TRIP13 Plays an Important Role in the Sensitivity of Leukemia Cell Response to Sulforaphane Therapy.

Sulforaphane is one of the most characterized isothiocyanate compounds in cruciferous vegetables and shows anticancer effects, especially antileukemia properties. However, the molecular mechanism of the growth inhibition effect of sulforaphane in acute myeloid leukemia (AML) has not been fully explored. In the present study, a proteomic analysis was performed on the AML cell line U937 responding to sulforaphane treatment to identify novel and efficient therapeutic targets of sulforaphane on AML cells. Key driver analysis was run on the leukemia network, and TRIP13 was identified as a key regulatory factor in sulforaphane-induced growth inhibition in U937 cells. Pretreatment with DCZ0415, an inhibitor of TRIP13, could significantly attenuate sulforaphane-induced cell apoptosis and cell cycle arrest in vitro through the PI3K/Akt/mTOR signaling pathway. In addition, the inhibitory effect of sulforaphane on the tumor volume could also be obviously attenuated by the pretreatment of DCZ0415 in vivo. These results indicate that TRIP13 plays an important role in the sensitivity of leukemia cell response to sulforaphane treatment, and these findings expand the understanding of the mechanism of the antileukemic effect of sulforaphane and provide a new target for the treatment of AML.

Comparison of proteomic landscape of extracellular vesicles in pleural effusions isolated by three strategies.

Extracellular vesicles (EVs) derived from pleural effusion (PE) is emerging as disease biomarkers. However, the methods for isolation of EVs from PE (pEVs) were rarely studied. In our study, three methods for isolating pEVs of lung cancer patients were compared, including ultracentrifugation (UC), a combination of UC and size exclusion chromatography (UC-SEC) and a combination of UC and density gradient ultracentrifugation (UC-DGU). The subpopulation of pEVs was identified by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), Western blotting (WB) and nano-flow cytometry (nFCM). Additionally, the proteomic landscape of pEVs was analyzed by Label-free proteomics. The results showed that, compared with UC and UC-DGU, the UC-SEC method separated pEVs with the highest purity. In the proteomic analysis, on average, 1595 proteins were identified in the pEVs isolated by UC-SEC, much more than pEVs isolated by UC (1222) or UC-DGU (807). Furthermore, approximately 90% of identified proteins in each method were found in the EVs public database ExoCarta. Consistent with this, GO annotation indicated that the core proteins identified in each method were mainly enriched in "extracellular exosome." Many of the top 100 proteins with high expression in each method were suggested as protein markers to validate the presence of EVs in the MISEV2018 guidelines. In addition, combined with lung tissue-specific proteins and vesicular membrane proteins, we screened out and validated several novel protein markers (CD11C, HLA DPA1 and HLA DRB1), which were enriched in pEVs rather than in plasma EVs. In conclusion, our study shows that the method of UC-SEC could significantly improve the purity of EVs and the performance of mass spectrometry-based proteomic profiling in analyzing pEVs. The exosomal proteins CD11C, HLA DPA1 and HLA DRB1 may act as potential markers of pEVs. The proteomic analysis of pEVs provides important information and new ideas for studying diseases complicated with PE.

FJX1 as a candidate diagnostic and prognostic serum biomarker for colorectal cancer

Purpose: Colorectal cancer (CRC) is one of the most common cancer worldwide. It is essential to identify non-invasive diagnostic and prognostic biomarkers of CRC. The aim of the present study was to screen candidate biomarkers in diagnosis and prognosis of CRC based on a novel strategy.Materials and methodsThe expression level of gene higher in cancer than in adjacent non-cancer tissue was defined as “positive”, and the top 10% genes with “positive rate” were filtered out as candidate diagnostic biomarkers in four Gene Expression Omnibus (GEO) datasets. Then, the prognostic value of candidate biomarkers was estimated Cox regression analysis. Moreover, the concentration of biomarker in serum was detected in CRC patients.ResultsEighteen candidate biomarkers were identified with efficient diagnostic value in CRC. As a prognostic biomarker, FJX1 (four-jointed box kinase 1) showed a good performance in predicting overall survivals in CRC patients. In serum levels, FJX1 showed high sensitivity and specificity in distinguishing CRC patients from controls, and the concentration of serum FJX1 was associated with distant metastasis in CRC. In addition, serum FJX1 was significantly decreased after surgery in CRC patients. Compared with traditional CRC biomarkers CEA and CA 19-9, FJX1 still showed good efficiency in diagnosis and prognosis. Moreover, inhibition of FJX1 expression by siRNA or neutralization of secreted FJX1 by antibody could suppress cell proliferation and migration in vitro.ConclusionOur findings provided a novel strategy to identify diagnostic biomarkers based on public datasets, and suggested that FJX1 was a candidate diagnostic and prognostic biomarker in CRC patients. (AU)

FJX1 as a candidate diagnostic and prognostic serum biomarker for colorectal cancer.

PURPOSE: Colorectal cancer (CRC) is one of the most common cancer worldwide. It is essential to identify non-invasive diagnostic and prognostic biomarkers of CRC. The aim of the present study was to screen candidate biomarkers in diagnosis and prognosis of CRC based on a novel strategy. MATERIALS AND METHODS: The expression level of gene higher in cancer than in adjacent non-cancer tissue was defined as "positive", and the top 10% genes with "positive rate" were filtered out as candidate diagnostic biomarkers in four Gene Expression Omnibus (GEO) datasets. Then, the prognostic value of candidate biomarkers was estimated Cox regression analysis. Moreover, the concentration of biomarker in serum was detected in CRC patients. RESULTS: Eighteen candidate biomarkers were identified with efficient diagnostic value in CRC. As a prognostic biomarker, FJX1 (four-jointed box kinase 1) showed a good performance in predicting overall survivals in CRC patients. In serum levels, FJX1 showed high sensitivity and specificity in distinguishing CRC patients from controls, and the concentration of serum FJX1 was associated with distant metastasis in CRC. In addition, serum FJX1 was significantly decreased after surgery in CRC patients. Compared with traditional CRC biomarkers CEA and CA 19-9, FJX1 still showed good efficiency in diagnosis and prognosis. Moreover, inhibition of FJX1 expression by siRNA or neutralization of secreted FJX1 by antibody could suppress cell proliferation and migration in vitro. CONCLUSION: Our findings provided a novel strategy to identify diagnostic biomarkers based on public datasets, and suggested that FJX1 was a candidate diagnostic and prognostic biomarker in CRC patients.

[A highly sensitive high performance liquid chromatography-electrochemical detection method for the determination of five phenolic compounds from Salvia miltiorrhiza].

The determination of antioxidants continues to be interested, since the oxidative damage is thought to be one of the main mechanisms involved in nearly all chronic renal pathologies. A highly sensitive high performance liquid chromatography-electrochemical detection (HPLC-ECD) method was developed for evaluating the antioxidant properties of Salvia miltiorrhiza (Dan Shen). The method was optimized with respect to selectivity and sensitivity. Chromatographic conditions, including mobile phase pH value, buffer concentration, buffer type, organic solvent type, gradient profile and flow rate, were systematically investigated. Low pH value (2.8), low buffer concentration (20 mmol/L NaH2PO4), a shallow water-acetonitrile gradient, and a flow rate of 0.2 mL/min were the determined optimal conditions for the quantitative analysis of aimed five antioxidants from 14 batches of Dan Shen samples. The described method provided a good recovery (>95%), a very wide linear range (up to 104 for all analytes), a good precision (RSDs<4.01%), and a high sensitivity (LOQ of caffeic acid, 1.5 µ g/L). Compared with UV detection, the described ECD method was also more effective for evaluating the antioxidant properties of Dan Shen, as it provided highly selective detection of electro-active antioxidants.

[High sensitive non-derivative determination of cyclovirobuxin D by high-performance liquid chromatography-electrochemical determination].

A high-performance liquid chromatography-electrochemical detection (HPLC-ECD) method was developed to determine cyclovirobuxin D (CVB-D) levels in tablets and human blood samples. A column with a positive charge-modified C18 stationary phase, C18HCE, was selected to analyze CVB-D, because it provided a sharper and more symmetric peak for CVB-D than conventional C18 stationary phase. Two types of working electrode materials, glassy carbon (GC) and boron-doped diamond (BDD), were evaluated. BDD was found to provide better sensitivity than GC owing to its lower background current and baseline noise. Utilizing the BDD electrode, C18HCE column, and optimized mobile phase composition, the developed HPLC-ECD method showed a much better sensitivity. The limit of detection and limit of quantification of the HPLC-ECD method for CVB-D were 0.198 and 0.297 µg/L, respectively. It was approximately 12727, 11481, and 2630 times more sensitive than ultraviolet (UV), evaporative light scattering detection, and charged aerosol detection, respectively. The sensitivity of the developed HPLC-ECD method was comparable or even better (16.8 times) than reported mass spectrometry (MS) methods for the determination of CVB-D. Additionally, it offered a much wider linear dynamic range (up to 4 orders of magnitude, 0.297-1891 µg/L) and was much less complicated than MS methods for determination of CVB-D. The developed HPLC-ECD method can be used for determination of CVB-D at both high and low concentrations. Good intra-day (relative standard deviation (RSD) of peak area<5.08%) and inter-day (RSD of peak area<5.57%) reproducibilities of the developed HPLC-ECD method were obtained even for a low mass concentration (59.1 µg/L) sample. After the optimized parameters were acquired, this method was applied to the quantitative analysis of CVB-D in CVB-D tablets and human blood samples. With a slight modification, the current HPLC-ECD method can also be applied to analyze many other basic compounds including basic drugs and environmental pollutants.

[Determination of mannose-6-phosphate in pharmaceutical intermediates by high performance ion-exchange chromatography-electrochemical detection].

A novel method was developed for the determination of mannose-6-phosphate and the phosphate in pharmaceutical intermediates by high performance ion-exchange chromatography (IC) and electrochemical detection. The sample was dissolved with purified water, filtrated by a 0.22 microm nylon filter membrane, and then separated by ion-exchange chromatography. The separation was performed on an IonPac AS18 column (250 mm x4 mm) with an IonPac AG18 (50 mm x4 mm) as guard column, and 25 mmol/L KOH solution as the eluent at a flow rate of 1.0 mL/min. The detection was performed with electrochemical detectors (an integrated pulsed amperometric detector and a suppressed conductivity detector in line). Mannose-6-phosphate was selectively determined with an integrated pulsed amperometric detector first. Then, both of mannose-6-phosphate and phosphate were determined with a suppressed conductivity detector after the background conductance of KOH was suppressed with the ASRS suppressor. The injection volume was 25 microL. The external standard calibration curve was used for quantitative analysis. With an amperometric detector, the linear range of the method for mannose-6-phosphate were 0.06 - 10.0 mg/L (r = 0.9998). The recoveries were 92.1% - 103.1% with the relative standard deviations (RSDs) less than 3%. The detection limit was 0.02 mg/L for mannose-6-phosphate. The method is simple, effective, sensitive and selective. It can be used to determine the quality of pharmaceutical intermediates.