CM-Dil Staining and SEC of Plasma as an Approach to Increase Sensitivity of Extracellular Nanovesicles Quantification by Bead-Assisted Flow Cytometry

The quantification of the specific disease-associated populations of circulating extracellular membrane nanovesicles (ENVs) has opened up new opportunities for liquid biopsy in cancer and other chronic diseases. However, the sensitivity of such methods is mediated by an optimal combination of the isolation and labeling approaches, and is not yet sufficient for routine clinical application. The presented study aimed to develop, characterize, and explore a new approach to non-specific ENV staining, followed by size-exclusive chromatography (SEC), which allows us to increase the sensitivity of bead-assisted flow cytometry. Plasma from healthy donors was purified from large components, stained with lipophilic CM-Dil dye, and fractionated by means of SEC. The obtained fractions were analyzed in terms of particle size and concentration using NTA, as well as vesicular markers and plasma protein content via dot-blotting. We characterized the process of CM-Dil-stained plasma fractionation in detail and indicated the fractions with optimal characteristics. Finally, we explored the sensitivity of on-bead flow cytometry for the analysis of specific populations of plasma ENVs and demonstrated the advantages and limitations of the proposed technique.

Constructing an Efficient Bacillus subtilis Spore Display by Using Cohesin−Dockerin Interactions

Bacillus subtilis spore display has become a field of increasing interest in the past two decades. To improve the efficiency of B. subtilis spore display, its directed modification was performed based on the cellulosome architecture by introducing onto them divergent cohesin (Coh) modules that can specifically bind to the target enzyme bearing the matching dockerins (Doc). In this study, five different pairs of cohesins and dockerins, selected from four cellulolytic microbes, were examined for their capabilities in displaying a tetrameric enzyme β-galactosidase from Bacillus stearothermophilus IAM11001 on the surface of B. subtilis WB600 spores. Immunofluorescence microscopy, western blotting, dot blotting, and enzyme assay was applied to confirm its surface expression. All the resultant five Coh–Doc based spore display can hydrolyze o-nitrophenyl-β-D-galactopyranoside. Further, the optimized Coh–Doc based spore display exhibited the highest display efficiency. Overall, the results of current study may open new perspectives on the use of Coh–Doc interaction, which will find application in improving the efficiency of B. subtilis spore display.

Detection and characterization of purified antigenic proteins from culture filtrate of Mycobacterium bovis strain AN5

Background and Objectives: Bovine tuberculosis diagnosis is usually performed by various tests with specific limitations. Mycobacterium bovis culture filtrate contains antigenic proteins that could be used to improve the sensitivity of bovine tuber- culosis diagnosis. The objective of this study was to identify and purify antigenic proteins from culture filtrates of M. bovis strain AN5 for use in immunological assays. Materials and Methods: Secreted proteins were purified from the heat-treated culture filtrate of M. bovis strain AN5. Pro- teins were precipitated with ammonium sulfate, fractionated by Sephadex G50 chromatography. The protein concentrations and the approximate molecular weight were determined by lowry method and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. Immunological methods, including dot-blotting and western blotting, as- sessed the quality of the isolated proteins. Results: The quantity of antigenic proteins in the culture medium was measured at far more than 15% of the amount of pro- teins secreted into medium. Three main chromatographic fractions obtained and showed concentrations of proteins ranging from 14 to 60 μg/μl with molecular weights in the 10 to 180 kDa range. The purified antigens showed positive reactions to the infected cattle serum throughout dot-blotting. Western blotting revealed a total of 15 to 70 kDa molecular weight proteins. Conclusion: Immunoblotting analysis made it possible to detect and recognize novel antigens that are useful for bovine tu- berculosis diagnosis improvement. This is significant since non-specific reactions were not observed when we utilized serum of cattle experimentally infected with M. bovis as a polyclonal antibody.

Fabrication of a paper strip for facile and rapid detection of bovine viral diarrhea virus via signal enhancement by copper polyhedral nanoshells

A rapid and simple affinity dot-blotting scheme via signal enhancement by copper nano-polyhedral shells on the surface of gold nanoparticles.

The possibility of identifying individual strains of glanders and melioidosis pathogens with a combination of immunoblotting and DNA amplification methods

Causative agents of melioidosis and glanders are among the most dangerous bacterial pathogens for human. Moreover, Burkholderia pseudomallei and Burkholderia mallei are considered to be potential bioterrorism agents. In connection with this, timely diagnostics of such bacteria is of high importance. In our study, we made an attempt to develop an approach for detecting pathogenic Burkholderia spp. by combining species-specific amplification and strain-specific dot blotting assay with monoclonal antibodies. The following pathogenic Burkholderia strains were used in experiments: B. mallei (C-4, C-5, t-12, B-120, P-1, Мuksuwar-11, Z-12,Zagreb, Ivanovich, 5534), and B. pseudomallei (100, 102, 115, 116, 132, 135, 301, 51274, 60913, 61503). Real-Time PCR (RT-PCR) and dot blotting with monoclonal antibodies against surface Burkholderia epitopes were used to detect such pathogens. RT-PCR was carried out by using primers designed to recognize DNA fragments in B. mallei IS407A-fliP and the gene Orf12 fromB. pseudomallei. For this, DNA was isolated from bacterial cells suspended at 1 × 104 microbial cells/ml. accumulation of the end reaction products was visualized by staining with dye SYBR Green I. Specificity of amplification reaction was determined by measuring melting temperature (Tm) for end products followed by running gel electrophoresis. It was demonstrated that all ten strains of eitherB. malleiorB. pseudomalleiexamined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. It was demonstrated that all ten strains of eitherB. malleiorB. pseudomalleiexamined in the study were detected by using primers against IS407A-fliP DNA fragment and the gene Orf12, respectively. Importantly, no signals specific to heterologous microbial DNA (isolated from bacterial cell suspension at concentration of 1 × 107 microbial cells/ml) were detected by using RT-PCR. Thus, RT-PCR provides an opportunity for assessing an inter-species diversity among pathogenic Burkholderia species. A genus-specificity was observed by using monoclonal antibodies 3D3 which bind to both Burkholderia strains, whereas antibodies 2D11 exhibited no selective binding to strain Р1 B. mallei and strain 100 B. pseudomallei, thereby displaying a strain-specific interaction. Thus, it allowed to conclude that combining a species-specific DNA amplification particularly RT-PCR together with immune-based assay such as dot blotting by using a panel of monoclonal antibodies seems to be a promising approach for assessing intra-species diversity among pathogenic Burkholderia.

New evidence on pectin-related instantaneous pressure softening mechanism of asparagus lettuce under high pressure processing

Evidence on mechanism of instantaneous pressure softening of asparagus lettuce under high pressure processing was explored with respect to pectin methylesterase activity, degree of methylation of pectin, degree of methylation patterns of pectin fractions, and pectin distribution in cell wall matrix. Instantaneous pressure softening was observed at 300 MPa, while texture recovery was obtained at 500 MPa. Pectin methylesterase activity was not significantly affected at 100 and 300 MPa, but dramatically activated at 500 MPa (p < 0.05). Correspondingly, the degree of methylation of pectin decreased as pressure rose. Results of in situ immuno-dot blotting and immunolabeling based on specific bindings of antipectin antibodies showed a significant reduction of chelator-soluble pectin at 300 MPa, in contrast to a remarkable increase at 500 MPa. High pressure processing-induced demethoxylation was further verified by the enhanced fluorescence intensity of LM19 (an antihomogalacturonan antibody specifically binds to nonmethoxylated pectin) immunolabeled pectin, which was mainly located in tricellular junctions at 300 MPa, but covered the full cell surface at 500 MPa. In conclusion, instantaneous pressure softening of asparagus lettuce is strongly associated with loss of chelator-soluble pectin at 300 MPa.