Protein Biomarker Discovery Studies on Urinary sEV Fractions Separated with UF-SEC for the First Diagnosis and Detection of Recurrence in Bladder Cancer Patients.

Urinary extracellular vesicles (EVs) are an attractive source of bladder cancer biomarkers. Here, a protein biomarker discovery study was performed on the protein content of small urinary EVs (sEVs) to identify possible biomarkers for the primary diagnosis and recurrence of non-muscle-invasive bladder cancer (NMIBC). The sEVs were isolated by ultrafiltration (UF) in combination with size-exclusion chromatography (SEC). The first part of the study compared healthy individuals with NMIBC patients with a primary diagnosis. The second part compared tumor-free patients with patients with a recurrent NMIBC diagnosis. The separated sEVs were in the size range of 40 to 200 nm. Based on manually curated high quality mass spectrometry (MS) data, the statistical analysis revealed 69 proteins that were differentially expressed in these sEV fractions of patients with a first bladder cancer tumor vs. an age- and gender-matched healthy control group. When the discriminating power between healthy individuals and first diagnosis patients is taken into account, the biomarkers with the most potential are MASP2, C3, A2M, CHMP2A and NHE-RF1. Additionally, two proteins (HBB and HBA1) were differentially expressed between bladder cancer patients with a recurrent diagnosis vs. tumor-free samples of bladder cancer patients, but their biological relevance is very limited.

Macrophages Release Extracellular Vesicles of Different Properties and Composition Following Exposure to Nanoparticles.

Extracellular vesicles are membrane-bound carriers with complex cargoes, which play a major role in intercellular communication, for instance, in the context of the immune response. Macrophages are known to release extracellular vesicles in response to different stimuli, and changes in their size, number, and composition may provide important insights into the responses induced. Macrophages are also known to be highly efficient in clearing nanoparticles, when in contact with them, and in triggering the immune system. However, little is known about how the nature and composition of the vesicles released by these cells may vary upon nanoparticle exposure. In order to study this, in this work, alveolar-like macrophages were exposed to a panel of nanoparticles with varying surface and composition, including amino-modified and carboxylated polystyrene and plain silica. We previously showed that these nanoparticles induced very different responses in these cells. Here, experimental conditions were carefully tuned in order to separate the extracellular vesicles released by the macrophages several hours after exposure to sub-toxic concentrations of the same nanoparticles. After separation, different methods, including high-sensitivity flow cytometry, TEM imaging, Western blotting, and nanoparticle tracking analysis, were combined in order to characterize the extracellular vesicles. Finally, proteomics was used to determine their composition and how it varied upon exposure to the different nanoparticles. Our results show that depending on the nanoparticles' properties. The macrophages produced extracellular vesicles of varying number, size, and protein composition. This indicates that macrophages release specific signals in response to nanoparticles and overall suggests that extracellular vesicles can reflect subtle responses to nanoparticles and nanoparticle impact on intercellular communication.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles.

Urine is commonly used for clinical diagnosis and biomedical research. The discovery of extracellular vesicles (EV) in urine opened a new fast-growing scientific field. In the last decade urinary extracellular vesicles (uEVs) were shown to mirror molecular processes as well as physiological and pathological conditions in kidney, urothelial and prostate tissue. Therefore, several methods to isolate and characterize uEVs have been developed. However, methodological aspects of EV separation and analysis, including normalization of results, need further optimization and standardization to foster scientific advances in uEV research and a subsequent successful translation into clinical practice. This position paper is written by the Urine Task Force of the Rigor and Standardization Subcommittee of ISEV consisting of nephrologists, urologists, cardiologists and biologists with active experience in uEV research. Our aim is to present the state of the art and identify challenges and gaps in current uEV-based analyses for clinical applications. Finally, recommendations for improved rigor, reproducibility and interoperability in uEV research are provided in order to facilitate advances in the field.

Determination of variability due to biological and technical variation in urinary extracellular vesicles as a crucial step in biomarker discovery studies.

Urinary extracellular vesicles (EVs) are an attractive source of biomarkers for urological diseases. A crucial step in biomarker discovery studies is the determination of the variation parameters to perform a sample size calculation. In this way, a biomarker discovery study with sufficient statistical power can be performed to obtain biologically significant biomarkers. Here, a variation study was performed on both the protein and lipid content of urinary EVs of healthy individuals, aged between 52 and 69 years. Ultrafiltration (UF) in combination with size exclusion chromatography (SEC) was used to isolate the EVs from urine. Different experimental variation set-ups were used in this variation study. The calculated standard deviations (SDs) of the 90% least variable peptides and lipids did not exceed 2 and 1.2, respectively. These parameters can be used in a sample size calculation for a well-designed biomarker discovery study at the cargo of EVs.

Bladder Cancer Diagnosis and Follow-Up: The Current Status and Possible Role of Extracellular Vesicles.

Diagnostic methods currently used for bladder cancer are cystoscopy and urine cytology. Cystoscopy is an invasive tool and has low sensitivity for carcinoma in situ. Urine cytology is non-invasive, is a low-cost method, and has a high specificity but low sensitivity for low-grade urothelial tumors. Despite the search for urinary biomarkers for the early and non-invasive detection of bladder cancer, no biomarkers are used at the present in daily clinical practice. Extracellular vesicles (EVs) have been recently studied as a promising source of biomarkers because of their role in intercellular communication and tumor progression. In this review, we give an overview of Food and Drug Administration (FDA)-approved urine tests to detect bladder cancer and why their use is not widespread in clinical practice. We also include non-FDA approved urinary biomarkers in this review. We describe the role of EVs in bladder cancer and their possible role as biomarkers for the diagnosis and follow-up of bladder cancer patients. We review recently discovered EV-derived biomarkers for the diagnosis of bladder cancer.

The challenges of peptidomics in complementing proteomics in a clinical context.

Naturally occurring peptides, including growth factors, hormones, and neurotransmitters, represent an important class of biomolecules and have crucial roles in human physiology. The study of these peptides in clinical samples is therefore as relevant as ever. Compared to more routine proteomics applications in clinical research, peptidomics research questions are more challenging and have special requirements with regard to sample handling, experimental design, and bioinformatics. In this review, we describe the issues that confront peptidomics in a clinical context. After these hurdles are (partially) overcome, peptidomics will be ready for a successful translation into medical practice.

Ultrafiltration and size exclusion chromatography combined with asymmetrical-flow field-flow fractionation for the isolation and characterisation of extracellular vesicles from urine.

Extracellular vesicles (EVs) have a great potential in clinical applications. However, their isolation from different bodily fluids and their characterisation are currently not optimal or standardised. Here, we report the results of examining the performance of ultrafiltration combined with size exclusion chromatography (UF-SEC) to isolate EVs from urine. The results reveal that UF-SEC is an efficient method and provides high purity. Furthermore, we introduce asymmetrical-flow field-flow fractionation coupled with a UV detector and multi-angle light-scattering detector (AF4/UV-MALS) as a characterisation method and compare it with current methods. We demonstrate that AF4/UV-MALS is a straightforward and reproducible method for determining size, amount and purity of isolated urinary EVs.

Adaptive tuning of mutation rates allows fast response to lethal stress in Escherichia coli.

While specific mutations allow organisms to adapt to stressful environments, most changes in an organism's DNA negatively impact fitness. The mutation rate is therefore strictly regulated and often considered a slowly-evolving parameter. In contrast, we demonstrate an unexpected flexibility in cellular mutation rates as a response to changes in selective pressure. We show that hypermutation independently evolves when different Escherichia coli cultures adapt to high ethanol stress. Furthermore, hypermutator states are transitory and repeatedly alternate with decreases in mutation rate. Specifically, population mutation rates rise when cells experience higher stress and decline again once cells are adapted. Interestingly, we identified cellular mortality as the major force driving the quick evolution of mutation rates. Together, these findings show how organisms balance robustness and evolvability and help explain the prevalence of hypermutation in various settings, ranging from emergence of antibiotic resistance in microbes to cancer relapses upon chemotherapy.