SALARECON connects the Atlantic salmon genome to growth and feed efficiency.

Atlantic salmon (Salmo salar) is the most valuable farmed fish globally and there is much interest in optimizing its genetics and rearing conditions for growth and feed efficiency. Marine feed ingredients must be replaced to meet global demand, with challenges for fish health and sustainability. Metabolic models can address this by connecting genomes to metabolism, which converts nutrients in the feed to energy and biomass, but such models are currently not available for major aquaculture species such as salmon. We present SALARECON, a model focusing on energy, amino acid, and nucleotide metabolism that links the Atlantic salmon genome to metabolic fluxes and growth. It performs well in standardized tests and captures expected metabolic (in)capabilities. We show that it can explain observed hypoxic growth in terms of metabolic fluxes and apply it to aquaculture by simulating growth with commercial feed ingredients. Predicted limiting amino acids and feed efficiencies agree with data, and the model suggests that marine feed efficiency can be achieved by supplementing a few amino acids to plant- and insect-based feeds. SALARECON is a high-quality model that makes it possible to simulate Atlantic salmon metabolism and growth. It can be used to explain Atlantic salmon physiology and address key challenges in aquaculture such as development of sustainable feeds.

The role of the cadmium-binding protein response of the digestive gland of the Yesso scallop Mizuhopecten yessoensis (Jay, 1857) for marine environmental assessments.

The ability of Pectinidae to accumulate heavy metals and store them in their tissues allows the use of scallops for biomonitoring marine pollution. High molecular weight metallothionein (MT)-like proteins (MTlps) play a central role in this process. Two major MTlps (72 and 43 kDa) have been identified in the digestive glands of Mizuhopecten yessoensis (Yesso scallop). These proteins have a very high affinity for the heavy metals cadmium, cobalt, and caesium. Additionally, these proteins can be deposited in large quantities in the digestive glands of this mollusc. It has been shown that 72 kDa MTlp is the main stress-response protein in areas polluted with cadmium or radioactive metals. Monitoring the amounts of MTlps in the digestive glands of the scallop M. yessoensis in areas with different anthropogenic pollutants has shown that these proteins are reliable biological markers of heavy-metal pollution in the marine environment.

Novel Extracellular Electron Transfer Channels in a Gram-Positive Thermophilic Bacterium.

Biogenic transformation of Fe minerals, associated with extracellular electron transfer (EET), allows microorganisms to exploit high-potential refractory electron acceptors for energy generation. EET-capable thermophiles are dominated by hyperthermophilic archaea and Gram-positive bacteria. Information on their EET pathways is sparse. Here, we describe EET channels in the thermophilic Gram-positive bacterium Carboxydothermus ferrireducens that drive exoelectrogenesis and rapid conversion of amorphous mineral ferrihydrite to large magnetite crystals. Microscopic studies indicated biocontrolled formation of unusual formicary-like ultrastructure of the magnetite crystals and revealed active colonization of anodes in bioelectrochemical systems (BESs) by C. ferrireducens. The internal structure of micron-scale biogenic magnetite crystals is reported for the first time. Genome analysis and expression profiling revealed three constitutive c-type multiheme cytochromes involved in electron exchange with ferrihydrite or an anode, sharing insignificant homology with previously described EET-related cytochromes thus representing novel determinants of EET. Our studies identify these cytochromes as extracellular and reveal potentially novel mechanisms of cell-to-mineral interactions in thermal environments.

Sensitivity analysis of a pathway with respect to fast and small temperature change.

Temperature affects all enzymes simultaneously in a metabolic system. The enzyme concentration in a biochemical system can be considered as invariant under fast and small temperature change. Therefore, the total sensitivity of a steady state flux through a pathway with respect to the temperature can be expressed as: the apparent activation energy of a steady state pathway flux equals the sum of weighted activation energies of the individual reactions contributing to the flux, where the weighting factors are the flux control coefficients of these reactions in the context of the network. Correspondingly, since the elasticity of any enzyme with respect to temperature is always nonzero, only the reactions with a nonzero flux control coefficient contribute accordingly to the temperature sensitivity of the pathway.

Using a Multi-compartmental Metabolic Model to Predict Carbon Allocation in Arabidopsis thaliana.

The molecular mechanism of loading/unloading of sucrose into/from the phloem plays an important role in sucrose translocation among plant tissues. Perturbation of this mechanism results in growth phenotypes of a plant. In order to better understand the coupling of sucrose translocation with metabolic processes a multi-compartmental metabolic network of Arabidopsis thaliana was reconstructed and optimized with respect to biomass growth, both in light and in dark conditions. The model can be used to perform flux balance analysis of metabolic fluxes through the central carbon metabolism and catabolic and anabolic pathways. Balances and turnover of energy (ATP/ADP) and redox metabolites (NAD(P)H/NAD(P)) as well as proton concentrations in different compartments can be estimated. Importantly, the model can be used to quantify the translocation of sucrose from source to sink tissues through phloem in association with an integral balance of protons, which in turn is defined by the operational modes of the energy metabolism (light and dark conditions). This chapter describes how a multi-compartmental model to predict carbon allocation is constructed and used.

Cell size and morphological properties of yeast Saccharomyces cerevisiae in relation to growth temperature.

Cell volume is an important parameter for modelling cellular processes. Temperature-induced variability of cellular size, volume, intracellular granularity, a fraction of budding cells of yeast Saccharomyces cerevisiae CEN.PK 113-7D (in anaerobic glucose unlimited batch cultures) were measured by flow cytometry and matched with the performance of the biomass growth (maximal specific growth rate (µmax), specific rate of glucose consumption, the rate of maintenance, biomass yield on glucose). The critical diameter of single cells was 7.94 µm and it is invariant at growth temperatures above 18.5°C. Below 18.5°C, it exponentially increases up to 10.2 µm. The size of the bud linearly depends on µmax, and it is between 50% at 5°C and 90% at 31°C of the averaged single cell. The intracellular granularity (side scatter channel (SSC)-index) negatively depends on µmax. There are two temperature regions (5-31°C vs. 33-40°C) where the relationship between SSC-index and various cellular parameters differ significantly. In supraoptimal temperature range (33-40°C), cells are less granulated perhaps due to a higher rate of the maintenance. There is temperature dependent passage through the checkpoints in the cell cycle which influences the µmax. The results point to the existence of two different morphological states of yeasts in these different temperature regions.

Metabolic model of central carbon and energy metabolisms of growing Arabidopsis thaliana in relation to sucrose translocation.

BACKGROUND: Sucrose translocation between plant tissues is crucial for growth, development and reproduction of plants. Systemic analysis of these metabolic and underlying regulatory processes allow a detailed understanding of carbon distribution within the plant and the formation of associated phenotypic traits. Sucrose translocation from 'source' tissues (e.g. mesophyll) to 'sink' tissues (e.g. root) is tightly bound to the proton gradient across the membranes. The plant sucrose transporters are grouped into efflux exporters (SWEET family) and proton-symport importers (SUC, STP families). To better understand regulation of sucrose export from source tissues and sucrose import into sink tissues, there is a need for a metabolic model that takes in account the tissue organisation of Arabidopsis thaliana with corresponding metabolic specificities of respective tissues in terms of sucrose and proton production/utilization. An ability of the model to operate under different light modes ('light' and 'dark') and correspondingly in different energy producing modes is particularly important in understanding regulatory modules. RESULTS: Here, we describe a multi-compartmental model consisting of a mesophyll cell with plastid and mitochondrion, a phloem cell, as well as a root cell with mitochondrion. In this model, the phloem was considered as a non-growing transport compartment, the mesophyll compartment was considered as both autotrophic (growing on CO2 under light) and heterotrophic (growing on starch in darkness), and the root was always considered as heterotrophic tissue dependent on sucrose supply from the mesophyll compartment. In total, the model includes 413 balanced compounds interconnected by 400 transformers. The structured metabolic model accounts for central carbon metabolism, photosynthesis, photorespiration, carbohydrate metabolism, energy and redox metabolisms, proton metabolism, biomass growth, nutrients uptake, proton gradient generation and sucrose translocation between tissues. Biochemical processes in the model were associated with gene-products (742 ORFs). Flux Balance Analysis (FBA) of the model resulted in balanced carbon, nitrogen, proton, energy and redox states under both light and dark conditions. The main H+-fluxes were reconstructed and their directions matched with proton-dependent sucrose translocation from 'source' to 'sink' under any light condition. CONCLUSIONS: The model quantified the translocation of sucrose between plant tissues in association with an integral balance of protons, which in turn is defined by operational modes of the energy metabolism.

From Phosphoproteome to Modeling of Plant Signaling Pathways.

Quantitative proteomic experiments in recent years became almost routine in many aspects of biology. Particularly the quantification of peptides and corresponding phosphorylated counterparts from a single experiment is highly important for understanding of dynamics of signaling pathways. We developed an analytical method to quantify phosphopeptides (pP) in relation to the quantity of the corresponding non-phosphorylated parent peptides (P). We used mixed-mode solid-phase extraction to purify total peptides from tryptic digest and separated them from most of the phosphorous-containing compounds (e.g., phospholipids, nucleotides) which enhances pP enrichment on TiO2 beads. Phosphoproteomic data derived with this designed method allows quantifying pP/P stoichiometry, and qualifying experimental data for mathematical modeling.

The new facile and straightforward method for the synthesis of 4H-1,2,3-thiadiazolo[5,4-b]indoles and determination of their antiproliferative activity.

A series of 4H-1,2,3-thiadiazolo[5,4-b]indoles were synthesized by novel tandem of oxidative cyclization of 3-alkoxycarbonylhydrazonoindoline-2-thiones, 1,5-H-shift and elimination of tert-butoxy(ethoxy)carbonyl group. The simple method for their modifications by the reactions with electrophilic agents were elaborated and as a result of the synthetic investigation a number of N-alkyl-, N-acyl- and N-sulfonyl-4H-1,2,3-thiadiazolo[5,4-b]indoles were prepared in good yields. Preliminary biological tests for the three examples of synthesized compounds with different substituents at the nitrogen atom indole ring have shown that the biological behavior of the investigated 1,2,3-thiadiazolo[5,4-b]indoles is substantially directed by this structural fragment.

Metabolic efficiency in yeast Saccharomyces cerevisiae in relation to temperature dependent growth and biomass yield.

Canonized view on temperature effects on growth rate of microorganisms is based on assumption of protein denaturation, which is not confirmed experimentally so far. We develop an alternative concept, which is based on view that limits of thermal tolerance are based on imbalance of cellular energy allocation. Therefore, we investigated growth suppression of yeast Saccharomyces cerevisiae in the supraoptimal temperature range (30-40°C), i.e. above optimal temperature (Topt). The maximal specific growth rate (µmax) of biomass, its concentration and yield on glucose (Yx/glc) were measured across the whole thermal window (5-40°C) of the yeast in batch anaerobic growth on glucose. Specific rate of glucose consumption, specific rate of glucose consumption for maintenance (mglc), true biomass yield on glucose (Yx/glc(true)), fractional conservation of substrate carbon in product and ATP yield on glucose (Yatp/glc) were estimated from the experimental data. There was a negative linear relationship between ATP, ADP and AMP concentrations and specific growth rate at any growth conditions, whilst the energy charge was always high (~0.83). There were two temperature regions where mglc differed 12-fold, which points to the existence of a 'low' (within 5-31°C) and a 'high' (within 33-40°C) metabolic mode regarding maintenance requirements. The rise from the low to high mode occurred at 31-32°C in step-wise manner and it was accompanied with onset of suppression of µmax. High mglc at supraoptimal temperatures indicates a significant reduction of scope for growth, due to high maintenance cost. Analysis of temperature dependencies of product formation efficiency and Yatp/glc revealed that the efficiency of energy metabolism approaches its lower limit at 26-31°C. This limit is reflected in the predetermined combination of Yx/glc(true), elemental biomass composition and degree of reduction of the growth substrate. Approaching the limit implies a reduction of the safety margin of metabolic efficiency. We hypothesize that a temperature increase above Topt (e.g. >31°C) triggers both an increment in mglc and suppression of µmax, which together contribute to an upshift of Yatp/glc from the lower limit and thus compensate for the loss of the safety margin. This trade-off allows adding 10 more degrees to Topt and extends the thermal window up to 40°C, sustaining survival and reproduction in supraoptimal temperatures. Deeper understanding of the limits of thermal tolerance can be practically exploited in biotechnological applications.

'Domino' systems biology and the 'A' of ATP.

We develop a strategic 'domino' approach that starts with one key feature of cell function and the main process providing for it, and then adds additional processes and components only as necessary to explain provoked experimental observations. The approach is here applied to the energy metabolism of yeast in a glucose limited chemostat, subjected to a sudden increase in glucose. The puzzles addressed include (i) the lack of increase in adenosine triphosphate (ATP) upon glucose addition, (ii) the lack of increase in adenosine diphosphate (ADP) when ATP is hydrolyzed, and (iii) the rapid disappearance of the 'A' (adenine) moiety of ATP. Neither the incorporation of nucleotides into new biomass, nor steady de novo synthesis of adenosine monophosphate (AMP) explains. Cycling of the 'A' moiety accelerates when the cell's energy state is endangered, another essential domino among the seven required for understanding of the experimental observations. This new domino analysis shows how strategic experimental design and observations in tandem with theory and modeling may identify and resolve important paradoxes. It also highlights the hitherto unexpected role of the 'A' component of ATP.

Simplified absolute metabolite quantification by gas chromatography-isotope dilution mass spectrometry on the basis of commercially available source material.

In the field of metabolomics, GC-MS has rather established itself as a tool for semi-quantitative strategies like metabolic fingerprinting or metabolic profiling. Absolute quantification of intra- or extracellular metabolites is nowadays mostly accomplished by application of diverse LC-MS techniques. Only few groups have so far adopted GC-MS technology for this exceptionally challenging task. Besides numerous and deeply investigated problems related to sample generation, the pronounced matrix effects in biological samples have led to the almost mandatory application of isotope dilution mass spectrometry (IDMS) for the accurate determination of absolute metabolite concentrations. Nevertheless, access to stable isotope labeled internal standards (ILIS), which are in many cases commercially unavailable, is quite laborious and very expensive. Here we present an improved and simplified gas chromatography-isotope dilution mass spectrometry (GC-IDMS) protocol for the absolute determination of intra- and extracellular metabolite levels. Commercially available (13)C-labeled algal cells were used as a convenient source for the preparation of internal standards. Advantages as well as limitations of the described method are discussed.

Miniaturized device for agitating a high-density yeast suspension that is suitable for in vivo nuclear magnetic resonance applications.

In vivo nuclear magnetic resonance (NMR) monitoring requires a high-density cell suspension, where cell precipitation should be avoided. We have designed a miniaturized cell agitator that fits entirely into an 8-mm NMR probe but that, being mounted into the instrument, is situated outside of the sensitive area. The device consists of two glass tubes connected in a way that, when gas flow is blown through them, creates influx of cell suspension into the device that returns through apertures. This flow creates continuous circular vortex of the cell suspension in the whole sample volume, whereas there are no moving mechanical parts or gas bubbles crossing the instrument's sensitive area. The gas flow controls conditions of the cell suspension and removes volatile waste metabolites.