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1.
Annu Rev Biochem ; 88: 487-514, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-31220978

RESUMO

Exosomes are small, single-membrane, secreted organelles of ∼30 to ∼200 nm in diameter that have the same topology as the cell and are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates. Exosomes contain an array of membrane-associated, high-order oligomeric protein complexes, display pronounced molecular heterogeneity, and are created by budding at both plasma and endosome membranes. Exosome biogenesis is a mechanism of protein quality control, and once released, exosomes have activities as diverse as remodeling the extracellular matrix and transmitting signals and molecules to other cells. This pathway of intercellular vesicle traffic plays important roles in many aspects of human health and disease, including development, immunity, tissue homeostasis, cancer, and neurodegenerative diseases. In addition, viruses co-opt exosome biogenesis pathways both for assembling infectious particles and for establishing host permissiveness. On the basis of these and other properties, exosomes are being developed as therapeutic agents in multiple disease models.


Assuntos
Exossomos/metabolismo , Animais , Transporte Biológico , Exossomos/imunologia , Exossomos/fisiologia , Exossomos/ultraestrutura , Matriz Extracelular/metabolismo , Humanos , Neoplasias , Doenças Neurodegenerativas , Multimerização Proteica , Transdução de Sinais
2.
Genes Dev ; 37(5-6): 243-257, 2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36810209

RESUMO

Transfer RNAs (tRNAs) are small adaptor RNAs essential for mRNA translation. Alterations in the cellular tRNA population can directly affect mRNA decoding rates and translational efficiency during cancer development and progression. To evaluate changes in the composition of the tRNA pool, multiple sequencing approaches have been developed to overcome reverse transcription blocks caused by the stable structures of these molecules and their numerous base modifications. However, it remains unclear whether current sequencing protocols faithfully capture tRNAs existing in cells or tissues. This is specifically challenging for clinical tissue samples that often present variable RNA qualities. For this reason, we developed ALL-tRNAseq, which combines the highly processive MarathonRT and RNA demethylation for the robust assessment of tRNA expression, together with a randomized adapter ligation strategy prior to reverse transcription to assess tRNA fragmentation levels in both cell lines and tissues. Incorporation of tRNA fragments not only informed on sample integrity but also significantly improved tRNA profiling of tissue samples. Our data showed that our profiling strategy effectively improves classification of oncogenic signatures in glioblastoma and diffuse large B-cell lymphoma tissues, particularly for samples presenting higher levels of RNA fragmentation, further highlighting the utility of ALL-tRNAseq for translational research.


Assuntos
Biossíntese de Proteínas , RNA de Transferência , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA Mensageiro/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos
3.
Nat Methods ; 18(9): 1013-1026, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34446922

RESUMO

Extracellular vesicles (EVs) are nano-sized lipid bilayer vesicles released by virtually every cell type. EVs have diverse biological activities, ranging from roles in development and homeostasis to cancer progression, which has spurred the development of EVs as disease biomarkers and drug nanovehicles. Owing to the small size of EVs, however, most studies have relied on isolation and biochemical analysis of bulk EVs separated from biofluids. Although informative, these approaches do not capture the dynamics of EV release, biodistribution, and other contributions to pathophysiology. Recent advances in live and high-resolution microscopy techniques, combined with innovative EV labeling strategies and reporter systems, provide new tools to study EVs in vivo in their physiological environment and at the single-vesicle level. Here we critically review the latest advances and challenges in EV imaging, and identify urgent, outstanding questions in our quest to unravel EV biology and therapeutic applications.


Assuntos
Vesículas Extracelulares , Microscopia/métodos , Animais , Corantes/química , Epitopos , Vesículas Extracelulares/química , Vesículas Extracelulares/patologia , Vesículas Extracelulares/fisiologia , Corantes Fluorescentes/química , Humanos
4.
J Pathol ; 261(3): 286-297, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37615198

RESUMO

Circulating tumor DNA (ctDNA) holds promise in resectable esophageal adenocarcinoma (EAC) to predict patient outcome but is not yet sensitive enough to be clinically applicable. Our aim was to combine ctDNA mutation data with shallow whole-genome sequencing (sWGS)-derived copy number tumor fraction estimates (ichorCNA) to improve pathological response and survival prediction in EAC. In total, 111 stage II/III EAC patients with baseline (n = 111), post-neoadjuvant chemoradiotherapy (nCRT) (n = 68), and pre-surgery (n = 92) plasma samples were used for ctDNA characterization. sWGS (<5× coverage) was performed on all time-point samples, and copy number aberrations were estimated using ichorCNA. Baseline and pre-surgery samples were sequenced using a custom amplicon panel for mutation detection. Detection of baseline ctDNA was successful in 44.3% of patients by amplicon sequencing and 10.5% by ichorCNA. Combining both, ctDNA could be detected in 50.5% of patients. Baseline ctDNA positivity was related to higher T stage (cT3, 4) (p = 0.017). There was no relationship between pathological response and baseline ctDNA positivity. However, baseline ctDNA metrics (variant allele frequency > 1% or ichorCNA > 3%) were associated with a high risk of disease progression [HR = 2.23 (95% CI 1.22-4.07), p = 0.007]. The non-clearance of a baseline variant or ichorCNA > 3% in pre-surgery samples was related to early progression [HR = 4.58 (95% CI 2.22-9.46), p < 0.001]. Multi-signal analysis improves detection of ctDNA and can be used for prognostication of resectable EAC patients. Future studies should explore the potential of multi-modality sequencing for risk stratification and treatment adaptation based on ctDNA results. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Assuntos
Adenocarcinoma , Ácidos Nucleicos Livres , DNA Tumoral Circulante , Neoplasias Esofágicas , Humanos , Ácidos Nucleicos Livres/genética , DNA Tumoral Circulante/genética , Adenocarcinoma/genética , Adenocarcinoma/terapia , Adenocarcinoma/diagnóstico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/terapia , Biomarcadores Tumorais/genética , Mutação
5.
Clin Chem ; 68(6): 803-813, 2022 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-35292813

RESUMO

BACKGROUND: Assays that account for the biological properties and fragmentation of cell-free DNA (cfDNA) can improve the performance of liquid biopsy. However, preanalytic and physiological differences between individuals on fragmentomic analysis are poorly defined. METHODS: We analyzed the impact of collection tube, plasma processing time, and physiology on the size distribution of cfDNA, their genome-wide representation, and sequence diversity at the cfDNA fragment ends using shallow whole-genome sequencing. RESULTS: Neither different stabilizing collection tubes nor processing times affected the cfDNA fragment sizes, but could impact the genome-wide fragmentation patterns and fragment-end sequences of cfDNA. In addition, beyond differences depending on the gender, the physiological conditions tested between 63 individuals (age, body mass index, use of medication, and chronic conditions) minimally influenced the outcome of fragmentomic methods. CONCLUSIONS: Fragmentomic approaches have potential for implementation in the clinic, pending clear traceability of analytical and physiological factors.


Assuntos
Ácidos Nucleicos Livres , Ácidos Nucleicos Livres/genética , Fragmentação do DNA , Humanos , Biópsia Líquida/métodos
6.
Circ Res ; 120(10): 1632-1648, 2017 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-28495994

RESUMO

Owing to the relationship between extracellular vesicles (EVs) and physiological and pathological conditions, the interest in EVs is exponentially growing. EVs hold high hopes for novel diagnostic and translational discoveries. This review provides an expert-based update of recent advances in the methods to study EVs and summarizes currently accepted considerations and recommendations from sample collection to isolation, detection, and characterization of EVs. Common misconceptions and methodological pitfalls are highlighted. Although EVs are found in all body fluids, in this review, we will focus on EVs from human blood, not only our most complex but also the most interesting body fluid for cardiovascular research.


Assuntos
Coleta de Amostras Sanguíneas/métodos , Coleta de Amostras Sanguíneas/normas , Vesículas Extracelulares/metabolismo , Biomarcadores/sangue , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/diagnóstico , Exossomos/metabolismo , Citometria de Fluxo/métodos , Humanos
7.
Proc Natl Acad Sci U S A ; 113(5): E587-96, 2016 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-26768848

RESUMO

Complex interactions between DNA herpesviruses and host factors determine the establishment of a life-long asymptomatic latent infection. The lymphotropic Epstein-Barr virus (EBV) seems to avoid recognition by innate sensors despite massive transcription of immunostimulatory small RNAs (EBV-EBERs). Here we demonstrate that in latently infected B cells, EBER1 transcripts interact with the lupus antigen (La) ribonucleoprotein, avoiding cytoplasmic RNA sensors. However, in coculture experiments we observed that latent-infected cells trigger antiviral immunity in dendritic cells (DCs) through selective release and transfer of RNA via exosomes. In ex vivo tonsillar cultures, we observed that EBER1-loaded exosomes are preferentially captured and internalized by human plasmacytoid DCs (pDCs) that express the TIM1 phosphatidylserine receptor, a known viral- and exosomal target. Using an EBER-deficient EBV strain, enzymatic removal of 5'ppp, in vitro transcripts, and coculture experiments, we established that 5'pppEBER1 transfer via exosomes drives antiviral immunity in nonpermissive DCs. Lupus erythematosus patients suffer from elevated EBV load and activated antiviral immunity, in particular in skin lesions that are infiltrated with pDCs. We detected high levels of EBER1 RNA in such skin lesions, as well as EBV-microRNAs, but no intact EBV-DNA, linking non-cell-autonomous EBER1 presence with skin inflammation in predisposed individuals. Collectively, our studies indicate that virus-modified exosomes have a physiological role in the host-pathogen stand-off and may promote inflammatory disease.


Assuntos
Células Dendríticas/virologia , Infecções por Vírus Epstein-Barr/genética , Exossomos/metabolismo , RNA Viral/metabolismo , Transporte Biológico , Infecções por Vírus Epstein-Barr/imunologia , Herpesvirus Humano 4/genética , Humanos , Proteoma
8.
Prostate ; 75(10): 1102-13, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25845829

RESUMO

INTRODUCTION: High-grade prostatic intraepithelial neoplasia (HGPIN) is a recognized precursor stage of PCa. Men who present HGPIN in a first prostate biopsy face years of active surveillance including repeat biopsies. This study aimed to identify non-invasive prognostic biomarkers that differentiate early on between indolent HGPIN cases and those that will transform into actual PCa. METHODS: We measured the expression of 21 candidate mRNA biomarkers using quantitative PCR in urine sediment samples from a cohort of 90 patients with initial diagnosis of HGPIN and a posterior follow up of at least two years. Uni- and multivariate statistical analyses were applied to analyze the candidate biomarkers and multiplex models using combinations of these biomarkers. RESULTS: PSMA, PCA3, PSGR, GOLM, KLK3, CDH1, and SPINK1 behaved as predictors for PCa presence in repeat biopsies. Multiplex models outperformed (AUC = 0.81-0.86) the predictive power of single genes, including the FDA-approved PCA3 (AUC = 0.70). With a fixed sensitivity of 95%, the specificity of our multiplex models was of 41-58%, compared to the 30% of PCA3. The PPV of our models (30-38%) was also higher than the PPV of PCA3 (27%), suggesting that benign cases could be more accurately identified. Applying statistical models, we estimated that 33% to 47% of repeat biopsies could be prevented with a multiplex PCR model, representing an easy applicable and significant advantage over the current gold standard in urine sediment. DISCUSSION: Using multiplex RTqPCR-based models in urine sediment it is possible to improve the current diagnostic method of choice (PCA3) to differentiate between benign HGPIN and PCa cases.


Assuntos
Biomarcadores Tumorais/urina , Neoplasia Prostática Intraepitelial/patologia , Neoplasia Prostática Intraepitelial/urina , Neoplasias da Próstata/urina , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Biópsia , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Prognóstico , Neoplasias da Próstata/patologia , RNA Mensageiro/urina , Sensibilidade e Especificidade
9.
EMBO J ; 30(11): 2115-29, 2011 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-21527913

RESUMO

The ubiquitous Epstein Barr virus (EBV) exploits human B-cell development to establish a persistent infection in ∼90% of the world population. Constitutive activation of NF-κB by the viral oncogene latent membrane protein 1 (LMP1) has an important role in persistence, but is a risk factor for EBV-associated lymphomas. Here, we demonstrate that endogenous LMP1 escapes degradation upon accumulation within intraluminal vesicles of multivesicular endosomes and secretion via exosomes. LMP1 associates and traffics with the intracellular tetraspanin CD63 into vesicles that lack MHC II and sustain low cholesterol levels, even in 'cholesterol-trapping' conditions. The lipid-raft anchoring sequence FWLY, nor ubiquitylation of the N-terminus, controls LMP1 sorting into exosomes. Rather, C-terminal modifications that retain LMP1 in Golgi compartments preclude assembly within CD63-enriched domains and/or exosomal discharge leading to NF-κB overstimulation. Interference through shRNAs further proved the antagonizing role of CD63 in LMP1-mediated signalling. Thus, LMP1 exploits CD63-enriched microdomains to restrain downstream NF-κB activation by promoting trafficking in the endosomal-exosomal pathway. CD63 is thus a critical mediator of LMP1 function in- and outside-infected (tumour) cells.


Assuntos
Antígenos CD/metabolismo , Endossomos/metabolismo , Exossomos/metabolismo , Herpesvirus Humano 4/imunologia , NF-kappa B/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Proteínas da Matriz Viral/metabolismo , Linhagem Celular , Herpesvirus Humano 4/patogenicidade , Humanos , Ligação Proteica , Transporte Proteico , Tetraspanina 30
10.
Blood ; 121(19): 3997-4006, S1-15, 2013 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-23532734

RESUMO

Signaling between endothelial cells, endothelial progenitor cells, and stromal cells is crucial for the establishment and maintenance of vascular integrity and involves exosomes, among other signaling pathways. Exosomes are important mediators of intercellular communication in immune signaling, tumor survival, stress responses, and angiogenesis. The ability of exosomes to incorporate and transfer messenger RNAs (mRNAs) encoding for "acquired" proteins or micro RNAs (miRNAs) repressing "resident" mRNA translation suggests that they can influence the physiological behavior of recipient cells. We demonstrate that miR-214, an miRNA that controls endothelial cell function and angiogenesis, plays a dominant role in exosome-mediated signaling between endothelial cells. Endothelial cell-derived exosomes stimulated migration and angiogenesis in recipient cells, whereas exosomes from miR-214-depleted endothelial cells failed to stimulate these processes. Exosomes containing miR-214 repressed the expression of ataxia telangiectasia mutated in recipient cells, thereby preventing senescence and allowing blood vessel formation. Concordantly, specific reduction of miR-214 content in exosome-producing endothelial cells abolishes the angiogenesis stimulatory function of the resulting exosomes. Collectively, our data indicate that endothelial cells release miR-214-containing exosomes to stimulate angiogenesis through the silencing of ataxia telangiectasia mutated in neighboring target cells.


Assuntos
Senescência Celular , Células Endoteliais/metabolismo , Exossomos/metabolismo , Exossomos/fisiologia , MicroRNAs/fisiologia , Neovascularização Fisiológica , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Células Cultivadas , Senescência Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Regulação para Baixo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Via Secretória/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/fisiologia
11.
Trends Mol Med ; 30(7): 660-672, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38692937

RESUMO

Diffuse large B cell lymphoma (DLBCL) exhibits significant biological and clinical heterogeneity that presents challenges for risk stratification and disease surveillance. Existing tools for risk stratification, including the international prognostic index (IPI), tissue molecular analyses, and imaging, have limited accuracy in predicting outcomes. The therapeutic landscape for aggressive lymphoma is rapidly evolving, and there is a pressing need to identify patients at risk of refractory or relapsed (R/R) disease in the context of personalized therapy. Liquid biopsy, a minimally invasive method for cancer signal detection, has been explored to address these challenges. We review advances in liquid biopsy strategies focusing on circulating nucleic acids in DLBCL patients and highlight their clinical potential. We also provide recommendations for biomarker-guided trials to support risk-adapted treatment modalities.


Assuntos
Biomarcadores Tumorais , Linfoma Difuso de Grandes Células B , Humanos , Biópsia Líquida/métodos , Linfoma Difuso de Grandes Células B/diagnóstico , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Prognóstico , Medicina de Precisão/métodos
12.
bioRxiv ; 2024 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-38765958

RESUMO

Small extracellular vesicles (sEVs) are heterogenous lipid membrane particles typically less than 200 nm in size and secreted by most cell types either constitutively or upon activation signals. sEVs isolated from biofluids contain RNAs, including small non-coding RNAs (ncRNAs), that can be either encapsulated within the EV lumen or bound to the EV surface. EV-associated microRNAs (miRNAs) are, despite a relatively low abundance, extensively investigated for their selective incorporation and their role in cell-cell communication. In contrast, the sorting of highly-structured ncRNA species is understudied, mainly due to technical limitations of traditional small RNA sequencing protocols. Here, we adapted ALL-tRNAseq to profile the relative abundance of highly structured and potentially methylated small ncRNA species, including transfer RNAs (tRNAs), small nucleolar RNAs (snoRNAs), and Y RNAs in bulk EV preparations. We determined that full-length tRNAs, typically 75 to 90 nucleotides in length, were the dominant small ncRNA species (>60% of all reads in the 18-120 nucleotides size-range) in all cell culture-derived EVs, as well as in human plasma-derived EV samples, vastly outnumbering 21 nucleotides-long miRNAs. Nearly all EV-associated tRNAs were protected from external RNAse treatment, indicating a location within the EV lumen. Strikingly, the vast majority of luminal-sorted, full-length, nucleobase modification-containing EV-tRNA sequences, harbored a dysfunctional 3' CCA tail, 1 to 3 nucleotides truncated, rendering them incompetent for amino acid loading. In contrast, in non-EV associated extracellular particle fractions (NVEPs), tRNAs appeared almost exclusively fragmented or 'nicked' into tRNA-derived small RNAs (tsRNAs) with lengths between 18 to 35 nucleotides. We propose that in mammalian cells, tRNAs that lack a functional 3' CCA tail are selectively sorted into EVs and shuttled out of the producing cell, offering a new perspective into the physiological role of secreted EVs and luminal cargo-selection.

13.
J Extracell Biol ; 3(7): e164, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38947877

RESUMO

Previously, we showed that quantification of lymphoma-associated miRNAs miR-155-5p, -127-3p and let-7a-5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV-based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV-miRNA quantitative real-time reverse-transcription PCR (q-RT-PCR) assay in an ISO-13485 certified quality-management system (QMS). Liposomes encapsulated with a synthetic (nematode-derived) miRNA spike-in controlled for EV isolation by automated size-exclusion chromatography (SEC). Additional miRNA spike-ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed-effect modelling with bootstrapping determined the diagnostic performance of the quality-controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76-0.92) compared to an AUC of 0.87 (CI: 0.80-0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality-controlled plasma EV-miRNA assay is technically robust, taking EV-miRNA as liquid biopsy assay an important step closer to clinical evaluation.

14.
Blood ; 118(13): 3680-3, 2011 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-21832279

RESUMO

Diagnostic platforms providing biomarkers that are highly predictive for diagnosing, monitoring, and stratifying cancer patients are key instruments in the development of personalized medicine. We demonstrate that tumor cells transfer (mutant) RNA into blood platelets in vitro and in vivo, and show that blood platelets isolated from glioma and prostate cancer patients contain the cancer-associated RNA biomarkers EGFRvIII and PCA3, respectively. In addition, gene-expression profiling revealed a distinct RNA signature in platelets from glioma patients compared with normal control subjects. Because platelets are easily accessible and isolated, they may form an attractive platform for the companion diagnostics of cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Plaquetas/metabolismo , Neoplasias/genética , RNA/metabolismo , Animais , Biomarcadores Tumorais/análise , Plaquetas/química , Neoplasias Encefálicas/sangue , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Glioma/sangue , Glioma/genética , Glioma/metabolismo , Glioma/patologia , Humanos , Masculino , Camundongos , Camundongos Nus , Análise em Microsséries , Neoplasias/metabolismo , Neoplasias da Próstata/sangue , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA/análise , RNA/sangue , RNA/genética , Transplante Heterólogo , Células Tumorais Cultivadas
15.
Proc Natl Acad Sci U S A ; 107(14): 6328-33, 2010 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-20304794

RESUMO

Noncoding regulatory microRNAs (miRNAs) of cellular and viral origin control gene expression by repressing the translation of mRNAs into protein. Interestingly, miRNAs are secreted actively through small vesicles called "exosomes" that protect them from degradation by RNases, suggesting that these miRNAs may function outside the cell in which they were produced. Here we demonstrate that miRNAs secreted by EBV-infected cells are transferred to and act in uninfected recipient cells. Using a quantitative RT-PCR approach, we demonstrate that mature EBV-encoded miRNAs are secreted by EBV-infected B cells through exosomes. These EBV-miRNAs are functional because internalization of exosomes by MoDC results in a dose-dependent, miRNA-mediated repression of confirmed EBV target genes, including CXCL11/ITAC, an immunoregulatory gene down-regulated in primary EBV-associated lymphomas. We demonstrate that throughout coculture of EBV-infected B cells EBV-miRNAs accumulate in noninfected neighboring MoDC and show that this accumulation is mediated by transfer of exosomes. Thus, the exogenous EBV-miRNAs transferred through exosomes are delivered to subcellular sites of gene repression in recipient cells. Finally, we show in peripheral blood mononuclear cells from patients with increased EBV load that, although EBV DNA is restricted to the circulating B-cell population, EBV BART miRNAs are present in both B-cell and non-B-cell fractions, suggestive of miRNA transfer. Taken together our findings are consistent with miRNA-mediated gene silencing as a potential mechanism of intercellular communication between cells of the immune system that may be exploited by the persistent human gamma-herpesvirus EBV.


Assuntos
Linfócitos B/metabolismo , Exossomos/metabolismo , Herpesvirus Humano 4/genética , MicroRNAs/metabolismo , RNA Viral/metabolismo , Linfócitos B/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Exossomos/ultraestrutura , Herpesvirus Humano 4/ultraestrutura , Humanos , MicroRNAs/genética , Microscopia Eletrônica , RNA Viral/genética , Internalização do Vírus
16.
STAR Protoc ; 4(4): 102645, 2023 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-37858475

RESUMO

Besides canonical microRNAs (miRNAs), sequence-based variations called isomiRs have biological relevance and diagnostic potential; however, accurate calling of these post-transcriptional modifications is challenging, especially for low input samples. Here, we present IsoSeek, a sequencing protocol that reduces ligation and PCR amplification bias and improves the accuracy of miRNA detection in low input samples. We describe steps for using randomized adapters combined with unique molecular identifiers (UMI), library quantification, and sequencing, followed by detailed procedures for data processing and analysis. For complete details on the use and execution of this protocol, please refer to C. Gómez-Martín et al. (2023)1 and Van Eijndhoven et al. (2021).2.


Assuntos
MicroRNAs , MicroRNAs/genética , Nucleotídeos/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de RNA/métodos , Biblioteca Gênica
17.
J Extracell Vesicles ; 12(2): e12302, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36788785

RESUMO

Human blood plasma prepared by centrifugation contains not only extracellular vesicles (EVs) but also platelets and erythrocyte ghosts (ery-ghosts). Here we studied whether analysis of miRNA associated with plasma EVs (EV-miRNA) is affected by the presence of platelets and ery-ghosts. EDTA blood was collected from healthy donors (n = 3), and plasma was prepared by the centrifugation protocol recommended by the International Society on Thrombosis and Haemostasis (ISTH), and by a centrifugation protocol from an EV-miRNA expert lab (non-ISTH protocol). EVs were isolated from plasma by size-exclusion chromatography CL-2B (SEC2B), and concentrations of platelets, activated platelets, ery-ghosts and EVs (150-1000 nm) were measured by calibrated flow cytometry. Two EV-associated miRNAs (let7a-5p and miR-21-5p), and one platelet-associated miRNA (miR-223-3p), were measured by qRT-PCR. Measurements were performed with and without filtration using 0.8 µm track-etched filters to remove platelets and ery-ghosts from plasma and EV-enriched SEC fractions. Plasma prepared by both centrifugation protocols contained platelets and ery-ghosts, which co-migrated with EVs into the EV-enriched SEC2B fractions. Filtration removed platelets and ery-ghosts (>97%; p ≤ 0.05) and did not affect the EV concentrations (p > 0.17). The miRNA concentrations were 2-4-fold overestimated due to the presence of platelets but not ery-ghosts. Thus, filtration of human plasma is expected to improve comparability and reproducibility of quantitative EV-miRNA studies. Therefore, we recommend to measure and report the plasma concentration of platelets for EV-miRNA studies, and to filter plasma before downstream analyses or storage in biobanks.


Assuntos
Vesículas Extracelulares , MicroRNAs , Humanos , MicroRNAs/genética , Reprodutibilidade dos Testes , Plaquetas , Plasma
18.
Cell Rep Methods ; 3(5): 100480, 2023 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-37323569

RESUMO

IsomiRs, sequence variants of mature microRNAs, are usually detected and quantified using high-throughput sequencing. Many examples of their biological relevance have been reported, but sequencing artifacts identified as artificial variants might bias biological inference and therefore need to be ideally avoided. We conducted a comprehensive evaluation of 10 different small RNA sequencing protocols, exploring both a theoretically isomiR-free pool of synthetic miRNAs and HEK293T cells. We calculated that, with the exception of two protocols, less than 5% of miRNA reads can be attributed to library preparation artifacts. Randomized-end adapter protocols showed superior accuracy, with 40% of true biological isomiRs. Nevertheless, we demonstrate concordance across protocols for selected miRNAs in non-templated uridyl additions. Notably, NTA-U calling and isomiR target prediction can be inaccurate when using protocols with poor single-nucleotide resolution. Our results highlight the relevance of protocol choice for biological isomiRs detection and annotation, which has key potential implications for biomedical applications.


Assuntos
MicroRNAs , Humanos , MicroRNAs/genética , Células HEK293 , Sequência de Bases , Análise de Sequência de RNA
19.
Genome Biol ; 24(1): 229, 2023 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-37828498

RESUMO

BACKGROUND: Existing methods to detect tumor signal in liquid biopsy have focused on the analysis of nuclear cell-free DNA (cfDNA). However, non-nuclear cfDNA and in particular mitochondrial DNA (mtDNA) has been understudied. We hypothesize that an increase in mtDNA in plasma could reflect the presence of cancer, and that leveraging cell-free mtDNA could enhance cancer detection. RESULTS: We survey 203 healthy and 664 cancer plasma samples from three collection centers covering 12 cancer types with whole genome sequencing to catalogue the plasma mtDNA fraction. The mtDNA fraction is increased in individuals with cholangiocarcinoma, colorectal, liver, pancreatic, or prostate cancer, in comparison to that in healthy individuals. We detect almost no increase of mtDNA fraction in individuals with other cancer types. The mtDNA fraction in plasma correlates with the cfDNA tumor fraction as determined by somatic mutations and/or copy number aberrations. However, the mtDNA fraction is also elevated in a fraction of patients without an apparent increase in tumor-derived cfDNA. A predictive model integrating mtDNA and copy number analysis increases the area under the curve (AUC) from 0.73 when using copy number alterations alone to an AUC of 0.81. CONCLUSIONS: The mtDNA signal retrieved by whole genome sequencing has the potential to boost the detection of cancer when combined with other tumor-derived signals in liquid biopsies.


Assuntos
Ácidos Nucleicos Livres , Neoplasias da Próstata , Masculino , Humanos , Biópsia Líquida , Mitocôndrias/genética , DNA Mitocondrial/genética , Neoplasias da Próstata/genética , Biomarcadores Tumorais/genética
20.
iScience ; 26(8): 107412, 2023 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-37575190

RESUMO

The human cytomegalovirus (HCMV)-encoded chemokine receptor US28 contributes to various aspects of the viral life cycle and promotes immune evasion by scavenging chemokines from the microenvironment of HCMV-infected cells. In contrast to the plasma membrane localization of most human chemokine receptors, US28 has a predominant intracellular localization. In this study, we used immunofluorescence and electron microscopy to determine the localization of US28 upon exogenous expression, as well as in HCMV-infected cells. We observed that US28 localizes to late endosomal compartments called multivesicular bodies (MVBs), where it is sorted in intraluminal vesicles. Live-cell total internal reflection fluorescence (TIRF) microscopy revealed that US28-containing MVBs can fuse with the plasma membrane, resulting in the secretion of US28 on exosomes. Exosomal US28 binds the chemokines CX3CL1 and CCL5, and US28-containing exosomes inhibited the CX3CL1-CX3CR1 signaling axis. These findings suggest that exosomal release of US28 contributes to chemokine scavenging and immune evasion by HCMV.

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