3,104 research outputs found
Heterologous overproduction of oviedomycin by refactoring biosynthetic gene cluster and metabolic engineering of host strain Streptomyces coelicolor
No full textBackground: Oviedomycin is one among several polyketides known for their potential as anticancer agents. The biosynthetic gene cluster (BGC) for oviedomycin is primarily found in Streptomyces antibioticus. However, because this BGC is usually inactive under normal laboratory conditions, it is necessary to employ systematic metabolic engineering methods, such as heterologous expression, refactoring of BGCs, and optimization of precursor biosynthesis, to allow efficient production of these compounds. Results: Oviedomycin BGC was captured from the genome of Streptomyces antibioticus by a newly constructed plasmid, pCBA, and conjugated into the heterologous strain, S. coelicolor M1152. To increase the production of oviedomycin, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system was utilized in an in vitro setting to refactor the native promoters within the ovm BGC. The target promoters of refactoring were selected based on examination of factors such as transcription levels and metabolite profiling. Furthermore, genome-scale metabolic simulation was applied to find overexpression targets that could enhance the biosynthesis of precursors or cofactors related to oviedomycin production. The combined approach led to a significant increase in oviedomycin production, reaching up to 670 mg/L, which is the highest titer reported to date. This demonstrates the potential of the approach undertaken in this study. Conclusions: The metabolic engineering approach used in this study led to the successful production of a valuable polyketide, oviedomycin, via BGC cloning, promoter refactoring, and gene manipulation of host metabolism aided by genome-scale metabolic simulation. This approach can be also useful for the efficient production of other secondary molecules encoded by ‘silent’ BGCs.
Understanding of altered N-glycosylation-related gene expression in recombinant Chinese hamster ovary cells subjected to elevated ammonium concentration by digital mRNA counting
No full textTo understand the effects of ammonium on N-glycosylation, recombinant Chinese hamster ovary (rCHO) cells that produce the Fc-fusion protein were cultivated in serum-free suspension cultures with 10mM ammonium addition. The addition of ammonium to the cultures reduced the relative proportion of acidic isoforms and sialic acid content of an Fc-fusion protein. Fifty two N-glycosylation-related gene expressions were assessed by the NanoString nCounter system, which provides a digital readout using custom-designed color-coded probes. Among these queried genes, thirteen genes (gale, nans, gpi, man2a1, b4galt5, b4galt7, st3gal2, st3gal5, glb1, hexa, hexb, neu1, and neu3) were up-regulated over 1.5 times in the culture with ammonium addition after 5 days of culture; however, none of the 54 genes were significantly different after 3 days of culture. In particular, the expression level of neu1 (sialidase-1) and neu3 (sialidase-3), which play a role in reduction of sialylation, increased over 2 times. Likewise, the protein expression levels of sialidase-1 and sialidase-3 determined by Western blot analysis were also increased significantly in the culture with ammonium addition. Transient transfection of neu-1 or neu3-targeted siRNAs significantly improved the sialic acid content of the Fc-fusion protein in the culture with ammonium addition, indicating that the decreased sialic acid content was in part due to the increased expression level of sialidase. Taken together, the results obtained in this study provide a better understanding of the detrimental effect of ammonium on N-glycosylation, especially sialylation, in rCHO cells.open
Self-Compensation of PZT Errors in White Light Interferometry
No full textOne of main error sources in white light scanning interferometry is the inaccuracy of scanning mechanisms in that PZT (piezoelectric transducer) micro-actuators are preferably used. We propose a new calibration method that is capable of identifying actual scanning errors directly by analyzing the spectral distribution of sampled interferograms. This calibration provides an effective means of self-compensation for the non-linearity errors caused by PZT hysteresis, enhancing the measurement uncertainty to a level of 5 nanometers over an entire measuring range of 100 ㎛
Machine learning-based discrete event dynamic surrogate model of communication systems for simulating the command, control, and communication system of systems
No full textCommand and control (C2) and communication are at the heart of successful military operations in network-centric warfare. Interoperable simulation of a C2 system model and a communication (C) system model may be employed to interactively analyze their detailed behaviors. However, such simulation would be inefficient in simulation time for analysis of combat effectiveness of the C2 model against possible input combinations while considering the communication effect in combat operations. This study proposes a discrete event dynamic surrogate model (DEDSM) for the C model, which would be integrated with the C2 model and simulated. The proposed integrated simulation reduces execution time markedly in analysis of combat effectiveness without sacrificing the accuracy reflecting the communication effect. We hypothesize the DEDSM as a probabilistic priority queuing model whose semantics is expressed in a discrete event systems specification model with some characteristic functions unknown. The unknown functions are identified by machine learning with a data set generated by interoperable simulation of the C2 and C models. The case study with the command, control, and communication system of systems first validates the proposed approach through an equivalence test between the interoperable simulation and the proposed one. It then compares the simulation execution times and the number of events exchanged between the two simulations.
Supp_data_2 – Supplemental material for Therapeutic effects of the novel Leucyl-tRNA synthetase inhibitor BC-LI-0186 in non-small cell lung cancer
No full textSupplemental material, Supp_data_2 for Therapeutic effects of the novel Leucyl-tRNA synthetase inhibitor BC-LI-0186 in non-small cell lung cancer by Eun Young Kim, Jin Gu Lee, Jung Mo Lee, Arum Kim, Hee Chan Yoo, Kibum Kim, Minji Lee, Chulho Lee, Gyoonhee Han, Jung Min Han and Yoon Soo Chang in Therapeutic Advances in Medical Oncology</p
Supp_data_1 – Supplemental material for Therapeutic effects of the novel Leucyl-tRNA synthetase inhibitor BC-LI-0186 in non-small cell lung cancer
No full textSupplemental material, Supp_data_1 for Therapeutic effects of the novel Leucyl-tRNA synthetase inhibitor BC-LI-0186 in non-small cell lung cancer by Eun Young Kim, Jin Gu Lee, Jung Mo Lee, Arum Kim, Hee Chan Yoo, Kibum Kim, Minji Lee, Chulho Lee, Gyoonhee Han, Jung Min Han and Yoon Soo Chang in Therapeutic Advances in Medical Oncology</p
Supp_data_4 – Supplemental material for Therapeutic effects of the novel Leucyl-tRNA synthetase inhibitor BC-LI-0186 in non-small cell lung cancer
No full textSupplemental material, Supp_data_4 for Therapeutic effects of the novel Leucyl-tRNA synthetase inhibitor BC-LI-0186 in non-small cell lung cancer by Eun Young Kim, Jin Gu Lee, Jung Mo Lee, Arum Kim, Hee Chan Yoo, Kibum Kim, Minji Lee, Chulho Lee, Gyoonhee Han, Jung Min Han and Yoon Soo Chang in Therapeutic Advances in Medical Oncology</p
Supp_data_3 – Supplemental material for Therapeutic effects of the novel Leucyl-tRNA synthetase inhibitor BC-LI-0186 in non-small cell lung cancer
No full textSupplemental material, Supp_data_3 for Therapeutic effects of the novel Leucyl-tRNA synthetase inhibitor BC-LI-0186 in non-small cell lung cancer by Eun Young Kim, Jin Gu Lee, Jung Mo Lee, Arum Kim, Hee Chan Yoo, Kibum Kim, Minji Lee, Chulho Lee, Gyoonhee Han, Jung Min Han and Yoon Soo Chang in Therapeutic Advances in Medical Oncology</p
Effect of reductive cyclic carbonate additives and linear carbonate cosolvents on fast chargeability of LiNi0.6Co0.2Mn0.2O2/graphite cells
No full textFor application to electric vehicles, the fast charging of lithium-ion batteries is required. However, lithium-ion batteries are faced with undesirable Li plating causing the capacity fading with low Coulombic efficiency at high charge rates. Here, we present the effects of solid electrolyte interphase structures of the graphite anode and linear carbonate solvents on fast charging capability of LiNi0.6Co0.2Mn0.2O2/graphite full cells. To control the nature of the interfacial layer on the graphite anode affecting the Li plating behavior, we exploit three kinds of additives, ethylene carbonate, fluoroethylene carbonate and vinylene carbonate, as anode solid electrolyte interphase formers. In addition, the effect of ethyl methyl carbonate and dimethyl carbonate on the solvation and transport of high concentrations of Li ions de-intercalated from the LiNi0.6Co0.2Mn0.2O2 cathode in a full cell at high charge rates is explored in fluoroethylene carbonate-based electrolytes. Our investigation reveals that the combination of fluoroethylene carbonate and dimethyl carbonate in the electrolyte enables fast-charging LiNi0.6Co0.2Mn0.2O2/graphite full cells showing the excellent capacity retention of 79% after 1000 cycles at a high charging current density of 6&#8239;mA&#8239;cm&#8722;2, corresponding to 2C, and a discharge rate of 1C without Li plating on the graphite anode
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