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With the constant accumulation of electronic waste, extracting precious metals contained therein is becoming a major challenge for sustainable development. Bacillus megaterium is currently one of the microbes used for the production of cyanide, which is the main leaching agent for gold recovery. The present study aimed to propose a strategy for metabolic engineering of B. megaterium to overproduce cyanide, and thus ameliorate the bioleaching process. For this, we employed constraint-based modeling, running in silico simulations on i JA1121, the genome-scale metabolic model of B. megaterium DSM319. Flux balance analysis (FBA) was initially used to identify amino acids to be added to the culture medium. Considering cyanide as the desired product, we used growth-coupled methods, constrained minimal cut sets (cMCSs) and OptKnock to identify gene inactivation targets. To identify gene overexpression targets, flux scanning based on enforced objective flux (FSEOF) was performed. Further analysis was carried out on the identified targets to determine compounds with beneficial regulatory effects. We have proposed a chemical-defined medium for accelerating cyanide production on the basis of microplate assays to evaluate the components with the greatest improving effects. Accordingly, the cultivation of B. megaterium DSM319 in a chemically-defined medium with 5.56 mM glucose as the carbon source, and supplemented with 413 μM cysteine, led to the production of considerably increased amounts of cyanide. Bioleaching experiments were successfully performed in this medium to recover gold and copper from telecommunication printed circuit boards. The results of inductively coupled plasma (ICP) analysis confirmed that gold recovery peaked out at around 55% after 4 days, whereas copper recovery continued to increase for several more days, peaking out at around 85%. To further validate the bioleaching results, FESEM, XRD, FTIR, and EDAX mapping analyses were performed. We concluded that the proposed strategy represents a viable route for improving the performance of the bioleaching processes., (Copyright © 2020 Aminian-Dehkordi, Mousavi, Marashi, Jafari and Mijakovic.)

Embryonic development is controlled by retinoids, and one approach that has been used to investigate the mechanisms for retinoid actions in developmental processes has been to study the effects of adding retinoids to cultures of pluripotent embryonic stem cells (ESC). To date, most in vitro retinoid research has been directed at deciphering the actions of all-trans retinoic acid (atRA). atRA is a derivative of all-trans retinol (a.k.a. vitamin A, VA), which mammals can generate via an enzyme-catalyzed pathway. atRA's effects on development result from its (1) activation of receptor complexes (RARs and RXRs) in the nucleus which then bind to and activate RA response elements (RAREs) in genes and (2) its interactions with processes that are initiated in the cytoplasm. While much work has focused on the impact of atRA on cell differentiation, VA, itself, has been shown to exert effects on the maintenance of ESC identity that are not dependent upon classic RA-signaling pathways. In this chapter, we present results from our laboratory and others using well-documented approaches for investigating the effects of retinoids on the differentiation of ESC in vitro and introduce a novel method that uses chemically-defined growth conditions. The merits of each approach are discussed., Competing Interests: Disclosures We have nothing to disclose., (© 2020 Elsevier Inc. All rights reserved.)

Large-scale production of human pluripotent stem cells (hPSCs) in an efficient and safe manner is crucial to the successful application of hPSCs in biomedical research and regenerative medicine. Three-dimensional culture methods for hPSCs have been extensively studied using single-cell passaging approaches; however, these techniques have been challenged by the induction of massive cell death and accumulation of genomic abnormalities. In this work, we developed and optimized a novel, simple clump-passaging method for in vitro hPSCs 3-dimensional (3D) culture that can be exploited for large-scale production. Fully grown hPSC spheroids were dissociated into smaller-sized spheroid clumps by simple treatment with enzyme-free dissociation buffer, and clumped hPSCs were inoculated and maintained for 3D suspension culture. Our clump-passaging method effectively increased the hPSCs survival rate after subculture and supported scalable hPSCs 3D expansion. We also tested and selected chemically defined media formulations that are suitable for 3D culture and commercially available. Overall, our clump-passaging and expansion method demonstrated high survival and expansion rates for hPSC spheroids compared with conventional methods and may also have the advantage of maintaining genomic stability., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The multipotency and anti-inflammatory effects of mesenchymal stem cells (MSCs) make them attractive for cell therapy in regenerative medicine. A large number of MSCs is required for efficient therapy owing to the low homing efficiency of MSCs to target sites. Furthermore, owing to limitations in obtaining sufficient amounts of MSCs, in vitro expansion of MSCs that preserves their differentiation and proliferative potential is essential. The animal factor included in culture media also limits clinical application. In this study, adipose-derived MSCs showed a significantly higher proliferation rate in STK2, a chemically-defined medium, than in DMEM/FBS. The expression of MSC surface markers was increased in the culture using STK2 compared to that using DMEM/FBS. Tri-lineage differentiation analyses showed that MSCs cultured in STK2 were superior to those cultured in DMEM/FBS. In addition, MSCs cultured in STK2 showed a reduced senescence rate, small and homogenous cell size, and were more genetically stable compared to those cultured in DMEM/FBS. Furthermore, secretome analysis showed that the expression of factors related to proliferation/migration, anti-inflammation, and differentiation were increased in STK2 culture medium compared to DMEM/FBS. Taken together, these results suggest that culture using STK2 medium offers many advantages through which it is possible to obtain safer, superior, and larger numbers of MSCs., Competing Interests: Myung-Suk Lee currently hold stock option in ABION Inc., and Young-Deug Kim, and Young Kee Shin currently hold stock in ABION Inc. Byung Jun Park is salaried employee of ABION Inc. Christine Youn and Sungyoul Hong: nothing to disclose. Sang Gyu Park currently hold stock in Xcell Therapeutics. Myung-Suk Lee, Jeong Hyun Kim, and Jongchan Ahn are salaried employees of Xcell therapeutics.