Hepatocellular carcinoma diagnosis using a novel electrochemiluminescence immunoassay targeting serum IgM-free AIM

Background Recent increases in the number of patients with non-alcoholic steatohepatitis (NASH) warrant the identification of biomarkers for early detection of hepatocellular carcinoma (HCC) associated with NASH (NASH-HCC). IgM-free apoptosis inhibitor of macrophage (AIM), which generally associates with IgM in blood and exerts its biological function by dissociation from IgM, may serve as an effective biomarker for NASH-HCC. Here, we established a fully automatic and high-throughput electrochemiluminescence immunoassay (ECLIA) to measure IgM-free AIM and investigated its efficacy in diagnosing NASH-HCC and viral HCC. Methods IgM-free AIM levels were measured in 212 serum samples from patients with, or without, HCC related to NASH, hepatitis B virus, and hepatitis C virus, using ECLIA. We also developed an ECLIA for measuring both IgM-free and IgM-bound AIM and investigated the existing form of AIM in blood by size-exclusion chromatography. Results IgM-free AIM levels were significantly higher in the HCC group than in the non-HCC group, regardless of the associated pathogenesis. Moreover, the area under the receiver operating curve for IgM-free AIM was greater than that for conventional HCC biomarkers, alpha-fetoprotein or des-γ-carboxy prothrombin, regardless of the cancer stage. ECLIA counts of IgM-free AIM derived from samples fractionated by size-exclusion chromatography were significantly higher in patients with NASH-HCC than in healthy volunteers and in patients with non-alcoholic fatty liver and NASH. Conclusions Serum IgM-free AIM may represent a universal HCC diagnostic marker superior to alpha-fetoprotein or des-γ-carboxy prothrombin. Our newly established ECLIA could contribute to further clinical studies on AIM and in vitro HCC diagnosis.

Influence of environment, temperature and time of the thermal modification of ash wood (Fraxinus excelsior L.) on the cellulose weight average degree of polymerization

Influence of environment, temperature and time of the thermal modification of ash wood (Fraxinus excelsior L.) on the cellulose weight average degree of polymerization . Using the size-exclusion chromatography (HPLC SEC) method, the weight average degree of cellulose polymerization was determined. The polymer was isolated by the Kürschner-Hoffer method from ash wood (Fraxinus excelsior L.). The wood was thermally modified in different environments (nitrogen, steam and air) at 190°C and modification times of 2, 6 and 10 hours. Depending on the anaerobic atmosphere used, the highest values of the weight average degree of cellulose polymerization were obtained for the nitrogen environment, followed by steam and air. The effect of modification time on the weight average degree of polymerization was observed. The highest values were obtained for wood modified at 2 hours, then 6 and 10 hours of modification. The native wood showed the highest degree of polymerization. On the basis of the results obtained, it can be concluded that for the material studied the oxidation and degradation reactions occurring depend on the environment and time for a given temperature of wood modification.

Characterization of DNA nanostructure stability by size exclusion chromatography

DNA-based nanostructures (DNs) are advantageous for the design of functional materials for biology and medicine due to the nanoscale control provided by their predictable self-assembly. However, the use of DNs in vivo has been limited due to structural instability in biofluids. As the stability of a particular DN sets the scope of its potential biological applications, efficient methods to characterize stability are required. Here, we apply size exclusion chromatography (SEC) to study the stability of a tetrahedron DNA nanostructure (TDN) and demonstrate the analytical capabilities of our method in characterizing degradation by enzymes and a diluted human serum matrix. We show that SEC analysis can reliably assay TDN degradation by a nuclease through direct injection and peak integration. Furthermore, data analysis using a ratio chromatogram technique enables TDN peak deconvolution from the matrix of serum proteins. Using our method, we found that TDNs exhibit half-lives of 23.9 hours and 10.1 hours in 20% and 50% diluted human serum, respectively, which is consistent with reported stability studies in 10% fetal bovine serum. We anticipate that this method could be broadly applicable to characterize a variety of DNs and serve as an efficient technique toward analysis of the stability of new DN designs in complex biological matrixes.

The Past, the Present, and the Future of the Size Exclusion Chromatography in Extracellular Vesicles Separation

Extracellular vesicles (EVs) are cell-derived membranous particles secreted by all cell types (including virus infected and uninfected cells) into the extracellular milieu. EVs carry, protect, and transport a wide array of bioactive cargoes to recipient/target cells. EVs regulate physiological and pathophysiological processes in recipient cells and are important in therapeutics/drug delivery. Despite these great attributes of EVs, an efficient protocol for EV separation from biofluids is lacking. Numerous techniques have been adapted for the separation of EVs with size exclusion chromatography (SEC)-based methods being the most promising. Here, we review the SEC protocols used for EV separation, and discuss opportunities for significant improvements, such as the development of novel particle purification liquid chromatography (PPLC) system capable of tandem purification and characterization of biological and synthetic particles with near-single vesicle resolution. Finally, we identify future perspectives and current issues to make PPLC a tool capable of providing a unified, automated, adaptable, yet simple and affordable particle separation resource.