RESUMO
Small extracellular vesicles isolated from urine (uEVs) are increasingly recognized as potential biomarkers. Meanwhile, different uEV preparation strategies exist. Conventionally, the performance of EV preparation methods is evaluated by single particle quantification, Western blot, and electron microscopy. Recently, we introduced imaging flow cytometry (IFCM) as a next-generation single EV analysis technology. Here, we analyzed uEV samples obtained with different preparation procedures using nanoparticle tracking analysis (NTA), semiquantitative Western blot, and IFCM. IFCM analyses demonstrated that urine contains a predominant CD9+ sEV population, which exceeds CD63+ and CD81+ sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9+ sEVs. Although overall reduced, the highest CD9+ sEV recovery was obtained from urine samples stored at -80 °C and the lowest from those stored at -20 °C. Upon comparing the yield of the different uEV preparations, incongruencies between NTA and IFCM data became apparent. Results obtained by both NTA and IFCM were consistent with Western blot analyses for EV marker proteins; however, NTA results correlated with the amount of the impurity marker uromodulin. Despite demonstrating that the combination of ultrafiltration and size exclusion chromatography appears as a reliable uEV preparation technique, our data challenge the soundness of traditional NTA for the evaluation of different EV preparation methods.
Assuntos
Vesículas Extracelulares/química , Citometria de Fluxo/métodos , Imagem Molecular/métodos , Urinálise/métodos , Adulto , Biomarcadores/urina , Cromatografia em Gel , Feminino , Voluntários Saudáveis , Humanos , Masculino , Nanopartículas/química , Nanopartículas/ultraestrutura , Tetraspanina 28/urina , Tetraspanina 29/urina , Tetraspanina 30/urina , Ultrafiltração , Urinálise/instrumentação , Urina/química , Uromodulina/urinaRESUMO
Vibroacoustic metamaterials (VAMMs) are artificial materials that are specifically designed to control, direct, and manipulate sound waves by creating a frequency gap, known as the stop band, which blocks free wave propagation. In this paper, a new power-based approach that relies on the active structural intensity (STI) for predicting the stop band behavior of finite VAMM structures is presented. The proposed method quantifies the power loss in a locally resonant finite VAMM plate in terms of percentage, such as STI99% and STI90%, for stop band prediction. This allows for the quantitative analysis of the vibration attenuation capabilities of a VAMM structure. This study is presented in the context of a two-dimensional VAMM plate with 25 resonators mounted in the middle section of the plate. It has been demonstrated that this method can predict the stop band limits of a finite VAMM plate more accurately than using negative effective mass, unit cell dispersion analysis, or the frequency response function methods. The proposed approach is then implemented to establish a framework for investigating the influence of parameter uncertainties on the stop band behavior of the VAMM plate. Based on the STI99% method, which aims for significant vibration reduction, stricter tolerances in the mass fabrication process are required to ensure the robustness of VAMM. Conversely, the STI90% method suggests that larger fabrication tolerances can be leveraged to achieve a broader stop band range while still meeting the desired performance level, leading to cost savings in manufacturing.
RESUMO
Tumor-derived extracellular vesicles (TEVs) are important regulators of the immune response in cancer; however, most research so far has been carried out using cell culture systems. Immune-competent murine tumor models currently provide the best platform to assess proposed roles of TEVs using in vivo animal models and therefore are important for examining interactions between TEVs and the immune system. In this review, we present the current knowledge on TEVs using in vivo tumor-bearing animal models, with a focus on the role of TEVs in mediating crosstalk between tumor cells and both adaptive and innate immune cells. In particular, we address the question how animal models can clarify the reported heterogeneity of TEV effects in both anti-tumor responses and evasion of immune surveillance. The potential of TEVs in mediating direct antigen-presenting functions supports their potential as cancer vaccine therapeutics, therefore, we provide an overview of key findings of TEV trials that have the potential as novel immunotherapies, and shed light on challenges in the path toward the first in-human trials. We also highlight the important updates on the methods that continue to enhance the rigor and reproducibility of EV studies, particularly in functional animal models.