Targeted mass spectrometric approach for biomarker discovery and validation with nonglycosylated tryptic peptides from N-linked glycoproteins in human plasma

A simple mass spectrometric approach for the discovery and validation of biomarkers in human plasma was developed by targeting nonglycosylated tryptic peptides adjacent to glycosylation sites in an N-linked glycoprotein, one of the most important biomarkers for early detection, prognoses, and disease therapies. The discovery and validation of novel biomarkers requires complex sample pretreatment steps, such as depletion of highly abundant proteins, enrichment of desired proteins, or the development of new antibodies. The current study exploited the steric hindrance of glycan units in N-linked glycoproteins, which significantly affects the efficiency of proteolytic digestion if an enzymatically active amino acid is adjacent to the N-linked glycosylation site. Proteolytic digestion then results in quantitatively different peptide products in accordance with the degree of glycosylation. The effect of glycan steric hindrance on tryptic digestion was first demonstrated using alpha-1-acid glycoprotein (AGP) as a model compound versus deglycosylated alpha-1-acid glycoprotein. Second, nonglycosylated tryptic peptide biomarkers, which generally show much higher sensitivity in mass spectrometric analyses than their glycosylated counterparts, were quantified in human hepatocellular carcinoma plasma using a label-free method with no need for N-linked glycoprotein enrichment. Finally, the method was validated using a multiple reaction monitoring analysis, demonstrating that the newly discovered nonglycosylated tryptic peptide targets were present at different levels in normal and hepatocellular carcinoma plasmas. The area under the receiver operating characteristic curve generated through analyses of nonglycosylated tryptic peptide from vitronectin precursor protein was 0.978, the highest observed in a group of patients with hepatocellular carcinoma. This work provides a targeted means of discovering and validating nonglycosylated tryptic peptides as biomarkers in human plasma, without the need for complex enrichment processes or expensive antibody preparations.     

Regulatory role of osteopontin in malignant transformation of endometrial cancer

Osteopontin (OPN) involves in the tumor-promoting or metastasis in human endometrial cancer. Depletion of OPN gene expression in endometrial cancer cells was significantly decreased in cell viability and the cells undergo apoptotic cell death. The status of OPN in THESC, RL95, Hec1A and Ishikawa cell lines were analyzed by RT-PCR and western blot. After OPN-siRNA transfection, mRNA and protein expression levels of OPN were determined in Hec1A and Ishikawa cells. Cell proliferation and cell cycle distribution were observed by MTT and flow cytometry analysis. DNA fragmentation assay was used to measure cell apoptosis. Cell migration was assessed by wound healing assay. Depletion of OPN gene expression in endometrial cancer cell lines (Hec1A and Ishikawa cells) reproducibly changed their ability of proliferation. Concomitant changes were seen in the expression of OPN binding cell surface receptors, cell cycle-regulatory genes, cell invasion and colony formation nature of the tumor cells. Decreased colonizing potential in the absence of OPN was reversed in the presence of recombinant OPN. Inhibition of anchorage-independent growth was observed in the presence of metabolic inhibitors of the PI3K, Src and integrin signaling cascades, which was ameliorated in the presence of exogenously added OPN. Our result showed the role of OPN in endometrial cancer, in particular on the malignancy-promoting aspects of OPN that may pave way for new approaches to the clinical management of endometrial cancer.     

A Simple Clearing Method for Improved Visualization of lacZ Expression in Fish Larvae

The protocol described in this report provides a simple and reliable method for improved visualization of lacZ expression upon X-gal staining. The simple treatments of KOH-H2O2 and glycerol removed all pigment from the whole body and made the demelanized fry transparent without any structural damage. Based on this method, the exogenous expression of lacZ could be easily identified and distinguished from endogenous background activity in pigmented transgenic fry.     

Inactivation of catalase and superoxide dismutase by singlet oxygen derived from photoactivated dye

Both superoxide dismutase (SOD) and catalase are key enzymes in the antioxidant system of the cells that work to maintain low steady-state concentrations of the reactive oxygen species. When exposed to a singlet oxygen-producing system composed of dye, such as methylene blue or rose bengal, and visible light both SOD and catalase were susceptible to oxidative modification and damage as indicated by the loss of activity, fragmentation and aggregation of peptide as well as by the formation of carbonyl groups. Histidine, a powerful quenching agent for singlet oxygen, and the polyamines, such as spermine and spermidine, were effective at protecting the activity loss mediated by illuminated dye, whereas spin traps were only mildly effective. The structural alterations of modified enzymes were indicated by the increase in susceptibility to proteases, the change in absorption spectra and in fluorescence spectra. The singlet oxygen-mediated damage to SOD and catalase may result in the perturbation of cellular antioxidant defense mechanisms and subsequently lead to a pro-oxidant condition.     

Harvesting of microalgae using flocculation combined with dissolved air flotation

This study explored efficient methods of harvesting the Tetraselmis sp. KCTC12236BP using flocculation and dissolved air flotation. Concentration ranges of flocculation agents were optimized using jar tests (batch flocculation experiments) using inorganic (aluminum sulfate, ferric sulfate) and organic (chitosan) flocculants in a pH range of 4 ～ 10. The optimal dosage and pH level were 1.2 g/L and pH 5 ～ 6 for aluminum sulfate, 0.7 g/L and pH 4 ～ 8 for ferric sulfate, and 5.0 mg/mL and pH 7 ～ 8 for chitosan. The highest harvesting efficiency achieved with each of the four compounds was 85.6, 92.6, 93, and 91.3%, respectively.     

Synergistic cytokine-induced nitric oxide production in human alveolar epithelial cells.

Nitric oxide (NO) is an important mediator molecule in regulating normal airway function, as well as in the pathophysiology of inflammatory airway diseases. In addition, cytokines are potent messenger molecules at sites of inflammation. The specific relationship among IL-1beta, TNF-alpha, and IFN-gamma on iNOS induction and NO synthesis in human alveolar epithelial cells has not been determined. In addition, rigorous methods to determine potential synergistic action between the cytokines have not been employed. We exposed monolayer cultures of A549 cells to a factorial combination of three cytokines (IL-1beta, TNF-alpha, and IFN-gamma) and three concentrations (0, 5, and 100 ng/mL). TNF-alpha alone does not induce NO production directly; however, it does have a stimulatory effect on IL-1beta-induced NO production. IL-1beta and INF-gamma both induce NO production alone, yet at different concentration thresholds, and act synergistically when present together. In the presence of all three cytokines, the net effect of NO production exceeds the predicted additive effect of each individual cytokine and the two-way interactions. Several plausible mechanisms of synergy among IL-1beta, TNF-alpha, and IFN-gamma in NO production from human alveolar epithelial cells (A549) are proposed. In order to verify the proposed mechanisms of synergy, future experimental and theoretical studies must address several molecular steps through which the iNOS gene is expressed and regulated, as well as the expression and regulation of enzyme cofactors and substrates.     

Development of an Accurate and Sensitive Diagnostic System Based on Conventional PCR for Detection of African Swine Fever Virus in Food Waste

African swine fever virus (ASFV), a highly contagious virus, can cause diseases with high mortality rates in pigs, making it a pathogen of social and economic significance. ASFV has been reported to show potential long-term survival in living livestock, such as pigs, but also in leftover cooking meat and undercooked pork meat. Hence, it is possible that there could be direct reinfection or secondary infection through feed produced from household food waste and treatment facilities. Many polymerase chain reaction (PCR)-based molecular diagnostic techniques to detect ASFV in clinical swine samples have been reported. However, those with applicability for food waste samples, which contain relatively low viral copy numbers and may contain various unknown inhibitors of PCR, are still lacking. In this study, we developed a conventional PCR-based diagnostic system that can detect ASFV with high sensitivity from food waste sample types. The technique shows a 10–100 times higher limit of detection compared to that of previously reported methods based on conventional PCR and quantitative real-time PCR. It is also capable of amplifying a sequence that is approximately 751 nucleotides, which is advantageous for similarity analysis and genotyping. Moreover, a ASFV-modified positive material different from ASFV that could synthesize 1400 nucleotide amplicons was developed to identify false-positive cases and thus enhance diagnostic accuracy. The method developed herein may be applicable for future ASFV monitoring, identification, and genotyping in food waste samples.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-022-01007-y.