Affinity purification of recombinant human plasminogen activator from transgenic rabbit milk using a novel polyolresponsive monoclonal antibody

Purpose: To develop processes for effective isolation and purification of recombinant human plasminogen activator (rhPA) from transgenic rabbit milk.Methods: Immunoaffinity chromatography was selected and improved by a special polyol-responsive monoclonal antibody (PR-mAb). Alteplase was used as immunogen because of its similarity to rhPA in terms of structure. The PR-mAb was prepared by hybridoma technology and screened by ELISA-elution assay. Screening antibody was performed using rhPA milk in an ELISA-elution assay. The antibody clone C4-PR-mAb was selected for immunoaffinity chromatography. The rhPA was effectively bound to immobilized C4-PR-mAb on the column and was eluted with Tris buffer comprising 0.75 mol/L ammonium sulfate and 40n% propanediol (pH7.9). The rhPA was further purified by passing through Chromdex75 gel filtration column.Results: There were 12 hybridoma strains selected into the polyol responsive mAbs screen step and three hybridoma strains were superior for producing PR-mAbs (C1, C4, C8). The rhPA can be purified from transgenic rabbit milk and maintained a higher thrombolytic activity in vitro by FAPA.Conclusion: The results demonstrate the suitability of the alternative approach used in this study. Using immunoaffinity chromatography and  gel filtration column is feasible and convenient for extracting rhPA from milk, and should be useful for purifying other tPA mutants or other novel recombinant milkderived proteins.Keywords: tPA, Immunoaffinity chromatography, PR-mAb, ELISA-elution, Antibody, Thrombolytic activit

BZML, a novel colchicine binding site inhibitor, overcomes multidrug resistance in A549/Taxol cells by inhibiting P-gp function and inducing mitotic catastrophe

Multidrug resistance (MDR) interferes with the efficiency of chemotherapy. Therefore, developing novel anti-cancer agents that can overcome MDR is necessary. Here, we screened a series of colchicine binding site inhibitors (CBSIs) and found that 5-(3, 4, 5-trimethoxybenzoyl)-4-methyl-2-(p-tolyl) imidazol (BZML) displayed potent cytotoxic activity against both A549 and A549/Taxol cells. We further explored the underlying mechanisms and found that BZML caused mitosis phase arrest by inhibiting tubulin polymerization in A549 and A549/Taxol cells. Importantly, BZML was a poor substrate for P-glycoprotein (P-gp) and inhibited P-gp function by decreasing P-gp expression at the protein and mRNA levels. Cell morphology changes and the expression of cycle- or apoptosis-related proteins indicated that BZML mainly drove A549/Taxol cells to die by mitotic catastrophe (MC), a p53-independent apoptotic-like cell death, whereas induced A549 cells to die by apoptosis. Taken together, our data suggest that BZML is a novel colchicine binding site inhibitor and overcomes MDR in A549/Taxol cells by inhibiting P-gp function and inducing MC. Our study also offers a new strategy to solve the problem of apoptosis-resistance. (C) 2017 Elsevier B.V. All rights reserved

Additional file 1: Table S1. of TGTT and AACA: two transcriptionally active LTR retrotransposon subfamilies with a specific LTR structure and horizontal transfer in four Rosaceae species

List of four species and their genomic sequence and DNA material source information used in this study. Table S2. Sequence identity between all TGTT and AACA consensus sequences in the four Rosaceae genomes. Table S3. Summary of TGTT and AACA elements identified in P. bretschneideri, M. domestcia, P. persica and P. mume. Table S4. Primers used for wet lab validations. Table S5. Sequence identity between all TGTT1 elements identified in the four Rosaceae genomes. Table S6. Sequence divergence of orthologous singletons between apple and peach. Table S7. Sequence identity of the orthologous singletons between apple and peach. Table S8. Primers used for Real time Quantitative PCR. (XLSX 142 kb

Assessment of three risk evaluation systems for patients aged ≥70 in East China: performance of SinoSCORE, EuroSCORE II and the STS risk evaluation system

Objectives To assess and compare the predictive ability of three risk evaluation systems (SinoSCORE, EuroSCORE II and the STS risk evaluation system) in patients aged ≥70, and who underwent coronary artery bypass grafting (CABG) in East China. Methods Three risk evaluation systems were applied to 1,946 consecutive patients who underwent isolated CABG from January 2004 to September 2016 in two hospitals. Patients were divided into two subsets according to their age: elderly group (age ≥70) with a younger group (age <70) used for comparison. The outcome of interest in this study was in-hospital mortality. The entire cohort and subsets of patients were analyzed. The calibration and discrimination in total and in subsets were assessed by the Hosmer–Lemeshow and the C statistics respectively. Results Institutional overall mortality was 2.52%. The expected mortality rates of SinoSCORE, EuroSCORE II and the STS risk evaluation system were 0.78(0.64)%, 1.43(1.14)% and 0.78(0.77)%, respectively. SinoSCORE achieved the best discrimination (the area under the receiver operating characteristic curve (AUC) = 0.829), followed by the STS risk evaluation system (AUC = 0.790) and EuroSCORE II (AUC = 0.769) in the entire cohort. In the elderly group, the observed mortality rate was 4.82% while it was 1.38% in the younger group. SinoSCORE (AUC = .829) also achieved the best discrimination in the elderly group, followed by the STS risk evaluation system (AUC = .730) and EuroSCORE II (AUC = 0.640) while all three risk evaluation systems all had good performances in the younger group. SinoSCORE, EuroSCORE II and the STS risk evaluation system all achieved positive calibrations in the entire cohort and subsets. Conclusion The performance of the three risk evaluation systems was not ideal in the entire cohort. In the elderly group, SinoSCORE appeared to achieve better predictive efficiency than EuroSCORE II and the STS risk evaluation system

Additional file 2: Figure S1. of TGTT and AACA: two transcriptionally active LTR retrotransposon subfamilies with a specific LTR structure and horizontal transfer in four Rosaceae species

Sequence alignment of the TGTT1 elements. Figure S2. Wet laboratory validation of the TGTT and AACA elements. Figure S3. Multiple alignments of PBS sites from TGTT, AACA and normal TGCA elements of the Ale lineage. Figure S4. INT phylogenetic relationships among TGTT, AACA and normal TGCA elements. Figure S5. Evidence of transcriptional activity in five TGTT and AACA elements. (PDF 2824 kb

Charge redistribution enhanced oxygen reduction of carbon-based electrocatalyst

The surface charge distribution is regarded as a key factor affecting the electrocatalytic performance. Herein, we report a strategy to regulate the surface charge distribution in carbon-based electrocatalyst to enhance its activity and stability in oxygen reduction reaction (ORR). The sample obtained in a sealed reactor N,S co-doped carbon encapsulate Co9S8 nanoparticles (Co9S8@CNS-S) exhibits excellent ORR performance. X-ray photoelectron spectroscopy, kelvin probe force microscopy and density functional theory simulation were used to investigate the mechanism of performance improvement. The enhanced ORR activity was due to the exposed more positive charge of surface carbon atoms by N, S elements doping synergistic with Co9S8 nanoparticles (NPs). The improved ORR stability was attributed to the carbon layer combination with Co9S8 NPs which greatly suppresses the generation of H2O2, thus avoiding the erosion of H2O2 on carbon layer during electrocatalysis. This work provides a strategy to regulate electrocatalyst surface charge distribution to achieve active and stable ORR electrocatalyst.The financial support from the National Natural Science Foundation of China (No. 22173072, 21676216, 21973074, 22273073), Innovation Capability Support Program of Shaanxi Province (No. 2022TD-32) and Preferential Funding Project for Scientific and Technological Activities of Overseas Chinese in Shaanxi Province (No. 2021008) are gratefully acknowledged

Additional file 4: Table S2. of Efficient TALEN-mediated myostatin gene editing in goats

Primers for PCR amplification of TALENs target regions. (DOC 29 kb

Additional file 3: Table S4. of Efficient TALEN-mediated myostatin gene editing in goats

Primer pairs for PCR amplification of off-target regions for MTAL-2. (DOC 32 kb