Combination of size-exclusion chromatography and ion exchange adsorption for improving the proteomic analysis of plasma-derived extracellular vesicles.

Extracellular vesicles (EVs) are lipid membrane vesicles released by live cells that carry a variety of biomolecules, including nucleic acids, lipids, and proteins. Recently, proteins in plasma-derived EVs have emerged as novel biomarkers with essential functions in the diagnosis and prognosis of human diseases. However, the current methods of isolating EVs from plasma often lead to coisolated impurities in biological fluids. Therefore, before performing any research protocol, the process of extracting EVs from plasma for proteomic analysis must be optimized. In this study, two EV isolation strategies, size exclusion chromatography (SEC) and SEC combined with ion exchange adsorption (SEC + IEA), were compared in terms of the purity and quantity of protein in EVs. Our results demonstrated that, compared to single-step SEC, SEC combined with IEA could produce plasma-derived EVs with a higher purity by decreasing the abundance of lipoprotein. Additionally, with MS analysis, we demonstrated that the combination approach maintained the stability and improved the purity of EVs in many plasma samples. Furthermore, by combining SEC with IEA, more cancer-associated proteins were detected in the plasma of various cancer samples.

Inhibition of human sperm function by an antibody against apolipoprotein A1: A protein located in human spermatozoa.

Apolipoproteins A1 (ApoA1), an important protein in initiating sperm capacitation, has been found in the oviduct liquid, whilst whether it expresses in human spermatozoa has not yet been defined. ApoA1 expression was detected by reverse transcription-polymerase chain reaction, Western blot and immunofluorescence in sperm cells. Sperms were incubated with different concentration of ApoA1 antibody to observe the changes of sperm motility and apoptosis including annexin V binding to the cell surface, mitochondrial membrane potential and ultrastructural changes. In addition, cholesterol levels of human spermatozoa and fertilization in vitro were completed to explore the role of ApoA1 in fertilization process. We identify ApoA1 mRNA is presented in spermatozoa, and the ApoA1 protein, the molecular weights of 31 kD, is located at the postacrosomal region and neck of human spermatozoa. ApoA1 antibody affects cholesterol efflux of human spermatozoa and inhibits fertilization in vitro. Moreover, ApoA1 antibody decreases sperm motility and increases sperm apoptosis. This work shows ApoA1 plays an important role in maintaining cholesterol stability and sperm motility, survival rate and fertilization process. Furthermore, ApoA1 implications in the clinical work and other reproductive areas are needed to be studied.

Increased NGAL level associated with iron store in chronic kidney disease with anemia.

An iron scarcity often occurs in chronic kidney disease (CKD). Neutrophil gelatinase-associated lipocalin (NGAL), a biomarker of acute kidney injury, is associated with iron metabolism. The present study determined the association between serum NGAL and iron status in chronic kidney disease with anemia. A total of 154 adult CKD patients were divided into anemia and without anemia groups. The anemia groups were further subdivided into two groups based on the presence or absence of iron deficiency, defined as a transferrin saturation (TSAT) < 20%. The NGAL was measured for all the 154 patients, and the possible relationships with iron status were analyzed. 27.7% patients with TSAT < 20% presented lower hemoglobin, serum iron, serum ferritin, and higher NGAL values than those without iron deficiency. NGAL was inversely correlated with hemoglobin, hematocrit, MCV, MCH, serum iron, and TSAT. NGAL adequately diagnosed the status of iron deficiency among CKD patients by ROC analysis. The optimal NGAL cutoff value able to identify iron deficiency was found to be > 244.8 ng/mL, with 73.01% sensitivity and 68.29% specificity. CKD patients with anemia presented altered NGAL values as this protein is involved in the maintenance of iron balance. Thus, NGAL might be proposed as a new tool for assessing the iron deficiency and in the management of iron therapy for CKD patients.