scholarly journals Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!

2019 ◽  
Vol 21 (1) ◽  
pp. 291 ◽  
Author(s):  
Fanny Ender ◽  
Piet Zamzow ◽  
Nikolas von Bubnoff ◽  
Frank Gieseler
Keyword(s):  
High Speed ◽  
Maximum Yield ◽  
Extracellular Vesicle ◽  
Carcinoma Cells ◽  
Specific Method ◽  
Liquid Biopsies ◽  
Pancreas Carcinoma ◽  
Threshold Adjustment ◽  
Membrane Shedding ◽  
Protocol Standardization

The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV subpopulations from supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000× g centrifugation, we reconstituted high-speed (hs) EVs from the pellet, directly labeled them with the membrane dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and performed flow cytometry based analysis. The aim was to optimize the conditions for EV labeling and detection and hence to obtain a maximum yield of intact hsEVs. We found that, for sufficient labeling of EVs, minimal temperature variations and short incubation times correlated with EV stability. Furthermore, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of CFSE labeled hsEVs. When cells were CFSE labeled, we observed a transition of fluorescence onto EVs that were reconstituted from the pellet but not onto those that remained in the supernatant after hs centrifugation, suggesting the indirect labeling of EVs based on the way of biogenesis as a specific method for the distinction of exosomes and ectosomes. Protocol standardization is of major importance for the use of EVs as diagnostic markers in liquid biopsies.

Protein biomarkers in breast cancer-derived extracellular vesicles for use in liquid biopsies

2021 ◽  
Author(s):  
Dan Li ◽  
Wenjia Lai ◽  
Di Fan ◽  
Qiaojun Fang
Keyword(s):  
Breast Cancer ◽  
Early Diagnosis ◽  
Extracellular Vesicles ◽  
Liquid Biopsy ◽  
Breast Cancer Diagnosis ◽  
Extracellular Vesicle ◽  
Detection Methods ◽  
Protein Markers ◽  
Liquid Biopsies ◽  
Diagnosis And Prognosis

Breast cancer is the most common malignant disease in women worldwide. Early diagnosis and treatment can greatly improve the management of breast cancer. Liquid biopsies are becoming convenient detection methods for diagnosing and monitoring breast cancer due to their non-invasiveness and ability to provide real-time feedback. A range of liquid biopsy markers, including circulating tumor proteins, circulating tumor cells, and circulating tumor nucleic acids, have been implemented for breast cancer diagnosis and prognosis, with each having its own advantages and limitations. Circulating extracellular vesicles are messengers of intercellular communication that are packed with information from mother cells and are found in a wide variety of bodily fluids; thus, they are emerging as ideal candidates for liquid biopsy biomarkers. In this review, we summarize extracellular vesicle protein markers that can be potentially used for the early diagnosis and prognosis of breast cancer or determining its specific subtypes.

scholarly journals Small extracellular vesicle-encapsulated miR-181b-5p, miR-222-3p and let-7a-5p: Next generation plasma biopsy-based diagnostic biomarkers for inflammatory breast cancer

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250642
Author(s):  
Sarah Hamdy Ahmed ◽  
Nancy A. Espinoza-Sánchez ◽  
Ahmed El-Damen ◽  
Sarah Atef Fahim ◽  
Mohamed A. Badawy ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
Inflammatory Breast Cancer ◽  
Roc Curves ◽  
Cost Effective ◽  
Extracellular Vesicle ◽  
Lymphatic Invasion ◽  
Precipitation Method ◽  
Liquid Biopsies ◽  
Diagnostic Biomarkers

Inflammatory breast cancer (IBC) is a rare, but aggressive entity of breast carcinoma with rapid dermal lymphatic invasion in young females. It is either poorly or misdiagnosed as mastitis because of the absence of a distinct lump. Small extracellular vesicles (sEVs) circulating in liquid biopsies are a novel class of minimally invasive diagnostic alternative to invasive tissue biopsies. They modulate cancer progression via shuttling their encapsulated cargo including microRNAs (miRNAs) into recipient cells to either trigger signaling or induce malignant transformation of targeted cells. Plasma sEVs < 200 nm were isolated using a modified cost-effective polyethylene glycol (PEG)-based precipitation method and compared to standard methods, namely ultracentrifugation and a commercial kit, where the successful isolation was verified by different approaches. We evaluated the expression levels of selected sEV-derived miR-181b-5p, miR-222-3p and let-7a-5p using quantitative real PCR (qPCR). Relative to non-IBC, our qPCR data showed that sEV-derived miR-181b-5p and miR-222-3p were significantly upregulated, whereas let-7a-5p was downregulated in IBC patients. Interestingly, receiver operating characteristic (ROC) curves analysis revealed that diagnostic accuracy of let-7a-5p alone was the highest for IBC with an area under curve (AUC) value of 0.9188, and when combined with miR-222-3p the AUC was improved to 0.973. Further, 38 hub genes were identified using bioinformatics analysis. Together, circulating sEV-derived miR-181b-5p, miR-222-3p and let-7a-5p serve as promising non-invasive diagnostic biomarkers for IBC.

Defect and Dopant Control During Silicon Epitaxy Using B and Ge

1987 ◽  
Vol 104 ◽  
Author(s):  
R. R. Kola ◽  
J. B. Posthill ◽  
A. S. M. Salih ◽  
G. A. Rozgonyi ◽  
K. E. Bean ◽  
...  
Keyword(s):  
High Speed ◽  
Maximum Yield ◽  
Dark Field ◽  
Electrical Performance ◽  
Epitaxial Layers ◽  
Threading Dislocation ◽  
Epitaxial Silicon ◽  
Cross Sectional ◽  
Plan View ◽  
Co Doped

ABSTRACTThe control of dopants, impurities and defects for VLSI of silicon integrated circuits requires a complex set of crystal and processing conditions to be satisfied simultaneously. In order to achieve the maximum yield and highest level of electrical performance for a given device design, we have manipulated the lattice constant and boron doping levels in CVD epitaxial silicon layers co-doped with germanium. By adjusting the ratios of germane and diborane in a dichlorosilane/hydrogen CVD reactor we have obtained buried high conducting layers which are strain-free and lattice matched to the Si substrate. Degenerate boron and boron and germanium codoped epitaxial layers on (100) p-type silicon substrates were investigated. Solubility, electrical activity limits and defect structure of boron in strained and strain-free silicon epitaxial layers were investigated by spreading resistance, SIMS profiling, X-ray and transmission electron microscopy techniques. Bright field and weak-beam dark field imaging of cross-sectional and plan-view specimens were used to confirm the presence or absence of precipitates and threading dislocations. A model has been proposed to describe the mechanism of threading dislocation formation in heavily boron-doped layers. We are now in a position to strategically locate co-doped Si(B, Ge) p++ layers as recombination zones or buried field plates to suit the needs of MOS latchup control, high speed and radiation hard devices, as well as the needs of defect free p++ etch stops for thin membranes and three-dimensional silicon structures.

scholarly journals Dynamic Modelling, Experimental Identification and Computer Simulations of Non-Stationary Vibration in High-Speed Elevators

2021 ◽  
Vol 67 (6) ◽  
Author(s):  
Radomir Đokić ◽  
Jovan Vladić ◽  
Milan Kljajin ◽  
Vesna Jovanović ◽  
Goran Marković ◽  
...  
Keyword(s):  
Dynamic Model ◽  
High Speed ◽  
Dynamic Behaviour ◽  
Dynamic Modelling ◽  
Control Program ◽  
Specific Method ◽  
Unique Method ◽  
Stationary Vibration ◽  
Vertical Vibrations ◽  
Stiffness And Damping

Modelling the dynamic behaviour of elevators with high lifting velocities (contemporary elevators in building construction and mine elevators) is a complex task and an important step in the design process and creating conditions for safe and reliable exploitation of these machines. Due to high heights and lifting velocities, the standard procedures for dynamic exploitation are not adequate. The study presents the method of forming a dynamic model to analyse nonstationary vibrations of a rope with time-varying length with nonholonomic boundary conditions in the position where the rope is connected with the cabin (cage) and in the upcoming point of its winding onto the pulley (drum). A unique method was applied to identify the basic parameters of the dynamic model (stiffness and damping) based on experimental measures for a concrete elevator. Due to the verification of this procedure, the experiment was conducted on a mine elevator in RTB Bor, Serbia. Using the obtained computer-experimental results, the simulations of the dynamic behaviour of an empty and loaded cage were shown. In addition, the study shows the specific method as the basis for forming a control program that would enable the decrease in vertical vibrations during an elevator starting and braking mode.

scholarly journals Blood-Derived Extracellular Vesicle-Associated miR-3182 Detects Non-Small Cell Lung Cancer Patients

Cancers ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 257
Author(s):  
Kekoolani S. Visan ◽  
Richard J. Lobb ◽  
Shu Wen Wen ◽  
Justin Bedo ◽  
Luize G. Lima ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Early Stage ◽  
Survival Rates ◽  
Cancer Biomarkers ◽  
Extracellular Vesicle ◽  
Lung Cells ◽  
Sequencing Analysis ◽  
Sample Collection ◽  
Liquid Biopsies ◽  
Lung Cancer Patients

With five-year survival rates as low as 3%, lung cancer is the most common cause of cancer-related mortality worldwide. The severity of the disease at presentation is accredited to the lack of early detection capacities, resulting in the reliance on low-throughput diagnostic measures, such as tissue biopsy and imaging. Interest in the development and use of liquid biopsies has risen, due to non-invasive sample collection, and the depth of information it can provide on a disease. Small extracellular vesicles (sEVs) as viable liquid biopsies are of particular interest due to their potential as cancer biomarkers. To validate the use of sEVs as cancer biomarkers, we characterised cancer sEVs using miRNA sequencing analysis. We found that miRNA-3182 was highly enriched in sEVs derived from the blood of patients with invasive breast carcinoma and NSCLC. The enrichment of sEV miR-3182 was confirmed in oncogenic, transformed lung cells in comparison to isogenic, untransformed lung cells. Most importantly, miR-3182 can successfully distinguish early-stage NSCLC patients from those with benign lung conditions. Therefore, miR-3182 provides potential to be used for the detection of NSCLC in blood samples, which could result in earlier therapy and thus improved outcomes and survival for patients.

An Area-Based Faulting Measurement Method Using Three-Dimensional Pavement Data

2018 ◽  
Vol 2672 (40) ◽  
pp. 41-49 ◽  
Author(s):  
Georgene M. Geary ◽  
Yichang (James) Tsai ◽  
Yiching Wu
Keyword(s):  
High Speed ◽  
Three Dimensional ◽  
Field Tests ◽  
Automatic Testing ◽  
Specific Method ◽  
Concrete Pavements ◽  
Federal Highway ◽  
Manual Testing ◽  
The Impact ◽  
Departments Of Transportation

Faulting is one of the three performance measures recognized by the Code of Federal Regulations (CFR) for jointed concrete pavements. U.S. Federal Highway Administration rules developed for the CFR require the state Departments of Transportation to use the AASHTO Standard R 36, Standard Method for Evaluating Faulting of Concrete Pavements, for measurement of faulting. R 36 allows both manual testing using a faultmeter and automatic testing using a high-speed inertial profiler (HSIP). There is a concern in the literature that the HSIP methods are not accurate enough for network level testing of faulting. Recent studies have also shown that three-dimensional (3-D) laser technology is showing promise for providing more accurate faulting measurements. Still, there currently is not a specific method in R 36 that utilizes 3-D data for gathering faulting measurements. This paper proposes a new method that takes full advantage of the full-coverage capabilities of 3-D to measure faulting more accurately and consistently. This method uses the comparison of smoothed areas on both sides of a joint instead of a single longitudinal profile to measure elevation differences. Field tests were performed to compare the 3-D method with manual readings taken with a Georgia faultmeter on two sections of interstate in Georgia. In addition, the impact of various footprints and measuring locations of faulting measurements using this 3-D technology were quantitatively evaluated using the proposed method. Based on this case study the new proposed 3-D method appears to be appropriate to be considered to be added as a 3-D automatic method in AASHTO R 36.

scholarly journals A Framework for Mitigating DDoS and DOS Attacks in IoT Environment Using Hybrid Approach

Electronics ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1282
Author(s):  
Abdulrahman Aminu Ghali ◽  
Rohiza Ahmad ◽  
Hitham Alhussian
Keyword(s):  
High Speed ◽  
Oil And Gas ◽  
Denial Of Service ◽  
Hybrid Approach ◽  
Social Engineering ◽  
Specific Method ◽  
Dos Attacks ◽  
Man In The Middle ◽  
Intelligent Devices ◽  
Iot Devices

The Internet of Things (IoT) has gained remarkable acceptance from millions of individuals. This is evident in the extensive use of intelligent devices such as smartphones, smart television, speakers, air conditioning, lighting, and high-speed networks. The general application area of IoT includes industries, hospitals, schools, homes, sports, oil and gas, automobile, and entertainment, to mention a few. However, because of the unbounded connection of IoT devices and the lack of a specific method for overseeing communication, security concerns such as distributed denial of service (DDoS), denial of service (DoS), replay, botnet, social engineering, man-in-the-middle, and brute force attacks have posed enormous challenges in the IoT environment. Regarding these enormous challenges, this study focuses on DDoS and DoS attacks. These two attacks have the most severe consequences in the IoT environment. The solution proposed in this study can also help future researchers tackle the expansion of IoT security threats. Moreover, the study conducts rigorous experiments to assess the efficiency of the proposed approach. In summary, the experimental results show that the proposed hybrid approach mitigates data exfiltration caused by DDoS and DoS attacks by 95.4%, with average network lifetime, energy consumption, and throughput improvements of 15%, 25%, and 60%, respectively.

scholarly journals Large Extracellular Vesicle Characterization and Association with Circulating Tumor Cells in Metastatic Castrate Resistant Prostate Cancer

Cancers ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 1056
Author(s):  
Anna S. Gerdtsson ◽  
Sonia M. Setayesh ◽  
Paymaneh D. Malihi ◽  
Carmen Ruiz ◽  
Anders Carlsson ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Extracellular Vesicle ◽  
Object Identification ◽  
Protein Profiling ◽  
Liquid Biopsies ◽  
Cancer Tumor ◽  
Castrate Resistant Prostate Cancer ◽  
Castrate Resistant

Liquid biopsies hold potential as minimally invasive sources of tumor biomarkers for diagnosis, prognosis, therapy prediction or disease monitoring. We present an approach for parallel single-object identification of circulating tumor cells (CTCs) and tumor-derived large extracellular vesicles (LEVs) based on automated high-resolution immunofluorescence followed by downstream multiplexed protein profiling. Identification of LEVs >6 µm in size and CTC enumeration was highly correlated, with LEVs being 1.9 times as frequent as CTCs, and additional LEVs were identified in 73% of CTC-negative liquid biopsy samples from metastatic castrate resistant prostate cancer. Imaging mass cytometry (IMC) revealed that 49% of cytokeratin (CK)-positive LEVs and CTCs were EpCAM-negative, while frequently carrying prostate cancer tumor markers including AR, PSA, and PSMA. HSPD1 was shown to be a specific biomarker for tumor derived circulating cells and LEVs. CTCs and LEVs could be discriminated based on size, morphology, DNA load and protein score but not by protein signatures. Protein profiles were overall heterogeneous, and clusters could be identified across object classes. Parallel analysis of CTCs and LEVs confers increased sensitivity for liquid biopsies and expanded specificity with downstream characterization. Combined, it raises the possibility of a more comprehensive assessment of the disease state for precise diagnosis and monitoring.

scholarly journals EV Translational Horizons as Viewed Across the Complex Landscape of Liquid Biopsies

2021 ◽  
Vol 9 ◽  
Author(s):  
Bryce Killingsworth ◽  
Joshua A. Welsh ◽  
Jennifer C. Jones
Keyword(s):  
Extracellular Vesicle ◽  
Current Evidence ◽  
Liquid Biopsies ◽  
Ribonucleoprotein Complexes ◽  
Biological Studies ◽  
Complex Landscape ◽  
Clinical Correlations ◽  
Treatment Responses ◽  
Therapeutic Tools ◽  
Monitoring Treatment

Extracellular Vesicle (EV)-based diagnostic and therapeutic tools are an area of intensive study and substantial promise, but EVs as liquid biopsies have advanced years ahead of EVs as therapeutic tools. EVs are emerging as a promising approach for detecting tumors, evaluating the molecular profiles of known disease, and monitoring treatment responses. Although correlative assays based on liquid biopsies are already having an impact on translational studies and clinical practice, much remains to be learned before these assays will be optimized for clinical correlations, functional biological studies, and therapeutic use. What follows is an overview of current evidence supporting the investigation and use of liquid biopsies, organized by specific liquid biopsy components available for analysis, along with a summary of what challenges must be overcome before these assays will provide functional biological insights into the pathogenesis and treatment of disease. The same challenges must also be overcome before it will be feasible to measure and monitor the dosing, distribution, pharmacokinetics, and delivery of EV therapeutics and their cargo in complex biofluids where EVs and circulate with and are co-isolated with a number of other nanoscale materials, including lipoproteins (LPPs), ribonucleoprotein complexes (RNPs), and cell free nucleic acids (cfNA).

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