Anti-KIT monoclonal antibody, clone 2D6

Green fluorescent protein (GFP) is a useful biomolecule in biotechnology; however, its price makes its widespread application prohibitive. To overcome this issue, recently, the use of aqueous two-phase systems (ATPS) for GFP purification was proposed as an alternative platform to reduce processing costs. Aligned with this goal, this study performed bioprocess modelling coupled with economic analysis using the software Biosolve to evaluate the potential and commercial applicability of ATPS for GFP purification. This work analysed a collection of fourteen ATPS to discriminate through production costs while also incorporating the concept of product purity into the calculations. The two best systems (a PEG-based and an ionic liquid (IL)-based ATPS) were placed in a full bioprocess at different scale models (1 to 100 L) to elucidate the viability of applying ATPS at large scale. Although the results showed that the PEG-based ATPS exhibit the lowest costs (between USD 3.5x10(3).g(-1) at 1 L and USD 0.33x10(3).g(-1) at 100 L), for further developments, the inclusion of an ATPS granting a higher purity is desired for the development of simpler bioprocesses. Therefore, as a third approach in this work, a sensitivity analysis was performed to determine the impact of varying different model parameters (recovery yield, material costs discount and production titre), to elucidate the circumstances under which the IL-based system can overcome the production costs of the traditional PEG-based ATPS. The results indicate that the best cost-effectiveness approach is to improve the production titre (although it can affect all ATPS studied), as an increase from 1.33 to 3.8 g/L is enough for the IL-based ATPS to be less expensive than the traditional system at all analysed scales. This study demonstrates that ATPS can greatly reduce GFP manufacturing costs, which can potentially help to popularize new applications of fluorescent proteins that are currently mostly restricted to research kits due to their high prices.

Purpose MicroRNA (miRNA) is known to be involved in the pathological process of congenital heart disease (CHD), and nodal modulator1 (NOMO1) is a critical determinant of heart formation. The present study aims to discover the effect of miR-33a-5p and NOMO1 on CHD. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect expressions of miR-33a-5p mimic or inhibitor and overexpressed NOMO1 plasmid orNOMO1 knockdown. Human cardiomyocyte progenitor cells (hCMPCs) proliferation was measured by cell counting kit-8 (CCK-8) at 24, 48 and 72 h. Flow cytometry was applied to investigate hCMPCs cell cycle progression and apoptosis. Expressions of cell apoptotic proteins Bax, Cleaved(C) caspase-3 and Bcl-2, and expressions of cardiomyocyte differentiation markers GATA4, troponin T (cTnT) and myocyte enhancer factor2C (MEF2C) in hCMPCs were identified by qRT-PCR and western blot. Target genes and potential binding sites of NOMO1 and miR-33a-5p were predicted with Targetscan 7.2, and was confirmed through dual-luciferase reporter assay. Results Up-regulation of miR-33a-5p inhibited hCMPCs proliferation, cell cycle G0/S transition but promoted hCMPCs apoptosis, which was partially mitigated by overexpressed NOMO1. NOMO1 was the target gene of miR-33a-5p. Expressions of Bax and C caspase-3 were enhanced but expressions of Bcl-2, GATA4, cTnT and MEF2C were reduced by up-regulation of miR-33a-5p, which was partially mitigated by overexpressed NOMO1. Conclusion Up-regulation of miR-33a-5p inhibited hCMPCs proliferation, cell cycle G0/S transition and differentiation into cardiomyocytes but promoted apoptosis via targeting NOMO1.