Simple urine storage protocol for extracellular vesicle proteomics compatible with at-home self-sampling

AbstractUrinary extracellular vesicles (EVs) have gained increased interest as a biomarker source. Clinical implementation on a daily basis requires protocols that inevitably includes short-term storage of the clinical samples, especially when collected at home. However, little is known about the effect of delayed processing on the urinary EVs concentration and proteome. We evaluated two storage protocols. First, urine stored at 4 °C. Secondly a protocol compatible with at-home collection, in which urine was stored with the preservative EDTA at room temperature (RT). EVs were isolated using the ME-kit (VN96-peptide). For both conditions we explored the effect of storage duration (0, 2, 4 and 8 days) on EV concentration and proteome using EVQuant and data-independent acquisition mass spectrometry, respectively. The urinary EV concentration and proteome was highly stable using both protocols, in terms of protein number and quantitative changes. Furthermore, EDTA does not affect the urinary EV concentration or global proteome. In conclusion, urine can be stored either at 4 °C or with EDTA at RT for up to 8 days without any significant decay in EV concentration or a notable effect on the EV-proteome. These findings open up biomarker studies in urine collected via self-sampling at home.

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Longitudinal stability of urinary extracellular vesicle protein patterns within and between individuals

Scientific Reports ◽

10.1038/s41598-021-95082-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Leyla A. Erozenci ◽

Sander R. Piersma ◽

Thang V. Pham ◽

Irene V. Bijnsdorp ◽

Connie R. Jimenez

Keyword(s):

Expression Patterns ◽

Interaction Network ◽

Extracellular Vesicle ◽

Protein Signaling ◽

Protein Patterns ◽

Specific Expression ◽

Non Invasive ◽

Data Independent Acquisition ◽

Time Period ◽

Using Data

AbstractThe protein content of urinary extracellular vesicles (EVs) is considered to be an attractive non-invasive biomarker source. However, little is known about the consistency and variability of urinary EV proteins within and between individuals over a longer time-period. Here, we evaluated the stability of the urinary EV proteomes of 8 healthy individuals at 9 timepoints over 6 months using data-independent-acquisition mass spectrometry. The 1802 identified proteins had a high correlation amongst all samples, with 40% of the proteome detected in every sample and 90% detected in more than 1 individual at all timepoints. Unsupervised analysis of top 10% most variable proteins yielded person-specific profiles. The core EV-protein-interaction network of 516 proteins detected in all measured samples revealed sub-clusters involved in the biological processes of G-protein signaling, cytoskeletal transport, cellular energy metabolism and immunity. Furthermore, gender-specific expression patterns were detected in the urinary EV proteome. Our findings indicate that the urinary EV proteome is stable in longitudinal samples of healthy subjects over a prolonged time-period, further underscoring its potential for reliable non-invasive diagnostic/prognostic biomarkers.

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Raman Spectral Signatures of Serum-Derived Extracellular Vesicle-Enriched Isolates May Support the Diagnosis of CNS Tumors

Cancers ◽

10.3390/cancers13061407 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1407

Author(s):

Matyas Bukva ◽

Gabriella Dobra ◽

Juan Gomez-Perez ◽

Krisztian Koos ◽

Maria Harmati ◽

...

Keyword(s):

Lumbar Disc ◽

Area Under The Curve ◽

Principal Component ◽

Extracellular Vesicle ◽

Classification Performance ◽

Cns Tumors ◽

Clinical Samples ◽

Support Vector ◽

Serum Samples ◽

Raman Spectroscopic

Investigating the molecular composition of small extracellular vesicles (sEVs) for tumor diagnostic purposes is becoming increasingly popular, especially for diseases for which diagnosis is challenging, such as central nervous system (CNS) malignancies. Thorough examination of the molecular content of sEVs by Raman spectroscopy is a promising but hitherto barely explored approach for these tumor types. We attempt to reveal the potential role of serum-derived sEVs in diagnosing CNS tumors through Raman spectroscopic analyses using a relevant number of clinical samples. A total of 138 serum samples were obtained from four patient groups (glioblastoma multiforme, non-small-cell lung cancer brain metastasis, meningioma and lumbar disc herniation as control). After isolation, characterization and Raman spectroscopic assessment of sEVs, the Principal Component Analysis–Support Vector Machine (PCA–SVM) algorithm was performed on the Raman spectra for pairwise classifications. Classification accuracy (CA), sensitivity, specificity and the Area Under the Curve (AUC) value derived from Receiver Operating Characteristic (ROC) analyses were used to evaluate the performance of classification. The groups compared were distinguishable with 82.9–92.5% CA, 80–95% sensitivity and 80–90% specificity. AUC scores in the range of 0.82–0.9 suggest excellent and outstanding classification performance. Our results support that Raman spectroscopic analysis of sEV-enriched isolates from serum is a promising method that could be further developed in order to be applicable in the diagnosis of CNS tumors.

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EVQuant; high-throughput quantification and characterization of extracellular vesicle (sub)populations

10.1101/2020.10.21.348375 ◽

2020 ◽

Author(s):

T.A. Hartjes ◽

J.A. Slotman ◽

M.S. Vredenbregt ◽

N. Dits ◽

R. Van der Meel ◽

...

Keyword(s):

Protein Content ◽

Size Distribution ◽

High Throughput ◽

Liquid Biopsy ◽

Ideal Liquid ◽

Extracellular Vesicle ◽

Cell Of Origin ◽

Clinical Implementation ◽

High Throughput Assay

AbstractExtracellular vesicles (EVs) reflect the cell of origin in terms of nucleic acids and protein content. They are found in biofluids and represent an ideal liquid biopsy biomarker source for many diseases. Unfortunately, clinical implementation is limited by available technologies for EV analysis. We have developed a simple, robust and sensitive microscopy-based high-throughput assay (EVQuant) to overcome these limitations and allow widespread use in the EV community. The EVQuant assay can detect individual immobilized EVs as small as 35 nm and determine their concentration in biofluids without extensive EV isolation or purification procedures. It can also identify specific EV subpopulations based on combinations of biomarkers and is used here to identify prostate-derived urinary EVs as CD9-/CD63+. Moreover, characterization of individual EVs allows analysis of their size distribution. The ability to identify, quantify and characterize EV (sub-)populations in high-throughput substantially extents the applicability of the EVQuant assay over most current EV quantification assays.

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Isolation of extracellular vesicles improves the detection of mutant DNA from plasma of metastatic melanoma patients

Scientific Reports ◽

10.1038/s41598-020-72834-6 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 3

Author(s):

Davide Zocco ◽

Simona Bernardi ◽

Mauro Novelli ◽

Chiara Astrua ◽

Paolo Fava ◽

...

Keyword(s):

Metastatic Melanoma ◽

Braf Inhibitor ◽

Extracellular Vesicle ◽

Therapeutic Monitoring ◽

Clinical Samples ◽

Outer Side ◽

Alternative Source ◽

Gene Copies ◽

Melanoma Patients ◽

Promising Source

Abstract Detection of BRAFV600E within cell free tumor DNA (ctDNA) is emerging as a promising means to improve patients’ stratification or enable BRAF inhibitor (BRAFi) therapeutic monitoring in a minimally invasive manner. Here, we investigated whether extracellular vesicle-(EV)-associated-DNA (EV-DNA) has value as an alternative source of circulating BRAFV600E. To do so, we identified a clinical practice-compatible protocol for the isolation of EV-DNA and assessed BRAF gene status on plasma samples from metastatic melanoma patients at the beginning and during BRAFi therapy. This protocol uses a peptide with high affinity for EVs and it has been found to recover more mutant DNA from plasma than standard ultracentrifugation. Molecular analyses revealed that mutant DNA is largely unprotected from nuclease digestion, interacting with the outer side of the EV membrane or directly with the peptide. When used on clinical samples, we found that the protocol improves the detection of BRAFV600E gene copies in comparison to the reference protocol for ctDNA isolation. Taken together, these findings indicate that EVs are a promising source of mutant DNA and should be considered for the development of next-generation liquid biopsy approaches.

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Low-Cost Home Automation System for Physical Disability and Limited Mobility People

Advances in Computational Intelligence and Robotics - Driving Innovation and Productivity Through Sustainable Automation ◽

10.4018/978-1-7998-5879-9.ch007 ◽

2021 ◽

pp. 151-172

Author(s):

José Irineu Ferreira Júnior ◽

Paulo César do Nascimento Cunha ◽

Vitor Gabriel Nunes Soares ◽

Álvaro Sobrinho

Keyword(s):

Latin American ◽

Low Income ◽

Physical Disability ◽

Low Cost ◽

Daily Basis ◽

Automation System ◽

Home Automation ◽

Limited Mobility ◽

Home Automation System ◽

At Home

The general Latin American population with a physical disability or limited mobility has faced the daily basis challenge of having autonomy in home activities, with low-income or almost no-income. Needy Brazilian communities are examples of poor populations suffering from the lack of autonomy at home, aggravated by scarce financial resources. The authors developed a low-cost home automation system, aiming to assist people who live in Palmeira dos Indios city and Arapiraca city, needy communities located in the Northeast of Brazil. The system is composed of hardware and software components. The hardware comprises of microcontrollers used to actuate over electrical devices at home, while an Android application provides a simple graphical user interface (GUI) to control the devices using touch and voice commands by Bluetooth communication. They evaluated the system by implementing a home model and providing the home for four physical disability persons and one limited mobility person. They considered the system`s effectiveness, the system`s usability, and users` perceptions during the evaluation.

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Orthogonal proteomic platforms and their implications for the stable classification of high-grade serous ovarian cancer subtypes

10.1101/793026 ◽

2019 ◽

Cited By ~ 2

Author(s):

Stefani N. Thomas ◽

Betty Friedrich ◽

Michael Schnaubelt ◽

Daniel W. Chan ◽

Hui Zhang ◽

...

Keyword(s):

Ovarian Cancer ◽

Large Scale ◽

Clinical Samples ◽

High Grade ◽

Serous Ovarian Cancer ◽

Cancer Subtypes ◽

Data Independent Acquisition ◽

Proteomic Approach ◽

Liquid Chromatography Tandem Mass ◽

Isobaric Tagging

SummaryThe National Cancer Institute (NCI) Clinical Proteomic Tumor Analysis Consortium (CPTAC) has established a two-dimensional liquid chromatography-tandem mass spectrometry (2DLC-MS/MS) workflow using isobaric tagging to compare protein abundance across samples. The workflow has been used for large-scale clinical proteomic studies with deep proteomic coverage within and outside of CPTAC. SWATH-MS, an instance of data-independent acquisition (DIA) proteomic methods, was recently developed as an alternate proteomic approach. In this study, we analyzed remaining aliquots of peptides using SWATH-MS from the original retrospective TCGA samples generated for the CPTAC ovarian cancer proteogenomic study (Zhang et al., 2016). The SWATH-MS results indicated that both methods confidently identified differentially expressed proteins in enriched pathways associated with the robust Mesenchymal subtype of high-grade serous ovarian cancer (HGSOC) and the homologous recombination deficient tumors also present in the original study. The results demonstrated that SWATH/DIA-MS presents a promising complementary or orthogonal alternative to the CPTAC harmonized proteomic method, with the advantages of simpler, faster, and cheaper workflows, as well as lower sample consumption. However, the SWATH/DIA-MS workflow resulted in shallower proteome coverage. Overall, we concluded that both analytical methods are suitable to characterize clinical samples such as in the high-grade serous ovarian cancer study, providing proteomic workflow alternatives for cancer researchers depending on the specific goals and context of the studies.

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Su Jok Therapy for Managing Chest Pain During COVID-19 Pandemic Period: A Case Report

BIO Web of Conferences ◽

10.1051/bioconf/20214103004 ◽

2021 ◽

Vol 41 ◽

pp. 03004

Author(s):

Intansari Nurjannah

Keyword(s):

Case Report ◽

Chest Pain ◽

Hospital Admission ◽

Alternative Medicine ◽

Alternative Therapy ◽

Daily Basis ◽

Difficulty Breathing ◽

At Home

The Covid-19 pandemic has led people who were non-COVID-19 patient to avoid hospital admission and to seek help from alternative medicine. The aim of this report is to describe the management of chest pain at home using Su Jok therapy. Case report: A male 47 of years old complained of chest pain with difficulty breathing and asked for help from the researcher who was a nurse as well as Su Jok therapist. His sclera profile was recorded for analysis (sclerology analysis). Su Jok therapy was applied directly by the researcher on daily basis. Researcher also monitored his condition progress through checking pain point on his hands and also from his sclera profile. After two weeks, the symptoms subsided and the eye’s profile of sclera shows healing progress. Conclusion: The case study shows that Su Jok therapy may become an alternative therapy for managing chest pain.

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Evidence of the Mechanism by Which Polyomaviruses Exploit the Extracellular Vesicle Delivery System during Infection

Viruses ◽

10.3390/v12060585 ◽

2020 ◽

Vol 12 (6) ◽

pp. 585 ◽

Cited By ~ 4

Author(s):

Simone Giannecchini

Keyword(s):

Viral Infection ◽

Biological Fluids ◽

Viral Persistence ◽

Antigen Expression ◽

Extracellular Vesicle ◽

T Antigen ◽

Viral Reactivation ◽

Human Polyomavirus ◽

Clinical Samples ◽

Viral Particles

Increasing evidence suggests that human viruses can hijack extracellular vesicles (EVs) to deliver proteins, mRNAs, microRNAs (miRNAs) and whole viral particles during viral persistence in the host. Human polyomavirus (PyV) miRNAs, which downregulate large T-antigen expression and target host factors, help the virus escape immune elimination and may have roles in the success of viral persistence/replication and the development of diseases. In this context, several investigations have detected PyV miRNAs in EVs obtained from cell culture supernatants after viral infection, demonstrating the ability of these vesicles to deliver miRNAs to uninfected cells, potentially counteracting new viral infection. Additionally, PyV miRNAs have been identified in EVs derived from the biological fluids of clinical samples obtained from patients with or at risk of severe PyV-associated diseases and from asymptomatic control healthy subjects. Interestingly, PyV miRNAs were found to be circulating in blood, urine, cerebrospinal fluid, and saliva samples from patients despite their PyV DNA status. Recently, the association between EVs and PyV viral particles was reported, demonstrating the ability of PyV viral particles to enter the cell without natural receptor-mediated entry and evade antibody-mediated neutralization or to be neutralized at a step different from that of the neutralization of naked whole viral particles. All these data point toward a potential role of the association between PyVs with EVs in viral persistence, suggesting that further work to define the implication of this interaction in viral reactivation is warranted.

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Extracellular vesicle (EV)-polyphenol nanoaggregates for microRNA-based cancer diagnosis

NPG Asia Materials ◽

10.1038/s41427-019-0184-0 ◽

2019 ◽

Vol 11 (1) ◽

Author(s):

Minjeong Jang ◽

Giwoong Choi ◽

Yoon Young Choi ◽

Jae Eun Lee ◽

Jik-Han Jung ◽

...

Keyword(s):

Molecular Beacon ◽

Diagnostic System ◽

Extracellular Vesicle ◽

Diagnostic Techniques ◽

Clinical Samples ◽

Human Blood Plasma ◽

Strongly Correlated ◽

Efficient Resource ◽

Diagnosis Of Cancer ◽

Geo Database

AbstractSmall extracellular vesicles (EVs), including exosomes, in body fluids have important applications in the noninvasive liquid biopsy-based diagnosis of cancer. Current EV-based diagnostic techniques still face practical challenges, such as inefficient EV isolation. Here, we report an efficient, resource-free pre-enrichment approach using (–)-epigallocatechin-3-gallate (EGCG), a polyphenolic biomolecule, to isolate and detect exosomal microRNAs (miRNAs) in human blood plasma samples. Our system comprises three steps: (1) EGCG-mediated EV aggregation, (2) filter-based EV isolation, and (3) molecular beacon-based detection of target miRNA in EVs. Using blood samples from cancer patients with gastric cancer or hepatocellular carcinoma, we constructed an EGCG-assisted miRNA diagnostic system. For both cancers, the levels of target miRNAs (miR-21, -27a, and -375) in EVs were strongly correlated with those in the publicly available GEO database. Our approach, an easy-to-use method for efficient EV isolation and the detection of miRNA in clinical samples, is applicable for molecular diagnostics in precision medicine.

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In-Depth Benchmarking of DIA-type Proteomics Data Analysis Strategies Using a Large-Scale Benchmark Dataset Comprising Inter-Patient Heterogeneity

10.21203/rs.3.rs-893982/v1 ◽

2021 ◽

Author(s):

Klemens Fröhlich ◽

Eva Brombacher ◽

Matthias Fahrner ◽

Daniel Vogele ◽

Lucas Kook ◽

...

Keyword(s):

Data Analysis ◽

Large Scale ◽

Statistical Tests ◽

Benchmark Dataset ◽

Clinical Samples ◽

Proteomics Data ◽

Data Independent Acquisition ◽

Analysis Strategies ◽

Spectral Libraries ◽

Differentially Abundant Proteins

Abstract An overwhelming number of proteomics software tools and algorithms have been published for different steps of Data Independent Acquisition analysis of clinical samples. Nonetheless, there is still a lack of comprehensive benchmark studies evaluating which combinations of those isolated components perform best. Here, we used 92 lymph nodes from distinct patients to create a unique benchmark dataset representing real-world inter-individual heterogeneity. The publicly available dataset comprises 118 LC-MS/MS runs with > 12 million MS2 spectra and allowed us to objectively evaluate how well different combinations of spectral libraries, DIA software, sparsity reduction, normalization and statistical tests can detect differentially abundant proteins, while also taking sample size into account. Evaluation of 2 million data analysis workflows showed that a gas phase fractionation refined spectral library in combination with DIA-NN and Significance Analysis of Microarrays reliably detected differentially abundant proteins. Furthermore, DIA-NN and Spectronaut robustly avoided the false detection of truly absent proteins.

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