Advances in biomarker detection: Alternative approaches for blood-based biomarker detection

Rosado, Miguel; Silva, Rafael; Bexiga, Mariana G.; Jones, John G.; Manadas, Bruno; Anjo, Sandra I.

doi:10.1016/bs.acc.2019.04.003

Cited by 23 publications

(20 citation statements)

References 148 publications

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“…The purity of the isolated exosomes can vary due to the presence of contaminating particles, other EVs, viscosity of the sample, the presence of milk proteins, and nucleic acids that are often precipitated alongside MDEs [ 35 – 37 ]. Density gradient ultracentrifugation (DG-UC), where exosomes are separated based on size, mass, and density in a sucrose or iodixanol gradient, is considered the gold standard for exosome isolations [ 38 ]. However, critical drawbacks of this technique include the requirement of large sample volumes (range of milliliters to liters) [ 39 , 40 ], vehicle damage or exosome aggregation [ 39 , 41 , 42 ] (especially exosomes originating from highly viscous solutions such as milk), standardization issues [ 21 , 43 ], and lipoprotein contamination, where high density lipids (HDLs) will sediment alongside MDEs due to similar densities [ 40 , 41 ].…”

Section: Introductionmentioning

confidence: 99%

Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk

et al. 2021

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Milk is a highly complex, heterogeneous biological fluid that contains non-nutritive, bioactive extracellular vesicles called exosomes. Characterization of milk-derived exosomes (MDEs) is challenging due to the lack of standardized methods that are currently being used for milk pre-processing, storage, and exosome isolation. In this study, we tested: 1) three pre-processing methods to remove cream, fat, cellular debris, and casein proteins from bovine milk to determine whether pre-processing of whole milk prior to long-term storage improves MDE isolations, 2) the suitability of two standard exosome isolation methods for MDE fractionation, and 3) four extraction protocols for obtaining high quality RNA from bovine and human MDEs. MDEs were characterized via Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and western immunoblotting for CD9, CD63, and Calnexin protein markers. We also present an optimized method of TEM sample preparation for MDEs. Our results indicate that: 1) Removal of cream and fat globules from unpasteurized bovine milk, prior to long-term storage, improves the MDE yield but not purity, 2) Differential ultracentrifugation (DUC) combined with serial filtration is better suited for bovine MDE isolation compared to ExoQuick (EQ) combined with serial filtration, however both methods were comparable for human milk, and 3) TRIzol LS is better suited for RNA extraction from bovine MDEs isolated by EQ and DUC methods. 4) TRIzol LS, TRIzol+RNA Clean and Concentrator, and TRIzol LS+RNA Clean and Concentrator methods can be used for RNA extractions from human MDEs isolated by EQ, yet the TRIzol LS method is better suited for human MDEs isolated by DUC. The QIAzol + miRNeasy Mini Kit produced the lowest RNA yield for bovine and human MDEs.

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Section: Introductionmentioning

confidence: 99%

Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk

et al. 2021

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“…Moreover, in 1959, Seal, observing that red blood cells and white blood cells have higher specific gravities (1.092 and 1.065, respectively) compared to cancer cells (1.056), exploited these differences to separate them using one of the first density-based techniques involving silicone floatation [ 51 ] . To date, it is possible to count on different density gradient media, such as Ficoll®, a synthetic polymer formed by copolymerization of sucrose and epichlorohydrin [ 12 ] , Ficoll-Paque® and Lymphoprep®, both composed of polysaccharides and diatrizoate [ 52 ] , and Percoll®, consisting in a colloidal silica particle suspension [ 45 ] . All these media basically allow the separation of CTCs from blood cells through centrifugation.…”

Section: Ctc Cultures: a Strenuous Challengementioning

confidence: 99%

In vitro cultures of circulating tumor cells: a potential tool to unravel drug sensitivity

Renzi¹,

Marco²,

Meo³

et al. 2022

CDR

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Since taking part as leading actors in driving the metastatic process, circulating tumor cells (CTCs) have displayed a wide range of potential applications in the cancer-related research field. Besides their well-proved prognostic value, the role of CTCs in both predictive and diagnostics terms might be extremely informative about cancer properties and therefore highly helpful in the clinical decision-making process. Unfortunately, CTCs are scarcely released in the blood circulation and their counts vary a lot among different types of cancer, therefore CTC detection and consequent characterization are still highly challenging. In this context, in vitro CTC cultures could potentially offer a great opportunity to expand the number of tumor cells isolated at different stages of the disease and thus simplify the analysis of their biological and molecular features, allowing a deeper comprehension of the nature of neoplastic diseases. The aim of this review is to highlight the main attempts to establish in vitro CTC cultures from patients harboring different tumor types in order to highlight how powerful this practice could be, especially in optimizing the therapeutic strategies available in clinical practice and potentially preventing or contrasting the development of treatment resistance.

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“…3 Centro de Investigação em Ciências da Saúde (CICS-UBI), Universidade da Beira Interior, Covilhã, Portugal. 4 Centro Hospitalar Cova da Beira, E.P.E, Covilhã, Portugal. 5 Neurology Department, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal.…”

Section: Supplementary Informationunclassified

“…Thus, to avoid missing out some critical candidates, a highly demanding sample processing is required to reduce the complexity of the samples. Also, large cohorts are needed to perform biomarker discovery directly from human samples in order to overcome the large biological variability intrinsic of these samples [1][2][3][4]. All these aspects combined result in a very challenging analysis, which may justify, in part, the low reproducibility and success of biomarker discovery studies.…”

Section: Introductionmentioning

confidence: 99%

A different vision of translational research in biomarker discovery: a pilot study on circulatory mitochondrial proteins as Parkinson’s disease potential biomarkers

Anjo

Santos

Rosado

et al. 2020

Transl Neurodegener

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Background: The identification of circulating biomarkers that closely correlate with Parkinson's Disease (PD) has failed several times in the past. Nevertheless, in this pilot study, a translational approach was conducted, allowing the evaluation of the plasma levels of two mitochondrial-related proteins, whose combination leads to a robust model with potential diagnostic value to discriminate the PD patients from matched controls. Methods: The proposed translational approach was initiated by the analysis of secretomes from cells cultured under control or well-defined oxidative stress conditions, followed by the identification of proteins related to PD pathologic mechanisms that were altered between the two states. This pipeline was further translated into the analysis of undepleted plasma samples from 28 control and 31 PD patients. Results: From the secretome analysis, several mitochondria-related proteins were found to be differentially released between control and stress conditions and to be able to distinguish the two secretomes. Similarly, two mitochondrial-related proteins were found to be significantly changed in a PD cohort compared to matched controls. Moreover, a linear discriminant model with potential diagnostic value to discriminate PD patients was obtained using the combination of these two proteins. Both proteins are associated with apoptotic mitochondrial changes, which may correspond to potential indicators of cell death. Moreover, one of these proteins, the VPS35 protein, was reported in plasma for the first time, and its quantification was only possible due to its previous identification in the secretome analysis.

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Advances in biomarker detection: Alternative approaches for blood-based biomarker detection

Cited by 23 publications

References 148 publications

Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk

Comparison of methods for pre-processing, exosome isolation, and RNA extraction in unpasteurized bovine and human milk

In vitro cultures of circulating tumor cells: a potential tool to unravel drug sensitivity

A different vision of translational research in biomarker discovery: a pilot study on circulatory mitochondrial proteins as Parkinson’s disease potential biomarkers

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