ART: A machine learning Automated Recommendation Tool for synthetic biology

Biology has changed radically in the last two decades, transitioning from a descriptive science into a design science. Synthetic biology allows us to bioengineer cells to synthesize novel valuable molecules such as renewable biofuels or anticancer drugs. However, traditional synthetic biology approaches involve ad-hoc engineering practices, which lead to long development times. Here, we present the Automated Recommendation Tool (ART), a tool that leverages machine learning and probabilistic modeling techniques to guide synthetic biology in a systematic fashion, without the need for a full mechanistic understanding of the biological system. Using sampling-based optimization, ART provides a set of recommended strains to be built in the next engineering cycle, alongside probabilistic predictions of their production levels. We demonstrate the capabilities of ART on simulated data sets, as well as experimental data from real metabolic engineering projects producing renewable biofuels, hoppy flavored beer without hops, and fatty acids. Finally, we discuss the limitations of this approach, and the practical consequences of the underlying assumptions failing

Differential modulation of insulin actions by dexamethasone: studies in primary cultures of adult rat hepatocytes

Background/Aims: Steroid diabetes is associated with hepatic insulin resistance; in hepatic cell models, however, mainly insulin-permissive effects have been described. Here we investigate modulation by dexamethasone of a larger number of insulin actions. Methods: Adult rat hepatocytes were cultured dexamethasone for 48 h; insulin actions were studied subsequently. Results: Stimulation of glycolysis by insulin but not by glucose required culture with dexamethasone. Activation of glycogen synthesis by insulin or glucose was strongly enhanced by dexamethasone, the insulin effects on glycogenolysis and amino acid uptake were not modulated. When dexamethasone was omitted from the culture, insulin was incapable to activate glycogen synthase, inactivate glycogen phosphorylase or elevate the level of fructose 2,6-bisphosphate. Dexamethasone did not alter insulin binding, insulin receptor number or kinase activity, insulin receptor substrate-1 and Akt protein expression/phosphorylation. Insulin-stimulated association of phosphatidylinositol 3-kinase with insulin receptor substrates-1 and -2 was increased with dexamethasone, the increased association with IRS-2 may, at least partially, be explained by higher IRS-2 protein expression. Conclusions: The steroid does not cause hepatic resistance in vitro. The differential attenuation under steroid deprivation points to defects in branches of the insulin signal chain and/or loss of hormonal regulation at the level of target enzymes. (C) 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved

The expression of mesenchymal, neural and haematopoietic stem cell markers in adult hepatocytes proliferating in vitro

Background/Aims: Cultured adult hepatocytes may be stimulated into clonal expansion. We raise the question whether adult hepatocytes proliferating in vitro recapitulate the early process of hepatic development. Methods: A non-enzymatic method was used to isolate hepatocytes free of contamination with non-parenchymal cells. Hepatocytes were stimulated into proliferation in the presence of mitogens and conditioned media from nonparenchymal cell and hepatocyte culture supernatants. Immunofluorescence methods and PCR analysis were used to demonstrate immunophenotypical characteristics and gene expression profiles similar to those of progenitor cells. Results: Rapid growth occurred during the first 7 days of culture. Cells continued to express hepatic markers (phosphoenolpyruvate carboxykinase, cytokeratin 18, transferrin and dipeptidylpeptidase IV), but the gap junction protein connexin 32 was down-regulated. In the early stage of proliferation, cells started to express biliary and extrahepatic progenitor markers (cytokeratin 19, CD49b, CD49f, nestin, vimentin, Thyl and c-kit), followed by cytokeratin 7, connexin 43, and neural cell adhesion molecule. Co-expression of the epithelial liver progenitor marker alpha-foetoprotein with either nestin (neural marker) or Thyl (mesenchymal marker) was also demonstrated. Conclusions: Mature, hepatocytes reveal their potential to regain a spectrum of progenitor markers from different germ layers, suggesting, enormous plasticity and differentiation potential of adult liver cells. (c) 2006 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

Hepatocyte-supported serum-free culture of rat liver sinusoidal endothelial cells

Background/Aims: A major problem in rat liver endothelial cell culture is the rapid loss of cells after 48 h, This study aimed to develop a protocol that allowed easy maintenance and proliferation of sinusoidal endothelial cells in serum-free culture for 5-6 days. Methods: Cells isolated from adult rat liver by collagenase digestion were purified by centrifugal elutriation and cultured on glutaraldehyde-crosslinked collagen, Results: At high plating densities cells could be maintained serum-free for 6 days in the presence of hydrocortisone and basic fibroblast growth factor; at lower plating densities medium had to be supplemented with additional growth-promoting factors. Conditioned medium of adult rat hepatocytes proved to be the most effective growth stimulus; it increased thymidine incorporation, DNA content and cell number per dish with a half-maximal effect at 20% (v/v), Cell proliferation was also observed with either vascular endothelial growth factor, phorbol ester or conditioned media from FAO or HEPG2 liver cell lines provided the cultures were additionally supplemented with 1% newborn calf serum. Vascular endothelial growth factor was detected in all conditioned media. In the absence of hepatocyte-conditioned medium, 1% serum helped to maintain cultures; it itself exerted a low proliferative effect. Higher serum concentrations (>5%), however, led to cell loss after 48 h, The numerous sieve plates of 6-h-old cells progressively disappeared during culture and were replaced by randomly distributed pores, which later grouped together at cell-cell borders. More than 90% of the cells endocytosed acetylated low-density lipoprotein, Conclusions: The study shows that cultured hepatocytes secrete growth-promoting substances that stimulate in vitro endothelial cell proliferation in the absence of serum; this effect could be mimicked by the combined addition of vascular endothelial growth factor and 1% serum. The new media formulations should facilitate future research on liver endothelial cells in mono- or coculture

Beyond Growth Rate 0.6: What Drives Corynebacterium glutamicum to Higher Growth Rates in Defined Medium

In a former study we showed that Corynebacterium glutamicum grows much faster in defined CGXII glucose medium when growth was initiated in highly diluted environments [Grunberger et al. (2013b) Biotechnol Bioeng]. Here we studied the batch growth of C. glutamicum in CGXII at a comparable low starting biomass concentration of OD approximate to 0.005 in more detail. During bioreactor cultivations a biphasic growth behavior with changing growth rates was observed. Initially the culture grew with mu = 0.61 +/- 0.02 h(-1) before the growth rate dropped to mu = 0.46 +/- 0.02 h(-1). We were able to confirm the elevated growth rate for C. glutamicum in CGXII and showed for the first time a growth rate beyond 0.6 in lab-scale bioreactor cultivations on defined medium. Advanced growth studies combining well-designed bioreactor and microfluidic single-cell cultivations (MSCC) with quantitative transcriptomics, metabolomics and integrative in silico analysis revealed protocatechuic acid as a hidden co-substrate for accelerated growth within CGXII. The presented approach proves the general applicability of MSCC to investigate and validate the effect of single medium components on microorganism growth during cultivation in liquid media, and therefore might be of interest for any kind of basic growth study. (C) 2013 Wiley Periodicals, Inc