Modeling anaerobic digestion of microalgae using ADM1

The coupling between a microalgal pond and an anaerobic digester is a promising alternative for sustainable energy production by transforming carbon dioxide into methane using solar energy. In this paper, we demonstrate the ability of the original ADM1 model and a modified version (based on Contois kinetics for the hydrolysis steps) to represent microalgae anaerobic digestion. Simulations were compared to experimental data of an anaerobic digester fed with Chlorella vulgaris. The modified ADM1 fits adequately the data for the considered 140 day experiment encompassing a variety of influent load and flow rates. It turns out to be a reliable predictive tool for optimising the coupling of microalgae with anaerobic digestion processes.     

Conformational temperature-dependent behavior of a histone H2AX: a coarse-grained Monte Carlo approach via knowledge-based interaction potentials

Histone proteins are not only important due to their vital role in cellular processes such as DNA compaction, replication and repair but also show intriguing structural properties that might be exploited for bioengineering purposes such as the development of nano-materials. Based on their biological and technological implications, it is interesting to investigate the structural properties of proteins as a function of temperature. In this work, we study the spatial response dynamics of the histone H2AX, consisting of 143 residues, by a coarse-grained bond fluctuating model for a broad range of normalized temperatures. A knowledge-based interaction matrix is used as input for the residue-residue Lennard-Jones potential.We find a variety of equilibrium structures including global globular configurations at low normalized temperature (T* = 0.014), combination of segmental globules and elongated chains (T* = 0.016,0.017), predominantly elongated chains (T* = 0.019,0.020), as well as universal SAW conformations at high normalized temperature (T* ≥ 0.023). The radius of gyration of the protein exhibits a non-monotonic temperature dependence with a maximum at a characteristic temperature (T(c)* = 0.019) where a crossover occurs from a positive (stretching at T* ≤ T(c)*) to negative (contraction at T* ≥ T(c)*) thermal response on increasing T*.     

A method for introduction of unmarked mutations in the genome of Paracoccus denitrificans: construction of strains with multiple mutations in the genes encoding periplasmic cytochromes c550, c551i, and c553i.

A new suicide vector, pRVS1, was constructed to facilitate the site-directed introduction of unmarked mutations in the chromosome of Paracoccus denitrificans. The vector was derived from suicide vector pGRPd1, which was equipped with the lacZ gene encoding beta-galactosidase. The reporter gene was found to be a successful screening marker for the discrimination between plasmid integrant strains and mutant strains which had lost the plasmid after homologous recombination. Suicide vectors pGRPd1 and pRVS1 were used in gene replacement techniques for the construction of mutant strains with multiple mutations in the cycA, moxG, and cycB genes encoding the periplasmic cytochromes c550, c551i, and c553i, respectively. Southern analyses of the DNA and protein analyses of the resultant single, double, and triple mutant strains confirmed the correctness of the mutations. The wild type and mutant strains were all able to grow on succinate and choline chloride. In addition, all strains grew on methylamine and displayed wild-type levels of methylamine dehydrogenase activities. cycA mutant strains, however, showed a decreased maximum specific growth rate on the methylamine substrate. The wild-type strain, cycA and cycB mutant strains, and the cycA cycB double mutant strain were able to grow on methanol and showed wild-type levels of methanol dehydrogenase activities. moxG mutant strains failed to grow on methanol and had low levels of methanol dehydrogenase activities. The maximum specific growth rate of the cycA mutant strain on methanol was comparable with that of the wild-type strain. The data indicate the involvement of the soluble cytochromes c in clearly defined electron transport routes.     

pCloDF13-encoded bacteriocin release proteins with shortened carboxyl-terminal segments are lipid modified and processed and function in release of cloacin DF13 and apparent host cell lysis.

By oligonucleotide-directed mutagenesis, stop codon mutations were introduced at various sites in the pCloDF13-derived bacteriocin release protein (BRP) structural gene. The expression, lipid modification (incorporation of [3H]palmitate), and processing (in the presence and absence of globomycin) of the various carboxyl-terminal shortened BRPs were analyzed by a special electrophoresis system and immunoblotting with an antiserum raised against a synthetic BRP peptide, and their functioning with respect to release of cloacin DF13, lethality, and apparent host cell lysis were studied in Sup-, supF, and supP strains of Escherichia coli. All mutant BRPs were stably expressed, lipid modified, and processed by signal peptidase II, albeit with different efficiencies. The BRP signal peptide appeared to be extremely stable and accumulated in induced cells. Full induction of the mutant BRPs, including the shortest containing only 4 amino acid residues of the mature polypeptide, resulted in phospholipase A-dependent and Mg2+-suppressible apparent cell lysis. The extent of this lysis varied with the mutant BRP used. Induction of all mutant BRPs also prevented colony formation, which appeared to be phospholipase A independent. One shortened BRP, containing 20 amino acid residues of the mature polypeptide, was still able to bring about the release of cloacin DF13. The results indicated that the 8-amino-acid carboxyl-terminal segment of the BRP contains a strong antigenic determinant and that a small segment between amino acid residues 17 and 21, located in the carboxyl-terminal half of the BRP, is important for release of cloacin DF13. Either the stable signal peptide or the acylated amino-terminal BRP fragments (or both) are involved in host cell lysis and lethality.     

BIOSYNTHESIS OF S-ADENOSYL-L-METHIONINE IN THE RAT PINEAL GLAND

This investigation provides direct evidence that rat pineal gland contains ATP:L-methionine S-adenosyltransferase. The specific activity of the enzyme is about 25 times greater than that of hydroxyindole-O-methyltransferase, the unique marker enzyme in this tissue. The major product of the reaction catalyzed by ATP:L-methionine S-adenosyltransferase has been identified as S-adenosyl-methionine which can function as a methyl group donor for the formation of melatonin. The specific activity of pineal ATP:L-methionine S-adenosyltransferase is at least eight times greater than that of brain, and one-half that of the liver enzyme.     

Fibroblast growth factor signalling and regional specification of the pharyngeal ectoderm

Branchial arch development involves dynamic interactions between neural crest cells as well as ectodermal, endodermal and mesodermal cell populations. Despite their importance and evolutionary conservation, the intercellular interactions guiding the early development of the branchial arches are still poorly understood. We have here studied fibroblast growth factor (FGF) signalling in early pharyngeal development. In mice homozygous for a hypomorphic allele of Fgfr1, neural crest cells migrating from the hindbrain mostly fail to enter the second branchial arch. This defect is non-cell-autonomous suggesting that Fgfr1 provides a permissive environment for neural crest cell migration. Here we demonstrate localized down-regulation of the expression of the FGF responsive gene, Sprouty1 in the epithelium covering the presumptive second branchial arch of hypomorphic Fgfr1 mutants. This appears to result in a failure to establish an ectodermal signalling center expressing Fgf3 and Fgf15. We also studied differentiation of the ectoderm in the second branchial arch region. Development of the geniculate placode as well as the VIIth cranial ganglion is affected in Fgfr1 hypomorphs. Our results suggest that Fgfr1 is important for localized signalling in the pharyngeal ectoderm and consequently for normal tissue interactions in the developing second branchial arch.     

Dynamics of intracellular metabolites of glycolysis and TCA cycle during cell-cycle-related oscillation in Saccharomyces cerevisiae

In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycolysis and TCA cycle during autonomous, cell-cycle synchronized oscillations in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. This study complements previously reported oscillations in carbon dioxide production rate, intracellular concentrations of trehalose and various free amino acids, and extracellular acetate and pyruvate in the same culture. Of the glycolytic intermediates, fructose 1,6-bisphosphate, 2- and 3-phosphoglycerate, and phosphoenolpyruvate show the most pronounced oscillatory behavior, the latter three compounds oscillating out of phase with the former. This agrees with previously observed metabolic control by phosphofructokinase and pyruvate kinase. Although individually not clearly oscillating, several intermediates of the TCA cycle, i.e., alpha-ketoglutarate, succinate, fumarate, and malate, exhibited increasing concentration during the cell cycle phase with high carbon flux through glycolysis and TCA cycle. The average mass action ratios of beta-phosphoglucomutase and fumarase agreed well with previously determined in vitro equilibrium constants. Minor differences resulted for phosphoglucose isomerase and enolase. Together with the observed close correlation of the pool sizes of the involved metabolites, this might indicate that, in vivo, these reactions are operating close to equilibrium, whereby care must be taken due to possible differences between in vivo and in vitro conditions. Combining the data with previously determined intracellular amino acid levels from the same culture, a few clear correlations between catabolism and anabolism could be identified: phosphoglycerate/serine and alpha-ketoglutarate/lysine exhibited correlated oscillatory behavior, albeit with different phase shifts. Oscillations in intracellular amino acids might therefore be, at least partly, following oscillations of their anabolic precursors.