Framework for rapid comparison of extracellular vesicle isolation methods

Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive, single molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays, all on one platform, to improve SEC isolation from plasma and CSF. Our results highlight the utility of quantifying EV proteins using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

Sodium Chloroacetate Modified Polyethyleneimine/Trimesic Acid Nanofiltration Membrane to Improve Antifouling Performance

Nanofiltration (NF) is a separation technology with broad application prospects. Membrane fouling is an important bottleneck-restricting technology development. In the past, we prepared a positively charged polyethyleneimine/trimesic acid (PEI/TMA) NF membrane with excellent performance. Inevitably, it also faces poor resistance to protein contamination. Improving the antifouling ability of the PEI/TMA membrane can be achieved by considering the hydrophilicity and chargeability of the membrane surface. In this work, sodium chloroacetate (ClCH2COONa) is used as a modifier and is grafted onto the membrane surface. Additionally, 0.5% ClCH2COONa and 10 h modification time are the best conditions. Compared with the original membrane (M0, 17.2 L m−2 h−1), the initial flux of the modified membrane (M0-e, 30 L m−2 h−1) was effectively increased. After filtering the bovine albumin (BSA) solution, the original membrane flux dropped by 47% and the modified membrane dropped by 6.2%. The modification greatly improved the antipollution performance of the PEI/TMA membrane.

A Simple Method for Extraction of Lipopolysaccharides from Brucella Melitensis

Background: Brucellosis is the most common zoonotic bacterial disease around the world, and its causative agent is Brucella, a gram-negative bacterium. Lipopolysaccharide (LPS) molecules are the most significant surface antigen of Brucella bacteria. This antigen is used in a high number of research and diagnostic fields. The present study was designed in order to introduce a simple method to extract LPS from Brucella melitensis. Materials and Methods: Smooth strains of B. melitensis were cultured in large quantity, and LPS was extracted through changes in the hot-phenol method. The LPS concentration was measured by using the dimethyl-methylene blue method. The contamination rates of proteins and nucleic acids were determined using the bicinchoninic acid (BCA) method and absorbance measurement at 260 nm, respectively. Results: The amount of the extracted LPS was 1.1% of the wet weight of the bacteria. The rate of contamination with nucleic acid was measured to be 0.2% of the LPS. The protein contamination was not detectable through the BCA method. Conclusion: The advantages of our newly-introduced method are considerable decrease in the time required to extract LPS; independence from nucleic acid and protein digestive enzymes; an extraction rate of LPS equivalent to other applied methods; nucleic acid contamination equal or lower than other methods, and no protein contamination; and independence from equipment such as ultracentrifuge and chromatography. [GMJ.2021;10:e1944]

Comparison of Extracellular Vesicle Isolation Methods from Human Biofluids using Single Molecule Arrays

Extracellular vesicles (EVs) are released by all cells into biofluids and hold great promise as reservoirs of disease biomarkers. One of the main challenges in studying EVs and using them for diagnostics is a lack of methods to quantify EVs that are sensitive enough and can differentiate EVs from similarly sized lipoproteins and protein aggregates. We demonstrate the use of ultrasensitive assays to quantify EVs by immuno-isolating and detecting EV transmembrane proteins in microwell arrays. We developed single molecule array (Simoa) assays for the quantification of EVs using three widely expressed transmembrane proteins: the tetraspanins CD9, CD63, and CD81. Using Simoa to measure these three EV markers, as well as albumin to measure protein contamination, we were able to compare the relative efficiency and purity of several commonly used EV isolation methods in plasma and cerebrospinal fluid (CSF): ultracentrifugation, precipitation, and size exclusion chromatography (SEC). We further used these assays to rapidly optimize EV isolation using SEC from plasma and CSF. Our results highlight the utility of quantifying EVs using Simoa and provide a rapid framework for comparing and improving EV isolation methods from biofluids.

Three Method-Combination Protocol for Improving Purity of Extracellular Vesicles

Extracellular vesicles (EVs) are nanosized structures able to carry proteins, lipids and genetic material from one cell to another with critical implications in intercellular communication mechanisms. Even though the rapidly growing EVs research field has sparked great interest in the last 20 years, many biological and technical aspects still remain challenging. One of the main issues that the field is facing is the absence of consensus regarding methods for EVs concentration from biofluids and tissue culture medium. Yet, not only can classic methods be time consuming, commercialized kits are also often quite expensive, especially when research requires analyzing numerous samples or concentrating EVs from large sample volumes. In addition, EV concentration often results in either low final yield or significant contamination of the vesicle sample with proteins and protein complexes of similar densities and sizes. Eventually, low vesicle yields highly limit any further application and data reproducibility while contamination greatly impacts extensive functional studies. Hence, there is a need for accessible and sustainable methods for improved vesicle concentration as this is a critical step in any EVs-related research study. In this brief report, we describe a novel combination of three well-known methods in order to obtain moderate-to-high yields of EVs with reduced protein contamination. We believe that such methods could be of high benefits for in vitro and in vivo functional studies.

Characterizing the Physical Properties and Cell Compatibility of Phytoglycogen Extracted from Different Sweet Corn Varieties

Owing to its unique structure and properties, the glucose dendrimer phytoglycogen is gaining interest for medical and biotechnology applications. Although many maize variants are available from commercial and academic breeding programs, most applications rely on phytoglycogen extracted from the common maize variant, sugary1. Here we characterized the solubility, hydrodynamic diameter, water-binding properties, protein contaminant concentration, and cytotoxicity of phytoglycogens from different maize sources, A632su1, A619su1, Wesu7, and Ia453su1, harboring various sugary1 mutants. A619su1-SW phytoglycogen was cytotoxic while A632su1-SW phytoglycogen was not. A632su1-Pu phytoglycogen promoted cell growth, whereas extracts from A632su1-NE, A632su1-NC, and A632su1-CM were cytotoxic. Phytoglycogen extracted from Wesu7su1-NE using ethanol precipitation was cytotoxic. Acid-treatment improved Wesu7 phytoglycogen cytocompatibility. Protease-treated Wesu7 extracts promoted cell growth. Phytoglycogen extracted from Ia453su1 21 days after pollination (“Ia435su1 21DAP”) was cytotoxic, whereas phytoglycogen extracted at 40 days (“Ia435su1 40DAP”) was not. In general, size and solubility had no correlation with cytocompatibility, whereas protein contaminant concentration and water-binding properties did. A632su1-CM had the highest protein contamination among A632 mutants, consistent with its higher cytotoxicity. Likewise, Ia435su1 21DAP phytoglycogen had higher protein contamination than Ia435su1 40DAP. Conversely, protease-treated Wesu7 extracts had lower protein contamination than the other Wesu7 extracts. A632su1-NE, A632su1-NC, and A632su1-CM had similar water-binding properties which differed from those of A632su1-Pu and A632su1-SW. Likewise, water binding differed between Ia435su1 21DAP and Ia435su1 40DAP. Collectively, these data demonstrate that maize phytoglycogen extracts are not uniformly cytocompatible. Rather, maize variant, plant genotype, protein contaminants, and water-binding properties are determinants of phytoglycogen cytotoxicity.

Morinda Officinalis Polysaccharides Attenuate Varicocele-Induced Spermatogenic Impairment through the Modulation of Angiogenesis and Relative Factors

Evidence supporting best treatment practices for varicocele is lacking. The effects of a water-soluble polysaccharide extracted from Morinda officinalis (MOP) on the progression of varicocele were evaluated in the present study. The extracted MOP was confirmed as having a high purity of 98% with scant protein contamination, and it mainly consisted of glucose, lactose, and xylose at a molar ratio of 7.63:1.23:0.95 glucose:lactose:xylose. MOPs were administered to experimental left varicocele rats immediately after surgery at doses ranging from 25 to 200 mg/kg. As detected by sperm analysis and histopathological staining, the intragastric administration of 100 mg/kg MOPs significantly improved the sperm parameters of bilateral cauda epididymis, attenuated seminiferous epithelial structures, and inhibited germ cell apoptosis. The results of immunofluorescence and immunoblot showed that administration of 100 mg/kg MOPs effectively inhibited angiogenesis in the bilateral testes but modulated the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase 2 (MMP2), and MMP9 mildly. These results indicate that inhibition of angiogenesis may be one of the mechanisms by which MOP exerts its inhibitive activities on the progression of varicocele, whereas a relative upregulation of VEGF and MMP-9 may be crucial for the spermatogenetic protective effects of 100 mg/kg MOP administration.