scholarly journals Small Extracellular Vesicles Isolated from Serum May Serve as Signal-Enhancers for the Monitoring of CNS Tumors

2020 ◽  
Vol 21 (15) ◽  
pp. 5359 ◽  
Author(s):  
Gabriella Dobra ◽  
Matyas Bukva ◽  
Zoltan Szabo ◽  
Bella Bruszel ◽  
Maria Harmati ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Serum Proteins ◽  
Lumbar Disc ◽  
Principal Component ◽  
Cns Tumors ◽  
Serum Samples ◽  
Force Microscopy ◽  
Isolation And Characterization ◽  
Atomic Force ◽  
Patient Groups

Liquid biopsy-based methods to test biomarkers (e.g., serum proteins and extracellular vesicles) may help to monitor brain tumors. In this proteomics-based study, we aimed to identify a characteristic protein fingerprint associated with central nervous system (CNS) tumors. Overall, 96 human serum samples were obtained from four patient groups, namely glioblastoma multiforme (GBM), non-small-cell lung cancer brain metastasis (BM), meningioma (M) and lumbar disc hernia patients (CTRL). After the isolation and characterization of small extracellular vesicles (sEVs) by nanoparticle tracking analysis (NTA) and atomic force microscopy (AFM), liquid chromatography -mass spectrometry (LC-MS) was performed on two different sample types (whole serum and serum sEVs). Statistical analyses (ratio, Cohen’s d, receiver operating characteristic; ROC) were carried out to compare patient groups. To recognize differences between the two sample types, pairwise comparisons (Welch’s test) and ingenuity pathway analysis (IPA) were performed. According to our knowledge, this is the first study that compares the proteome of whole serum and serum-derived sEVs. From the 311 proteins identified, 10 whole serum proteins and 17 sEV proteins showed the highest intergroup differences. Sixty-five proteins were significantly enriched in sEV samples, while 129 proteins were significantly depleted compared to whole serum. Based on principal component analysis (PCA) analyses, sEVs are more suitable to discriminate between the patient groups. Our results support that sEVs have greater potential to monitor CNS tumors, than whole serum.

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

Cancers ◽  
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.

Study of NSCLC cell migration promoted by NSCLC-Derived Extracellular Vesicle using atomic force microscopy

2021 ◽  
Author(s):  
Shuwei Wang ◽  
Jiajia Wang ◽  
Tuoyu Ju ◽  
Kaige Qu ◽  
Fan Yang ◽  
...  
Keyword(s):  
Atomic Force Microscopy ◽  
Cell Migration ◽  
Cancer Cells ◽  
Extracellular Vesicles ◽  
Extracellular Vesicle ◽  
Cancer Microenvironment ◽  
Biological Processes ◽  
Force Microscopy ◽  
Atomic Force ◽  
Cellular Components

Extracellular Vesicles (EVs) secreted by cancer cells have a key role in the cancer microenvironment and progression. Previous studies have mainly focused on molecular functions, cellular components and biological processes...

scholarly journals Extracellular Vesicles Analysis in the COVID-19 Era: Insights on Serum Inactivation Protocols towards Downstream Isolation and Analysis

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 544
Author(s):  
Roberto Frigerio ◽  
Angelo Musicò ◽  
Marco Brucale ◽  
Andrea Ridolfi ◽  
Silvia Galbiati ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Analytical Techniques ◽  
Heat Inactivation ◽  
Force Microscopy ◽  
Atomic Force ◽  
Droplet Pcr ◽  
Detergent Treatment ◽  
Routine Procedures ◽  
The Impact ◽  
Mirna Content

Since the outbreak of the COVID-19 crisis, the handling of biological samples from confirmed or suspected SARS-CoV-2-positive individuals demanded the use of inactivation protocols to ensure laboratory operators’ safety. While not standardized, these practices can be roughly divided into two categories, namely heat inactivation and solvent-detergent treatments. These routine procedures should also apply to samples intended for Extracellular Vesicles (EVs) analysis. Assessing the impact of virus-inactivating pre-treatments is therefore of pivotal importance, given the well-known variability introduced by different pre-analytical steps on downstream EVs isolation and analysis. Arguably, shared guidelines on inactivation protocols tailored to best address EVs-specific requirements will be needed among the analytical community, yet deep investigations in this direction have not yet been reported. We here provide insights into SARS-CoV-2 inactivation practices to be adopted prior to serum EVs analysis by comparing solvent/detergent treatment vs. heat inactivation. Our analysis entails the evaluation of EVs recovery and purity along with biochemical, biophysical and biomolecular profiling by means of a set of complementary analytical techniques: Nanoparticle Tracking Analysis, Western Blotting, Atomic Force Microscopy, miRNA content (digital droplet PCR) and tetraspanin assessment by microarrays. Our data suggest an increase in ultracentrifugation (UC) recovery following heat treatment; however, it is accompanied by a marked enrichment in EVs-associated contaminants. On the other hand, solvent/detergent treatment is promising for small EVs (<150 nm range), yet a depletion of larger vesicular entities was detected. This work represents a first step towards the identification of optimal serum inactivation protocols targeted to EVs analysis.

scholarly journals Isolation and Characterization of Nanofibrillar Cellulose from Agave tequilana Weber Bagasse

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Hasbleidy Palacios Hinestroza ◽  
Javier A. Hernández Diaz ◽  
Marianelly Esquivel Alfaro ◽  
Guillermo Toriz ◽  
Orlando J. Rojas ◽  
...  
Keyword(s):  
Cellulose Nanofibers ◽  
Average Diameter ◽  
Added Value ◽  
Advanced Materials ◽  
Degree Of Crystallinity ◽  
Agave Tequilana ◽  
Force Microscopy ◽  
Nanofibrillar Cellulose ◽  
Isolation And Characterization ◽  
Atomic Force

The bagasse of Agave tequilana Weber is one of the most abundant agroindustrial wastes in the state of Jalisco. However, at the present time, there is no technical use for this waste, and its high availability makes it an environmental problem. The objective of this research was to take advantage of this waste and give it an added value to be used in the elaboration of advanced materials. In this sense, the agave bagasse cellulose was obtained using an organosolv method. To obtain the nanofibrils, the cellulose was passed through 6 cycles of a microfluidizer. The material was classified by FTIR, confirming the presence of the functional groups (O-H, C-H, C-C, and C-O-C), characteristics of cellulose, and the elimination of hemicellulose and lignin present in agave bagasse without treatment. The X-ray diffraction technique allowed the determination of the degree of crystallinity of the cellulose nanofibers, which was 68.5%, with a negative zeta potential of −42 mV. The images from the atomic force microscopy helped for the observation of the degree of fibrillation in the cellulose, and with the software ImageJ, the average diameter of the nanofibers was determined to be 75 ± 5 nm with a relatively uniform length of 1.0–1.2 μm. Finally, by means of thermogravimetric analysis, it was found that the obtained cellulose nanofibers (CNFs) supported high temperatures of thermal decomposition, so it was concluded that due to the diameter of the fibrils, the high resistance to pressure, and elasticity, the nanofibrils obtained in this investigation can be used in the elaboration of advanced materials.

scholarly journals Ultrafiltration Segregates Tissue Regenerative Stimuli Harboured Within and Independent of Extracellular Vesicles

2020 ◽  
Author(s):  
TT Cooper ◽  
SE Sherman ◽  
T Dayarathna ◽  
GI Bell ◽  
Jun Ma ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Limb Ischemia ◽  
Murine Models ◽  
Label Free ◽  
Tubule Formation ◽  
Simple Method ◽  
Force Microscopy ◽  
Atomic Force

AbstractThe release of extracellular vesicles (EVs) from human multipotent stromal cells (MSC) has been proposed as a mechanism by which MSC mediate regenerative functions in vivo. Our recent work has characterized MSC derived from human pancreatic tissues (Panc-MSC) that generated a tissue regenerative secretome. Despite these advancements, it remains unknown whether regenerative stimuli are released independent or within extracellular vesicles. Herein, this study demonstrates ultrafiltration is a simple method to enrich for EVs which can be injected in murine models of tissue regeneration. The enrichment of EVs from Panc-MSC conditioned media (CM) was validated using nanoscale flow cytometry and atomic force microscopy; in addition to the exclusive detection of classical EV-markers CD9, CD81, CD63 using label-free mass spectrometry. Additionally, we identified several pro-regenerative stimuli, such as WNT5A or ANGPT1, exclusive to EV-enriched CM. Endothelial cell tubule formation was enhanced in response to both Panc-MSC CM fractions in vitro yet only intramuscular injection of EV-enriched CM demonstrated vascular regenerative functions in NOD/SCID mice with unilateral hind-limb ischemia (*<p<0.05). Furthermore, both EV-depleted and EV-enriched CM reduced hyperglycemia following intrapancreatic injection in hyperglycemic mice (**p<0.01). Collectively, understanding the functional synergy between compartments of the secretome is required to advance cell-free biotherapeutics into applications of regenerative medicine.

scholarly journals Structural Insights on Fusion Mechanisms of Extracellular Vesicles with Model Plasma Membrane

2020 ◽  
Author(s):  
Fabio Perissinotto ◽  
Valeria Rondelli ◽  
Beatrice Senigagliesi ◽  
Paola Brocca ◽  
László Almásy ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Extracellular Vesicles ◽  
Small Angle ◽  
Biochemical Characterization ◽  
The Body ◽  
Supported Lipid Bilayers ◽  
Force Microscopy ◽  
X Ray Scattering ◽  
Atomic Force ◽  
Liquid Ordered

AbstractExtracellular vesicles (EVs) represent a potent intercellular communication system. Within a lipid bilayer such small vesicles transport biomolecules between cells and throughout the body, strongly influencing the fate of recipient cells. Due to their specific biological functions they have been proposed as biomarkers for various diseases and as optimal candidates for therapeutic applications. Despite of their extreme biological relevance, the small size (30 to a few hundred nanometers in diameter) of EVs still poses a great challenge for their isolation, quantification and biophysical/biochemical characterization, therefore the complex network of EVs and cells as well as their interaction remains to be further revealed. Here we propose a multiscale platform based on Atomic Force Microscopy, Small Angle X-ray Scattering, Small Angle Neutron Scattering and Neutron Reflectometry to reveal structure-function correlations of purified EVs through the analysis of their interaction with model membrane systems, in form of both supported lipid bilayers and suspended unilamellar vesicles of variably complex composition. The analysis reveals a strong interaction of EVs with the model membranes and preferentially with liquid ordered raft-like lipid domains, and opens the way to understand uptake mechanisms in different vesicle to cell membrane relative compositions.

Bioactive Ceramic And Model Surfaces Adsorb Ligands and Factors That Stimulate Cellular Function

2000 ◽  
Vol 6 (S2) ◽  
pp. 992-993
Author(s):  
Russell J. Composto ◽  
Paul Ducheyne ◽  
Elsie Effah Kaufman
Keyword(s):  
Bioactive Glass ◽  
Serum Proteins ◽  
Rutherford Backscattering Spectrometry ◽  
Ionic Composition ◽  
Aqueous Media ◽  
Force Microscopy ◽  
Atomic Force ◽  
Kinetics Of Formation ◽  
Physical And Chemical

Microscopy and microanalysis techniques have played an important role in our understanding of how biomaterials interact with their environment. In first part of this study, we will focus on the behavior of bioactive glass, whereas in the second a model surface will be investigated. Upon implantation bioactive glass undergoes a series of reactions that leads to the formation of a calcium phosphate-rich layer. Most in vitro studies of the changes that occur on the surface of bioactive glass have employed the use of buffer solutions with compositions reflecting the ionic composition of interstitial fluid. Although these studies have documented the physical and chemical changes associated with bioactive glass immersed in aqueous media, they do not reveal the effect of serum proteins and cells which are present at the implantation site. In the present study, we document, using atomic force microscopy (AFM) and Rutherford backscattering spectrometry (RBS), significant differences in reaction layer composition, thickness, morphology and kinetics of formation arising from the presence of serum proteins.

Epitope Peptide Amphiphile-Based Nanofiber as an Effective Vaccine for Viral Infectious Diseases

2020 ◽  
Vol 20 (9) ◽  
pp. 5329-5332 ◽  
Author(s):  
Yu-Gyeong Kim ◽  
Yunsu Lee ◽  
Jong-Wha Jung ◽  
Hyo-Eon Jin
Keyword(s):  
Immune Responses ◽  
Enterovirus 71 ◽  
Foot And Mouth Disease ◽  
Effective Vaccine ◽  
Epitope Peptide ◽  
Serum Samples ◽  
Peptide Amphiphile ◽  
Force Microscopy ◽  
Self Assembling ◽  
Atomic Force

Peptide-based vaccines are relatively safe but have weak immune responses even with an adjuvant. In order to overcome the limitations of peptide-based vaccines, we developed peptide amphiphile (PA)-based nanofibers to enhance the immune responses for preventing enterovirus 71 (EV71) infectious disease (i.e., Hand, Foot, and Mouth Disease). PAs are peptides conjugated with fatty acid alkyl chain and able to self-assemble into various structures including high-aspectratio nanofibers. We designed PAs by coupling EV71 virus particle 1 (VP1) epitope peptides and spacer-crosslinker to the N-terminal of long-chain fatty acids (VP1-PA). PAs then self-assembled into nanofibers at physiological pH (pH 7.4). PA nanofibers were characterized by atomic force microscopy (AFM). For the immunization studies, C57BL/6 mice were injected intraperitoneally (i.p.) with recombinant VP1 with adjuvant (alum), VP1 epitope peptide with or without adjuvant, VP1-PA nanofibers with or without adjuvant, and PBS. To assess the immunogenecity of the VP1-PA nanofibers on serum samples from the immunized mice was analyzed by Western blot for the evaluation of VP1-specific IgG. The PA group showed a higher immune response than the peptide group. We expect that self-assembling VP1-PA based nanofibers as an immune stimulator could enhance immune responses effectively against EV71 infection and overcome the limitations of peptide-based vaccine.

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