiO2 spacer/Au nanohole sensor, fabricated by an economical polystyrene nanosphere self-assembly method. The SiO2 spacer can act as an optical cavity and induce mode hybridization, leading to excellent optimization of both sensitivity and full width at half maximum compared with normal single layer Au nanohole sensors. When modified with CD63 or EpCAM aptamers, a detection of limit (LOD) of as low as 600 particles/µL was achieved. The sensors showed good capability to distinguish between non-tumor derived L02 exosomes and tumor derived HepG2 exosomes. Additionally, high reproducibility was also achieved in detection of artificial serum samples with RSD as low as 2%, making it feasible for clinical applications. This mode hybridization plasmonic sensor provides an effective approach to optimize the detection sensitivity of exosomes, pushing SPR sensing one step further towards cancer diagnosis.


Assuntos
Exossomos , Ouro , Limite de Detecção , Dióxido de Silício , Ressonância de Plasmônio de Superfície , Exossomos/química , Humanos , Ouro/química , Dióxido de Silício/química , Aptâmeros de Nucleotídeos/química , Molécula de Adesão da Célula Epitelial , Tetraspanina 30 , Células Hep G2 , Técnicas Biossensoriais/métodos , Técnicas Biossensoriais/instrumentação , Reprodutibilidade dos Testes , Desenho de Equipamento , Nanosferas/química , Hibridização de Ácido Nucleico , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/análise
8.
Commun Biol ; 7(1): 677, 2024 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-38830977

RESUMO

We present a quantitative sandwich immunoassay for CD63 Extracellular Vesicles (EVs) and a constituent surface cargo, EGFR and its activity state, that provides a sensitive, selective, fluorophore-free and rapid alternative to current EV-based diagnostic methods. Our sensing design utilizes a charge-gating strategy, with a hydrophilic anion exchange membrane functionalized with capture antibodies and a charged silica nanoparticle reporter functionalized with detection antibodies. With sensitivity and robustness enhancement by the ion-depletion action of the membrane, this hydrophilic design with charged reporters minimizes interference from dispersed proteins, thus enabling direct plasma analysis without the need for EV isolation or sensor blocking. With a LOD of 30 EVs/µL and a high relative sensitivity of 0.01% for targeted proteomic subfractions, our assay enables accurate quantification of the EV marker, CD63, with colocalized EGFR by an operator/sample insensitive universal normalized calibration. We analysed untreated clinical samples of Glioblastoma to demonstrate this new platform. Notably, we target both total and "active" EGFR on EVs; with a monoclonal antibody mAb806 that recognizes a normally hidden epitope on overexpressed or mutant variant III EGFR. Analysis of samples yielded an area-under-the-curve (AUC) value of 0.99 and a low p-value of 0.000033, surpassing the performance of existing assays and markers.


Assuntos
Receptores ErbB , Vesículas Extracelulares , Glioblastoma , Tetraspanina 30 , Humanos , Glioblastoma/sangue , Glioblastoma/diagnóstico , Glioblastoma/metabolismo , Tetraspanina 30/metabolismo , Receptores ErbB/metabolismo , Vesículas Extracelulares/metabolismo , Imunoensaio/métodos , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/diagnóstico
9.
Anal Methods ; 16(26): 4262-4267, 2024 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-38884118

RESUMO

Exosomes have significant functions in intercellular communication, as well as in tumor migration and invasion. Nevertheless, the precise identification of exosomes poses a significant obstacle due to their low abundance in biofluids and potential disruption caused by free protein molecules, such as CD63 protein. In this study, we have developed a signal amplification method for precise detection of exosomes using a proximity ligation hybridization triggered structure-switching approach. The method involves the dual-recognition of exosomes by two probes: an aptamer probe that recognizes the exosomal surface protein CD63 (L1 probe), and a cholesterol probe that targets the biolipid layer of the exosomes (L2 probe). Based on the dual-recognition of exosomes, we have successfully developed an accurate and sensitive approach that integrates the proximity ligation hybridization technique with a structure-switching based signal cycle. This approach allows for the simultaneous analysis of two biomarkers, enabling both quantification and tracing of exosomes without the need for enzymes. Eventually, the proposed method exhibits a wide detection range of 5 orders of magnitude and a low limit of detection of 36 particles per µL, making it suitable for a wide range of applications in the fields of biological science, biomedical engineering, and personalized medicine.


Assuntos
Exossomos , Hibridização de Ácido Nucleico , Tetraspanina 30 , Exossomos/química , Exossomos/metabolismo , Humanos , Tetraspanina 30/metabolismo , Aptâmeros de Nucleotídeos/química , Limite de Detecção , Técnicas Biossensoriais/métodos
10.
ACS Sens ; 9(6): 3244-3252, 2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-38785322

RESUMO

Extracellular vesicles (EVs) are small cellular organelles involved in intracellular signaling and cell-to-cell interactions. Recent studies suggested that exosomes may have potential applications in the diagnosis and treatment of cancer and neurodegenerative diseases. In this study, extracellular vesicles of the human nonsmall cell lung cancer cell line H1299 and the unlabeled antiCD63 antibody were imaged using a new label-free terahertz chemical microscopy (TCM) technique to detect changes in the terahertz wave amplitude. To verify the high specificity of the protein biomarkers and the sensitivity of the biosensor surface, we also confirmed the selective binding of the antibody to the antigen, bovine serum albumin, and cancer cells. We also performed real-time measurements of the interaction between EVs from the H1299 cell and the antiCD63 antibody, which showed that the amount of change in the terahertz intensity increased with increasing concentration and the time to saturation decreased. Finally, to reuse the used biosensors (sensing plates), plasma-oxygen cleaning was used, and the activity of the biosensor surface was confirmed by terahertz microscopy and atomic force microscopy and was found to be reusable after less than 3 min of cleaning. Consequently, terahertz chemical microscopy was able to detect the presence or absence of antigen-antibody binding and its reaction rate and binding strength.


Assuntos
Técnicas Biossensoriais , Vesículas Extracelulares , Tetraspanina 30 , Humanos , Tetraspanina 30/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Linhagem Celular Tumoral , Técnicas Biossensoriais/métodos , Microscopia/métodos , Soroalbumina Bovina/química
11.
Biosens Bioelectron ; 259: 116380, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38754193

RESUMO

Exosomes, as novel biomarker for liquid biopsy, exhibit huge important potential value for cancer diagnosis. However, various proteins show different expression levels on exosomal membrane, and the absolute concentration of exosomes in clinical samples is easily influenced by a number of factors. Here, we developed a CRISPR/Cas12a and aptamer-chemiluminescence based analysis (CACBA) for the relative abundance determination of tumor-related protein positive exosomes in plasma for breast cancer diagnosis. The total concentration of exosomes was determined through captured CD63 using a CRISPR/Cas12a-based method with the LoD of 8.97 × 103 particles/µl. Meanwhile, EpCAM and MUC1 positive exosomes were quantitatively detected by aptamer-chemiluminescence (ACL) based method with the LoD of 1.45 × 102 and 3.73 × 102 particles/µl, respectively. It showed that the percentages of EpCAM and MUC1 positive exosomes offered an excellent capability to differentiate breast cancer patients and healthy donors. The high sensitivity, strong specificity, outstanding anti-interference capability, and steady recovery rate of this approach offered higher accuracy and robustness than the commercialized method in clinical trial. In addition with good stability, easy preparation and low cost, this method not only provides a new approach to rapid analysis of exosome proteins, it may be quickly extended to the diagnoses of various cancers.


Assuntos
Aptâmeros de Nucleotídeos , Biomarcadores Tumorais , Técnicas Biossensoriais , Neoplasias da Mama , Sistemas CRISPR-Cas , Molécula de Adesão da Célula Epitelial , Exossomos , Mucina-1 , Humanos , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Exossomos/química , Exossomos/genética , Feminino , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Mucina-1/sangue , Mucina-1/genética , Mucina-1/análise , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Molécula de Adesão da Célula Epitelial/genética , Medições Luminescentes/métodos , Tetraspanina 30 , Limite de Detecção
12.
Biomolecules ; 14(5)2024 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-38785998

RESUMO

Small extracellular vesicles (sEVs) have emerged as promising therapeutic agents and drug delivery vehicles. Targeted modification of sEVs and their contents using genetic modification strategies is one of the most popular methods. This study investigated the effects of p53 fusion with arrestin domain-containing protein 1 (ARRDC1) and CD63 on the generation of sEVs, p53 loading efficiency, and therapeutic efficacy. Overexpression of either ARRDC1-p53 (ARP) or CD63-p53 (CDP) significantly elevated p53 mRNA and protein levels. The incorporation of ARRDC1 and CD63 significantly enhanced HEK293T-sEV biogenesis, evidenced by significant increases in sEV-associated proteins TSG101 and LAMP1, resulting in a boost in sEV production. Importantly, fusion with ARRDC1 or CD63 substantially increased the efficiency of loading both p53 fusion proteins and its mRNA into sEVs. sEVs equipped with ARP or CDP significantly enhanced the enrichment of p53 fusion proteins and mRNA in p53-null H1299 cells, resulting in a marked increase in apoptosis and a reduction in cell proliferation, with ARP-sEVs demonstrating greater effectiveness than CDP-sEVs. These findings underscore the enhanced functionality of ARRDC1- and CD63-modified sEVs, emphasizing the potential of genetic modifications in sEV-based therapies for targeted cancer treatment.


Assuntos
Apoptose , Vesículas Extracelulares , Tetraspanina 30 , Proteína Supressora de Tumor p53 , Humanos , Tetraspanina 30/metabolismo , Tetraspanina 30/genética , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Células HEK293 , Linhagem Celular Tumoral , Proliferação de Células , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteína 1 de Membrana Associada ao Lisossomo
13.
Talanta ; 275: 126182, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38701706

RESUMO

Exosomes, extracellular vesicles secreted by cells, play a crucial role in intercellular communication by transferring information from source cells to recipient cells. These vesicles carry important biomarkers, including nucleic acids and proteins, which provide valuable insights into the parent cells' status. As a result, exosomes have emerged as noninvasive indicators for the early diagnosis of cancer. Colorimetric biosensors have garnered significant attention due to their cost-effectiveness, simplicity, rapid response, and reproducibility. In this study, we employ sporopollenin microcapsules (SP), a natural biopolymer material derived from pollen, as a substrate for gold nanoparticles (AuNPs). By modifying the SP-Au complex with CD63 aptamers, we develop a label-free colorimetric biosensor for exosome detection. In the absence of exosomes, the SP-Au complex catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB), resulting in a color change from colorless to blue. However, the addition of exosomes inhibits the catalytic activity of the SP-Au complex due to coverage of exosomes on AuNPs. This colorimetric biosensor exhibits high sensitivity and selectivity for exosome detection, with a detection limit of 10 particles/µL and a wide linear range of 10 - 108 particles/µL. Additionally, the SP-Au biosensor demonstrates remarkable resistance to serum protein adsorption and excellent catalytic stability even in harsh environments, making it highly suitable for clinical diagnostics.


Assuntos
Técnicas Biossensoriais , Colorimetria , Exossomos , Ouro , Nanopartículas Metálicas , Colorimetria/métodos , Exossomos/química , Técnicas Biossensoriais/métodos , Humanos , Ouro/química , Nanopartículas Metálicas/química , Tetraspanina 30/metabolismo , Tetraspanina 30/análise , Biopolímeros/química , Biopolímeros/análise , Limite de Detecção , Benzidinas/química , Aptâmeros de Nucleotídeos/química , Cápsulas/química , Carotenoides
14.
Sci Adv ; 10(19): eadi9156, 2024 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-38718108

RESUMO

Exosomes are secreted vesicles of ~30 to 150 nm diameter that play important roles in human health and disease. To better understand how cells release these vesicles, we examined the biogenesis of the most highly enriched human exosome marker proteins, the exosomal tetraspanins CD81, CD9, and CD63. We show here that endocytosis inhibits their vesicular secretion and, in the case of CD9 and CD81, triggers their destruction. Furthermore, we show that syntenin, a previously described exosome biogenesis factor, drives the vesicular secretion of CD63 by blocking CD63 endocytosis and that other endocytosis inhibitors also induce the plasma membrane accumulation and vesicular secretion of CD63. Finally, we show that CD63 is an expression-dependent inhibitor of endocytosis that triggers the vesicular secretion of lysosomal proteins and the clathrin adaptor AP-2 mu2. These results suggest that the vesicular secretion of exosome marker proteins in exosome-sized vesicles occurs primarily by an endocytosis-independent pathway.


Assuntos
Endocitose , Exossomos , Tetraspanina 30 , Exossomos/metabolismo , Humanos , Tetraspanina 30/metabolismo , Biomarcadores/metabolismo , Sinteninas/metabolismo , Sinteninas/genética , Tetraspanina 28/metabolismo , Membrana Celular/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Tetraspanina 29/metabolismo
15.
Biosensors (Basel) ; 14(5)2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38785707

RESUMO

Exosomal biomarker detection holds great importance in the field of in vitro diagnostics, offering a non-invasive and highly sensitive approach for early disease detection and personalized treatment. Here, we proposed an "APPROACH" strategy, combining aptamer-mediated proximity ligation assay (PLA) with rolling circle amplification (RCA) and time-resolved Förster resonance energy transfer (TR-FRET) for the sensitive and semi-homogenous detection of exosomal biomarkers. PLA probes consisted of a cholesterol-conjugated oligonucleotide, which anchored to the membrane of an exosome, and a specific aptamer oligonucleotide that recognized a target protein of the exosome; the proximal binding of pairs of PLA probes to the same exosome positioned the oligonucleotides in the vicinity of each other, guiding the hybridization and ligation of two subsequently added backbone and connector oligonucleotides to form a circular DNA molecule. Circular DNA formed from PLA underwent rolling circle amplification (RCA) for signal amplification, and the resulting RCA products were subsequently quantified by TR-FRET. The limits of detection provided by APPROACH for the exosomal biomarkers CD63, PD-L1, and HER2 were 0.46 ng∙µL-1, 0.77 ng∙µL-1, and 1.1 ng∙µL-1, respectively, demonstrating excellent analytical performance with high sensitivity and quantification accuracy. Furthermore, the strategy afforded sensitive detection of exosomal CD63 with a LOD of 1.56 ng∙µL-1 in complex biological matrices, which underscored its anti-interference capability and potential for in vitro detection. The proposed strategy demonstrates wide-ranging applicability in quantifying diverse exosomal biomarkers while exhibiting robust analytical characteristics, including high sensitivity and accuracy.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , Exossomos , Transferência Ressonante de Energia de Fluorescência , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Biomarcadores , Técnicas de Amplificação de Ácido Nucleico/métodos , Tetraspanina 30
16.
Genes Chromosomes Cancer ; 63(5): e23246, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38747331

RESUMO

Dermatofibroma (DF) is a benign tumor that forms pedunculated lesions ranging in size from a few millimeters to 2 cm, usually affecting the extremities and trunks of young adults. Histopathologically, DF is characterized by the storiform proliferation of monomorphic fibroblast-like spindle cells. In addition to neoplastic cells, secondary elements such as foamy histiocytes, Touton-type giant cells, lymphoplasmacytes, and epidermal hyperplasia are characteristic histological features. Several histological variants, including atypical, cellular, aneurysmal, and lipidized variants, have been reported; cases with variant histologies are sometimes misdiagnosed as sarcomas. We present a case of metastasizing aneurysmal DF that was initially diagnosed as an angiosarcoma on biopsy. A 26-year-old woman was referred to our hospital with a gradually enlarging subcutaneous mass in her lower left leg. Positron emission tomography-computed tomography revealed high fluorodeoxyglucose uptake not only in the tumor but also in the left inguinal region. On biopsy, ERG and CD31-positive atypical spindle cells proliferated in slit-like spaces with extravasation, leading to the diagnosis of angiosarcoma. Histology of the wide-resection specimen was consistent with DF, and lymph node metastasis was also observed. Nanopore DNA sequencing detected CD63::PRKCD fusion and copy number gain, although CD63 was not included in the target region of adaptive sampling. This report highlights the importance of recognizing the unusual clinical, radiological, and pathological features of DF to avoid misdiagnosis, and the potential diagnostic utility of nanopore sequencer.


Assuntos
Hemangiossarcoma , Histiocitoma Fibroso Benigno , Sequenciamento por Nanoporos , Proteínas de Fusão Oncogênica , Adulto , Feminino , Humanos , Hemangiossarcoma/genética , Hemangiossarcoma/diagnóstico , Hemangiossarcoma/patologia , Histiocitoma Fibroso Benigno/genética , Histiocitoma Fibroso Benigno/diagnóstico , Histiocitoma Fibroso Benigno/patologia , Sequenciamento por Nanoporos/métodos , Proteínas de Fusão Oncogênica/análise , Proteínas de Fusão Oncogênica/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/diagnóstico , Tetraspanina 30/genética , Tetraspanina 30/metabolismo
17.
Int J Gynaecol Obstet ; 166(1): 99-106, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38650387

RESUMO

OBJECTIVE: Tubal ectopic pregnancy (EP) is a leading cause of maternal morbidity and mortality. Studies have suggested that infection-induced inflammatory responses are major risk factors for EP. The aim of the present study was to find an association between MMP2 and CD63 gene variants and risk of EP during Chlamydia trachomatis infection in an Indian population. METHODS: Fallopian tube samples of 120 EP and 120 tubal ligation women were collected. C. trachomatis was detected by PCR. The genotyping of MMP2 (rs17859882 G/T, rs7201A/C) and CD63(rs2231464 C/T, rs376086542 A/G) gene variants was done by qualitative real-time PCR using allelic discrimination method (VIC- and FAM-labeled). RESULTS: The frequency of GG or GT genotype of MMP2 G/T polymorphism (rs17859882) was 66.6% in infected EP and 36.7% in uninfected EP and 22% in tubal ligation controls (P < 0.0001), while the frequency of AC or CC genotype of MMP2 A/C polymorphism (rs7201) was 66.6% in infected EP and 20.6% in uninfected EP and 13.5% in tubal ligation controls (P < 0.0001). The frequency of CT or TT genotype of CD63 C/T polymorphism (rs2231464) was 74% in infected EP and 21.8% in uninfected EP and 11.8% tubal ligation controls (P < 0.0001), while the frequency of AG or GG genotype of CD63 A/G polymorphism (rs376086542) was 48.1% in infected EP and 41.3% in uninfected EP and 18.6% tubal ligation controls (P < 0.0001). CONCLUSIONS: The present study revealed a strong association between the presence of gene variants MMP2 (rs17859882 G/T, rs7201A/C) and CD63 (rs2231464 C/T, rs376086542 A/G) and risk of tubal EP during C. trachomatis infection.


Assuntos
Infecções por Chlamydia , Chlamydia trachomatis , Metaloproteinase 2 da Matriz , Polimorfismo de Nucleotídeo Único , Gravidez Tubária , Tetraspanina 30 , Humanos , Feminino , Adulto , Infecções por Chlamydia/genética , Chlamydia trachomatis/genética , Gravidez , Metaloproteinase 2 da Matriz/genética , Tetraspanina 30/genética , Gravidez Tubária/genética , Estudos de Casos e Controles , Genótipo , Índia , Predisposição Genética para Doença , Adulto Jovem
18.
Analyst ; 149(11): 3195-3203, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38651605

RESUMO

Extracellular vesicles (EVs) originating from cancer cells incorporate various critical biomolecules that can aid in early cancer diagnosis. However, the rapid analysis of these micro vesicles remains challenging due to their nano-scale size and overlapping dimensions, hindering sufficient capture in terms of quantity and purity. In this study, an acoustofluidic device was developed to enhance the yield of immune-captured EVs. The channel of the device was modified with degradable gelatin nanoparticles (∼220 nm) to increase the surface roughness, and subsequently treated with CD63 antibodies. The acoustic-induced streaming would prolong the rotation time of the EVs in the targeted continuous flow area, improving their aggregation towards the surrounding pillars and subsequent capture by the specific CD63 antibodies. Consequently, the capture efficiency of the device was improved when the signal was on, as evidenced by enhanced fluorescence intensity in the main channel. It is demonstrated that the acoustofluidic device could enhance the immune capture of EVs through acoustic mixing, showcasing great potential in the rapid and fast detection of EVs in liquid biopsy applications.


Assuntos
Vesículas Extracelulares , Gelatina , Nanopartículas , Tetraspanina 30 , Gelatina/química , Vesículas Extracelulares/química , Vesículas Extracelulares/imunologia , Nanopartículas/química , Humanos , Tetraspanina 30/metabolismo , Acústica , Dispositivos Lab-On-A-Chip
19.
J Cell Biol ; 223(8)2024 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-38668767

RESUMO

The microtubule cytoskeleton consists of microtubule subsets with distinct compositions of microtubule-associated proteins, which instruct the position and traffic of subcellular organelles. In the endocytic pathway, these microtubule-associated cues are poorly understood. Here, we report that in MDCK cells, endosomes with multivesicular body (MVB) and late endosome (LE) markers localize preferentially to microtubules coated with septin GTPases. Compared with early endosomes, CD63-containing MVBs/LEs are largely immotile on septin-coated microtubules. In vitro reconstitution assays revealed that the motility of isolated GFP-CD63 endosomes is directly inhibited by microtubule-associated septins. Quantification of CD63-positive endosomes containing the early endosome antigen (EEA1), the Rab7 effector and dynein adaptor RILP or Rab27a, showed that intermediary EEA1- and RILP-positive GFP-CD63 preferentially associate with septin-coated microtubules. Septin knockdown enhanced GFP-CD63 motility and decreased the percentage of CD63-positive MVBs/LEs with lysobiphosphatidic acid without impacting the fraction of EEA1-positive CD63. These results suggest that MVB maturation involves immobilization on septin-coated microtubules, which may facilitate multivesiculation and/or organelle-organelle contacts.


Assuntos
Microtúbulos , Corpos Multivesiculares , Septinas , Animais , Cães , Células Madin Darby de Rim Canino , Microtúbulos/química , Microtúbulos/metabolismo , Corpos Multivesiculares/química , Corpos Multivesiculares/metabolismo , Septinas/química , Septinas/metabolismo , Tetraspanina 30/metabolismo , Citoesqueleto/química , Citoesqueleto/metabolismo , Endocitose
20.
Anal Methods ; 16(17): 2751-2759, 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38634398

RESUMO

Exosomes have gained recognition as valuable reservoirs of biomarkers, holding immense potential for early cancer detection. Consequently, there is a pressing need for the development of an economical and highly sensitive exosome detection methodology. In this work, we present a fluorescence method for breast cancer-derived exosome detection based on Cu-triggered click reaction of azide-modified CD63 aptamer and alkyne functionalized Pdots. The detection threshold for the exosomes obtained from the breast cancer serum was determined to be 6.09 × 107 particles per µL, while the measurable range spanned from 6.50 × 107 to 1.30 × 109 particles per µL. The employed methodology achieved notable success in accurately distinguishing breast cancer patients from healthy individuals through serum analysis. The application of this method showcases the significant potential for early exosome analysis in the clinical diagnosis of breast cancer patients.


Assuntos
Alcinos , Aptâmeros de Nucleotídeos , Azidas , Técnicas Biossensoriais , Neoplasias da Mama , Química Click , Exossomos , Tetraspanina 30 , Humanos , Neoplasias da Mama/sangue , Feminino , Exossomos/química , Tetraspanina 30/metabolismo , Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Azidas/química , Alcinos/química , Corantes Fluorescentes/química , Polímeros/química
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