SE162:/S1/M2/D1

Sample Set Information

ID	TSE1323
Title	A U-system approach for predicting metabolic behaviors and responses based on an alleged metabolic reaction network.
Description	Background Progress in systems biology offers sophisticated approaches toward a comprehensive understanding of biological systems. Yet, computational analyses are held back due to difficulties in determining suitable model parameter values from experimental data which naturally are subject to biological fluctuations. The data may also be corrupted by experimental uncertainties and sometimes do not contain all information regarding variables that cannot be measured for technical reasons. Results We show here a streamlined approach for the construction of a coarse model that allows us to set up dynamic models with minimal input information. The approach uses a hybrid between a pure mass action system and a generalized mass action (GMA) system in the framework of biochemical systems theory (BST) with rate constants of 1, normal kinetic orders of 1, and -0.5 and 0.5 for inhibitory and activating effects, named Unity (U)-system. The U-system model does not necessarily fit all data well but is often sufficient for predicting metabolic behavior of metabolites which cannot be simultaneously measured, identifying inconsistencies between experimental data and the assumed underlying pathway structure, as well as predicting system responses to a modification of gene or enzyme. The U-system approach was validated with small, generic systems and implemented to model a large-scale metabolic reaction network of a higher plant, Arabidopsis. The dynamic behaviors obtained by predictive simulations agreed with actually available metabolomic time-series data, identified probable errors in the experimental datasets, and estimated probable behavior of unmeasurable metabolites in a qualitative manner. The model could also predict metabolic responses of Arabidopsis with altered network structures due to genetic modification. Conclusions The U-system approach can effectively predict metabolic behaviors and responses based on structures of an alleged metabolic reaction network. Thus, it can be a useful first-line tool of data analysis, model diagnostics and aid the design of next-step experiments.
Authors	Sriyudthsak K, Sawada Y, Chiba Y, Yamashita Y, Kanaya S, Onouchi H, Fujiwara T, Naito S, Voit EO, Shiraishi F, Hirai MY.
Reference	BMC Syst Biol. 2014;8 Suppl 5:S4. doi: 10.1186/1752-0509-8-S5-S4. Epub 2014 Dec 12.
Comment

PubMed

Sample Information

ID	S1
Title	Arabidopsis callus
Organism - Scientific Name	Arabidopsis thaliana
Organism - ID	NCBI taxonomy:3702
Compound - ID
Compound - Source
Preparation	Arabidopsis thaliana liquid callus culture derived from accession Col-0 was prepared as described in Murota et al. with slight modifications. For callus induction, minced seedlings were incubated in RM28 medium under constant light. The medium was changed every 6 days. For a metabolic perturbation experiment, RM28 medium supplemented with 10 mM L-lysine and 1 mM L-threonine was used at the third medium change. For a control experiment, RM28 without supplementation was used. Sucrose in RM28 medium was a sole carbon source for callus culture. The experiments were carried out in triplicate. For both metabolome and amino acid analyses, calli were collected prior to lysine and threonine treatment (0 h), and 2, 6, 12, 24, 36, 48, 60, 72, 84 and 96 h after the treatment. The calli were immediately frozen in liquid nitrogen and stored at -80°C. Prior to analyses, the frozen samples were lyophilized using a freeze dryer (FDU-2100, EYELA) in a vacuum.
Sample Preparation Details ID
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Analytical Method Information

ID	M2
Title	Amino acid analysis (GC-MS)
Method Details ID	MS2
Sample Amount	1 μL
Comment

Analytical Method Details Information

ID	MS2
Title	Amino acid analysis (GC-MS)
Instrument	GCMS-QP2010 Plus (Shimadzu)
Instrument Type
Ionization	EI
Ion Mode	Positive
Description	Metabolites were extracted by homogenizing lyophilized callus in 500 uL of the extraction solution (methanol: milliQ water = 4:1) per 2 mg dry weight in 2.0 mL sampling tubes using Mixer Mill MM300. After centrifugation at 15,000 r.p.m. at 25°C for 10 min, the 400 uL supernatant was used for the analysis performed according to the protocol based on the EZ:faast amino acid derivatization technique for GC-MS (Phenomenex, Torrance, CA) with slight modification. The reagent 1 (internal standard solution) was diluted to 10-fold while the reagent 6 (re-dissolution solvent) was added only 50uL to concentrate amino acid concentrations in samples. Then, 1 uL of solution was subjected to amino acid analysis by GC-MS using GCMS-QP2010 Plus (Shimadzu, Kyoto, Japan).
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Data Analysis Information

ID	D1
Title	Statistical evaluation
Data Analysis Details ID	DS2
Recommended decimal places of m/z
Comment

Data Analysis Details Information

ID	DS2
Title	Statistical evaluation
Description	The correlation coefficients, testing for the significance of the correlation coefficients, and student's t continuous random variable based on a survival function were calculated for comparing model simulations with experimental data.
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