Endotoxins are common exogenous pyrogens. Excessive endotoxins in medical devices and injections can lead to serious consequences such as sepsis, septic shock, and even death. Therefore, endotoxin detection plays a crucial role in medical, pharmaceutical, and food sectors. The wide application of Limulus amebocyte lysate (LAL) has led to a sharp decline in the number of horseshoe crabs. Moreover, the LAL assay has limitations such as interbatch variations and difficulty in quantification. The recombinant factor C (rFC) assay is stable between batches, highly sensitive, and capable of quantitation, and thus it can be used as an alternative for the LAL assay. However, the high cost and complex procedures involved in producing recombinant factor C have limited the widespread application of this method. In order to simplify the preparation and reduce the production cost of recombinant factor C, this study focuses on the production of recombinant factor C based on the baculovirus expression system. Multiple measures such as a high-yield and anti-apoptotic vector qBac-IIIG, the optimal signal peptide, and the optimized codon were used to reach the goal of endotoxin detection with cell supernatant. This method simplifies the steps of protein purification. The sensitivity of the supernatant reached 0.05 EU/mL in a 1-L fermentation system, and 500 000 detecting reactions can be supported per liter of fermentation broth. This study increases the yield and activity of recombinant factor C, simplifies the procedures of protein purification, and reduces the cost, laying a foundation for the promotion and application of recombinant factor C in endotoxin detection.
Animals ; Recombinant Proteins/genetics* ; Horseshoe Crabs/chemistry* ; Baculoviridae/metabolism* ; Endotoxins/analysis* ; Protein C/biosynthesis* ; Genetic Vectors/genetics* ; Arthropod Proteins/genetics* ; Enzyme Precursors ; Serine Endopeptidases

Objective:To evaluate the diagnostic performance of the prostate imaging reporting and data system version 2.1 (PI-RADS v2.1) based on multiparametric MRI (mpMRI) in the detection of clinically significant prostate cancer (csPCa).Methods:A total of 561 patients who underwent prostate mpMRI in the First Affiliated Hospital of Guangxi Medical University from June 2015 to December 2020 due to elevated prostate specific antigen were collected ambispectively. The patients were divided into csPCa group (276 cases) and non-csPCa group (285 cases) according to pathological findings. Prostate were scored according to the PI-RADS v2.1 scoring standard by a junior and a senior radiologist. The prostate volume was measured and the prostate specific antigen density (PSAD) was calculated. The diffusion-weighted imaging and dynamic contrast-enhanced MRI images were processed to measure the quantitative parameters of the index lesion, including apparent diffusion coefficient (ADC), volume transfer constant (K trans) and rate constant (K ep) values. The Mann-Whitney U test was used to compare the difference in parameters between the two groups. The predictors of csPCa were screened by logistic regression analysis. Predictive model of multi-parameter was established. The receiver operator characteristic curves were used to evaluate the efficacy of PI-RADS v2.1 and the model in diagnosing csPCa, and the comparisons of area under the curve (AUC) were conducted by DeLong test. Results:Compared with non-csPCa group, the patients in csPCa group had higher PI-RADS score of senior physician, PSAD, K trans and K ep value, lower ADC value ( Z=-16.69, -12.49, -3.43, -4.67, 13.91, all P<0.001). The PI-RADS scores of senior physician (OR=3.064, 95%CI 2.428-3.866, P<0.001), PSAD (OR=1.554, 95%CI 1.170-2.064, P=0.002) and ADC value (OR=0.095, 95%CI 0.032-0.288, P<0.001) were the predictors of csPCa. The AUC of junior, senior physician PI-RADS and combined prediction model were 0.861 (95%CI 0.830-0.892), 0.895 (95%CI 0.868-0.922) and 0.923 (95%CI 0.898-0.944). The pairwise difference was statistically significant (the PI-RADS score between the junior and senior physicians Z=3.24, P=0.001, the difference between the PI-RADS score of junior physician and prediction model Z=5.54, P<0.001, the difference between the PI-RADS score of senior physician and prediction model Z=4.20, P<0.001). Conclusion:Based on mpMRI, the application of PI-RADS v2.1 by junior and senior radiologists has the high diagnostic efficacy for csPCa, and the multi-parameter model has the best diagnostic efficacy for csPCa.

To investigate the effects of gossypol acetic acid (GA) on proliferation and apoptosis of the macrophage cell line RAW264.7 and further understand the possible underlying mechanism responsible for GA-induced cell apoptosis, RAW264.7 cells were treated with GA (25~35 micromol/L) for 24 h and the cytotoxicity was determined by MTT assay, while apoptotic cells were identified by TUNEL assay, acridine orange/ethidium bromide staining and flow cytometry. Moreover, mitochondrial membrane potential (DeltaPsi(m)) with Rhodamine 123 and reactive oxygen species (ROS) with DCFH-DA were analyzed by fluorescence spectrofluorometry. In addition, the expression of caspase-3 and caspase-9 was assessed by Western Blot assay. Finally, the GA-induced cell apoptosis was evaluated by flow cytometry in the present of caspase inhibitors Z-VAD-FMK and Ac-LEHD-FMK, respectively. GA significantly inhibited the proliferation of RAW264.7 cells in a dose-dependent manner, and caused obvious cell apoptosis and a loss of DeltaPsi(m) in RAW264.7 cells. Moreover, the ROS production in cells was elevated, and the levels of activated caspase-3 and caspase-9 were up-regulated in a dose-dependent manner. Notably, GA-induced cell apoptosis was markedly inhibited by caspase inhibitors. These results suggest that GA-induced RAW264.7 cell apoptosis may be mediated via a caspase-dependent mitochondrial signaling pathway.
Animals ; Antineoplastic Agents, Phytogenic/*pharmacology ; Apoptosis/*drug effects ; Cell Line ; Cell Proliferation/*drug effects ; Dose-Response Relationship, Drug ; Gossypol/*analogs & derivatives/pharmacology ; Membrane Potential, Mitochondrial/*drug effects ; Mice ; Mice, Inbred BALB C ; Reactive Oxygen Species/*metabolism ; Signal Transduction/*drug effects

Background Hypoxia can increase the secretion of vascular endothelial growth factor-A (VEGF A) by retinal pigment epithelium (RPE) cells and initiate choroidal neovascularization (CNV) consequently.Two VEGF-A isoforms,the angiogenic VEGF-A family (VEGFxxx) and the anti-angiogenic VEGF-A family (VEGFxxxb),are found and formed by alternative splicing.However,the expressing changes and its effect in human RPE cells under the normoxia and hypoxia are unclear.Objective This study was to investigate the expression of VEGFxxx b in human RPE cells under hypoxia.Methods ARPE-19,a human RPE cell line,were cultured.CoCl2 150 μmol/L was added into the medium to mimic the hypoxia environment for 0 hour (normoxia group),3,12 and 24 hours to induce the hypoxia cells.The cells or suspension was harvested at various time points.The expressions of VEGFxxx mRNA and VEGFxxxb mRNA in the cells were detected,and the expressions of the total VEGF-A protein and VEGFxxxb protein in the cells were assayed by Western blot.ELISA was used to determine the contents of total VEGF-A and VEGFxxxb proteins in suspension.Results VEGFxxx mRNA and VEGFxxxb mRNA were expressed in the ARPE-19 cells in both normoxia and hypoxia,and multiple VEGFxxx mRNA isoforms appeared,including VEGF121 mRNA,VEGF165 mRNA and extremely faint VEGF189 mRNA,but only VEGF165b band was seen in VEGFxxxb mRNA.With the prolong of hypoxia culture,the expression of VEGFxxx mRNA showed gradual increase,while VEGFxxx b mRNA appeared gradual decrease.Western blot exhibited that VEGFxxxb was expressed in the cells cultured by normoxia and hypoxia with the strongest expression in VEGF165b.The VEGFxxxb content in suspension of medium was (166.82± 2.55) pg/ml under the normoxia environment and (125.35 ±2.10) pg/ml in 24 hours under the hypoxia,and the total VEGF-A protein elevated from (294.27 ± 11.97) pg/ml under the normoxia environment to (582.26 ± 12.98) pg/ml in 24 hours under the hypoxia.In addition,the proportion of VEGFxxxb to total VEGF-A in the cells lowed from (56.71 ± 1.02) ％ under the normoxia environment to (21.53 ±0.08) ％ in 24 hours under the hypoxia.Conclusions VEGFxxxb isoforms are expressed in both normoxia and hypoxia ARPE-19 cells.VEGFxxxb isoform is a predominant isoform in normoxia ARPE-19 cells.In hypoxia ARPE-19 cells,however,the expression of VEGF A is significantly stronger than that of VEGFxxxb.