Tune Wulff

A Consensus Genome-scale Reconstruction of Chinese Hamster Ovary Cell Metabolism

Chinese hamster ovary (CHO) cells dominate biotherapeutic protein production and are widely used in mammalian cell line engineering research. To elucidate metabolic bottlenecks in protein production and to guide cell engineering and bioprocess optimization, we reconstructed the metabolic pathways in CHO and associated them with >1,700 genes in the Cricetulus griseus genome. The genome-scale metabolic model based on this reconstruction, iCHO1766, and cell-line-specific models for CHO-K1, CHO-S, and CHO-DG44 cells provide the biochemical basis of growth and recombinant protein production. The models accurately predict growth phenotypes and known auxotrophies in CHO cells. With the models, we quantify the protein synthesis capacity of CHO cells and demonstrate that common bioprocess treatments, such as histone deacetylase inhibitors, inefficiently increase product yield. However, our simulations show that the metabolic resources in CHO are more than three times more efficiently utilized for growth or recombinant protein synthesis following targeted efforts to engineer the CHO secretory pathway. This model will further accelerate CHO cell engineering and help optimize bioprocesses.

Predictable tuning of protein expression in bacteria

We comprehensively assessed the contribution of the Shine-Dalgarno sequence to protein expression and used the data to develop EMOPEC (Empirical Model and Oligos for Protein Expression Changes; http://emopec.biosustain.dtu.dk). EMOPEC is a free tool that makes it possible to modulate the expression level of any Escherichia coli gene by changing only a few bases. Measured protein levels for 91% of our designed sequences were within twofold of the desired target level.

Time-dependent changes in protein expression in rainbow trout muscle following hypoxia

Adaptation to hypoxia is a complex process, and individual proteins will be up- or down-regulated in order to address the main challenges at any given time. To investigate the dynamics of the adaptation, rainbow trout (Oncorhynchus mykiss) was exposed to 30% of normal oxygen tension for 1, 2, 5 and 24 h respectively, after which muscle samples were taken. The successful investigation of numerous proteins in a single study was achieved by selectively separating the sarcoplasmic proteins using 2-DE. In total 46 protein spots were identified as changing in abundance in response to hypoxia using one-way ANOVA and multivariate data analysis. Proteins of interest were subsequently identified by MS/MS following tryptic digestion. The observed regulation following hypoxia in skeletal muscle was determined to be time specific, as only a limited number of proteins were regulated in response to more than one time point. The cellular response to hypoxia included regulation of proteins involved in maintaining iron homeostasis, energy levels and muscle structure. In conclusion, this proteome-based study presents a comprehensive investigation of the expression profiles of numerous proteins at four different time points. This increases our understanding of timed changes in protein expression in rainbow trout muscle following hypoxia.

Dietary tools to modulate glycogen storage in gilthead seabream muscle: glycerol supplementation.

The quality and shelf life of fish meat products depend on the skeletal muscles energetic state at slaughter, as meat decomposition processes can be exacerbated by energy depletion. In this study, we tested dietary glycerol as a way of replenishing muscle glycogen reserves of farmed gilthead seabream. Two diets were tested in duplicate (n = 42/tank). Results show 5% inclusion of crude glycerol in gilthead seabream diets induces increased muscle glycogen, ATP levels and firmness, with no deleterious effects in terms of growth, proximate composition, fatty acid profile, oxidative state, and organoleptic properties (aroma and color). Proteomic analysis showed a low impact of glycerol-supplementation on muscle metabolism, with most changes probably reflecting increased stress coping capacity in glycerol-fed fish. This suggests inclusion of crude glycerol in gilthead seabream diets (particularly in the finishing phase) seems like a viable strategy to increase glycogen deposition in muscle without negatively impacting fish welfare and quality.

Nutritional mitigation of winter thermal stress in gilthead seabream associated metabolic pathways and potential indicators of nutritional state

A trial was carried out with gilthead seabream juveniles, aiming to investigate the ability of an enhanced dietary formulation (diet Winter Feed, WF, containing a higher proportion of marine-derived protein sources and supplemented in phospholipids, vitamin C, vitamin E and taurine) to assist fish in coping with winter thermal stress, compared to a low-cost commercial diet (diet CTRL). In order to identify the metabolic pathways affected by WF diet, a comparative two dimensional differential in-gel electrophoresis (2D-DIGE) analysis of fish liver proteome (pH 4–7) was undertaken at the end of winter. A total of 404 protein spots, out of 1637 detected, were differentially expressed between the two groups of fish. Mass spectrometry analysis of selected spots suggested that WF diet improved oxidative stress defense, reduced endoplasmic reticulum stress, enhanced metabolic flux through methionine cycle and phenylalanine/tyrosine catabolism, and induced higher aerobic metabolism and gluconeogenesis. Results support the notion that WF diet had a positive effect on fish nutritional state by partially counteracting the effect of thermal stress and underlined the sensitivity of proteome data for nutritional and metabolic profiling purposes. Intragroup variability and co-measured information were also used to pinpoint which proteins displayed a stronger relation with fish nutritional state. SIGNIFICANCE Winter low water temperature is a critical factor for gilthead seabream farming in the Mediterranean region, leading to a reduction of feed intake, which often results in metabolic and immunological disorders and stagnation of growth performances. In a recent trial, we investigated the ability of an enhanced dietary formulation (diet WF) to assist gilthead seabream in coping with winter thermal stress, compared to a standard commercial diet (diet CTRL). Within this context, in the present work, we identified metabolic processes that are involved in the stress-mitigating effect observed with diet WF, by undertaking a comparative analysis of fish liver proteome at the end of winter. This study brings information relative to biological processes that are involved in gilthead seabream winter thermal stress and shows that these can be mitigated through a nutritional strategy, assisting gilthead seabream to deal better with winter thermal conditions. Furthermore, the results show that proteomic information not only clearly distinguishes the two dietary groups from each other, but also captures heterogeneities that reflect intra-group differences in nutritional state. This was exploited in this work to refine the variable selection strategy so that protein spots displaying a stronger correlation with “nutritional state” could be identified as possible indicators of gilthead seabream metabolic and nutritional state. Finally, this study shows that gel-based proteomics seems to provide more reliable information than transmissive FT-IR spectroscopy, for the purposes of nutritional and metabolic profiling.

Identification of meat products by shotgun spectral matching

A new method, based on shotgun spectral matching of peptide tandem mass spectra, was successfully applied to the identification of different food species. The method was demonstrated to work on raw as well as processed samples from 16 mammalian and 10 bird species by counting spectral matches to spectral libraries in a reference database with one spectral library per species. A phylogenetic tree could also be constructed directly from the spectra. Nearly all samples could be correctly identified at the species level, and 100% at the genus level. The method does not use any genomic information and unlike targeted methods, no prior knowledge of genetic variation within a genus or species is necessary.

Triton X-114 cloud point extraction to subfractionate blood plasma proteins for two-dimensional gel electrophoresis

A simple and reproducible procedure for enrichment of a plasma protein subfraction suitable for two-dimensional polyacrylamide gel electrophoresis (2DE) was developed, using a Triton X-114-based cloud point extraction (CPE). Appropriate conditions for such a CPE procedure were found by SDS-PAGE to be a plasma protein concentration of about 10mg/ml in 3% (w/v) Triton X-114. 2DE of proteins obtained by CPE of 400 μl of human plasma revealed about 200 spots constituting a spot pattern very different from the pattern of total plasma. The CPE procedure only had a limited contribution to the technical variation. Identification of about 60 spots, representing only 22 proteins, revealed that several proteins in the obtained subfraction were present in more isoforms or modifications. Among these were apolipoproteins (A-1, D, E, L1, and M), haptoglobin-related protein, phosphatidylcholine-sterol acyltransferase, serum amyloid A, and serum paraoxonase/arylesterase 1, which are proteins of a hydrophobic nature, as in plasma they relate to lipoprotein particles. Thus, Triton X-114-based CPE is a simple plasma prefractionation tool, attractive for detailed 2DE studies of hydrophobic plasma proteins and their isoforms or modifications.

Proteome Analysis of Pyloric Ceca: A Methodology for Fish Feed Development?

Changing the protein source of fish feed from fish meal to alternative sources of protein will affect traits such as fish growth, quality, and feed utilization. The present investigation was initiated to introduce a two-dimensional gel electrophoresis based proteomic workflow as a tool to investigate feed effects on fish by analyzing protein changes in the fish gut. The workflow was used to study the effect of substituting fish meal in fish feed by alternative sources of protein. Rainbow trout divided into five groups were fed for 72 days with feeds varying in protein composition. By two-dimensional gel electrophoresis proteins extracted from the pyloric ceca were separated, making it possible to measure the abundance of more than 440 protein spots. The expression of 41 protein spots was found to change due to differences in feed composition. By mass spectrometry 31 of these proteins were identified, including proteins involved in digestion (trypsinogen, carboxylic ester hydrolase, and aminopeptidase). The many expression changes indicated that the trout, when adapting to differences in feed formulation, alter the protein composition of the gut.

Effect of α-lactalbumin and β-lactoglobulin on the oxidative stability of 10% fish oil-in-water emulsions depends on pH

The objective of this study was to investigate the influence of pH on lipid oxidation and protein partitioning in 10% fish oil-in-water emulsions prepared with different whey protein isolates with varying ratios of α-lactalbumin and β-lactoglobulin. Results showed that an increase in pH increased lipid oxidation irrespective of the emulsifier used. At pH 4, lipid oxidation was not affected by the type of whey protein emulsifier used or the partitioning of proteins between the interface and the water phase. However, at pH 7 the emulsifier with the highest concentration of β-lactoglobulin protected more effectively against oxidation during emulsion production, whereas the emulsions with the highest concentration of α-lactalbumin were most stable to oxidation during storage. These differences were explained by differences in the pressure and adsorption induced unfolding of the individual protein components.

Drug-Driven Phenotypic Convergence Supports Rational Treatment Strategies of Chronic Infections

Summary Chronic Pseudomonas aeruginosa infections evade antibiotic therapy and are associated with mortality in cystic fibrosis (CF) patients. We find that in vitro resistance evolution of P. aeruginosa toward clinically relevant antibiotics leads to phenotypic convergence toward distinct states. These states are associated with collateral sensitivity toward several antibiotic classes and encoded by mutations in antibiotic resistance genes, including transcriptional regulator nfxB. Longitudinal analysis of isolates from CF patients reveals similar and defined phenotypic states, which are associated with extinction of specific sub-lineages in patients. In-depth investigation of chronic P. aeruginosa populations in a CF patient during antibiotic therapy revealed dramatic genotypic and phenotypic convergence. Notably, fluoroquinolone-resistant subpopulations harboring nfxB mutations were eradicated by antibiotic therapy as predicted by our in vitro data. This study supports the hypothesis that antibiotic treatment of chronic infections can be optimized by targeting phenotypic states associated with specific mutations to improve treatment success in chronic infections.