Application of response surface methodology in medium optimization for spore production of <Emphasis Type="Italic">Coniothyrium minitans</Emphasis> in solid-state fermentation

Summary Spore production of Coniothyrium minitans was optimized by using response surface methodology (RSM), which is a powerful mathematical approach widely applied in the optimization of fermentation process. In the first step of optimization, with Plackett–Burman design, soluble starch, urea and KH²PO⁴ were found to be the important factors affecting C. minitans spore production significantly. In the second step, a 2³ full factorial central composite design and RSM were applied to determine the optimal concentration of each significant variable. A second-order polynomial was determined by the multiple regression analysis of the experimental data. The optimum values for the critical components for the maximum were obtained as follows: soluble starch 0.643 (36.43 g. l⁻¹), urea −0.544 (3.91 g l⁻¹) and KH²PO⁴ 0.049 (1.02 g l⁻¹) with a predicted value of maximum spore production of 9.94 × 10⁹ spores/g IDM. Under the optimal conditions, the practical spore production was 1.04 × 10¹⁰ spores/g IDM. The determination coefficient (R²) was 0.923, which ensure an adequate credibility of the model.     

Heterokaryon formation and parasexual recombination between vegetatively incompatible lineages in a population of the chestnut blight fungus, Cryphonectria parasitica

Heterokaryosis was recently reported in the chestnut blight fungus, Cryphonectria parasitica, in which individuals contain nuclei that are isogenic except at the mating-type locus (MAT). MAT heterokaryons were found in several natural populations, including a putatively clonal population in West Salem, Wisconsin, providing an opportunity to address the question of how heterokaryons arise. We represented relationships among RFLP fingerprint haplotypes as networks in which loop formation is considered evidence of recombination. From 1990 to 1995, this population was clonal, as indicated by a simple haplotype network without loops, and the correlation of vegetative compatibility (vc) types and mating types with haplotype lineages. By 1999, we observed loops in the haplotype network involving isolates of two vc types (WS-2 and WS-3). Isolates with haplotypes in the loops were either MAT heterokaryons, carried the opposite mating type from other isolates of the same vc type, and/or had two alleles at two or more codominant SCAR (sequence-characterized amplified region) loci. Segregation of markers and recombination were evident among single-spore isolates from one heterokaryon; these single-spore isolates had novel fingerprint haplotypes, also within the loops. In contrast, vc type WS-1, which comprises 85% of the population, was represented by a simple network with no loops, indicating a clonal lineage varying only by mutation. Almost all isolates of WS-1 had the same mating type; the exceptions were five isolates that were MAT heterokaryons. These results are consistent with the hypothesis that heterokaryons formed between vegetatively incompatible individuals, and recombination occurred by a parasexual process.     

Optimization of conditions for the enzymatic hydrolysis of phytoestrogen conjugates in urine and plasma

Optimal pH, temperature, and concentration of enzyme conditions for the rate of hydrolysis of five isoflavone conjugates (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone) from two biological matrices (urine and plasma) were studied using beta-glucuronidase from Helix pomatia. In addition, the use of mixtures of beta-glucuronidase and sulfatase enzymes from different sources was investigated to find enzyme preparations that contained lower amounts of naturally present phytoestrogens. Quantification of aglycones spiked with (13)C(3)-labeled internal standards was carried out by LC-MS/MS. In urine, all of the phytoestrogen conjugates hydrolyzed within 2h under standard hydrolysis conditions (24mul H. pomatia, pH 5, 37 degrees C). Hydrolysis rates were improved at 45 degrees C and by doubling the enzyme concentration and may be used to further reduce hydrolysis times down to 100min. In plasma, a 16-h hydrolysis was required to ensure complete hydrolysis of all conjugates. As with urine, the use of increased temperature or increased enzyme concentration reduced hydrolysis times for most analytes. However, the rate of hydrolysis in plasma was significantly slower than that in urine for all analytes except enterodiol, for which the reverse was true. Neither increased temperature nor increased enzyme concentration increased the rate of hydrolysis of enterolactone. Hydrolysis at pH 6 proved to be detrimental to hydrolysis of phytoestrogen conjugates, especially those in plasma. Other enzyme preparations from different sources, such as beta-glucuronidase from Escherichia coli, were found to contain lower amounts of contaminating phytoestrogens and showed increased enzyme activity for isoflavones, but lower activity for lignans, when used with other sulfatase enzymes. In addition, this involved complicating the analytical procedure through using mixtures of enzymes. Therefore, the use of beta-glucuronidase from H. pomatia combined with an enzyme "blank" to correct for phytoestrogen contamination was shown to be a suitable method for hydrolysis of phytoestrogens.     

Plasmodium falciparum: discovery of peroxidase active organelles

Staining with 3,3' diaminobenzidine tetrahydrochloride (DAB) is a common method used for the detection of peroxidases. Using this histochemical staining method in conjunction with transmission electron microscopy, we observed oxidation of DAB that was localized to a discrete set of organelles displaying morphological similarity to small (75-90 nm diameter) versions of higher eukaryotic microbodies or peroxisomes. These single membrane bounded organelles were characterized by an asymmetrical matrix capable of oxidizing DAB to an electron dense inclusion. Oxidation of DAB was further found to be dependent upon hydrogen peroxide (H2O2) as a substrate. Given a lack of peroxisomal import proteins and enzymes, it is unlikely that these represent conventional peroxisomes. Rather, they likely represent specialized organelles containing endogenous peroxidase or pseudo-peroxidase activity.     

Modelling gene networks at different organisational levels

Approaches to modelling gene regulation networks can be categorized, according to increasing detail, as network parts lists, network topology models, network control logic models, or dynamic models. We discuss the current state of the art for each of these approaches. There is a gap between the parts list and topology models on one hand, and control logic and dynamic models on the other hand. The first two classes of models have reached a genome-wide scale, while for the other model classes high throughput technologies are yet to make a major impact.     

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.     

Neurogenesis and neuronal communication on micropatterned neurochips

Neural networks are formed by accurate connectivity of neurons and glial cells in the brain. These networks employ a three-dimensional bio-surface that both assigns precise coordinates to cells during development and facilitates their connectivity and functionality throughout life. Using specific topographic and chemical features, we have taken steps towards the development of poly(dimethylsiloxane; PDMS) neurochips that can be used to generate and study synthetic neural networks. These neurochips have micropatterned structures that permit adequate cell positioning and support cell survival. Within days of plating, cells differentiate into neurons displaying excitability and communication, as evidenced by intracellular calcium oscillations and action potentials. The structural and functional capacities of such simple neural networks open up new opportunities to study synaptic communication and plasticity.     

Optimization of human serum albumin production in methylotrophic yeast Pichia pastoris by repeated fed-batch fermentation

An optimization method for repeated fed-batch fermentation was established with the aim of improving the recombinant human serum albumin (rHSA) production in Pichia pastoris. A simulation model for fed-batch fermentation was formulated and the optimal methanol-feeding policy calculated by dynamic programming method using five different methanol-feeding periods. The necessary state variables were collected from the calculated results and used for further optimization of repeated fed-batch fermentation. The optimal operation policy was investigated using the pre-collected state variables by estimating the overall profit per total methanol-feeding time. The calculated results indicated that the initial cell mass from the 2nd fed-batch fermentation on should be set at 35 or 40 g and methanol-feeding time at 264 h. In repeated fed-batch fermentation using the optimal operation policy, actual culture volume was in good agreement with the values simulated by model equations, but some discrepancy was observed in rHSA production. Minimum experiments were therefore carried out to re-evaluate rHSA production levels, which were then applied in re-calculations to determine the optimal operation policy. The optimal policy for repeated fed-batch fermentation established in the present study (i.e., 4-times-repeated fed-batch fermentation) achieved a 47% increase in annual rHSA production. Optimization of the culture period also brought about a 28% increase in annual rHSA production even in simple (not repeated) fed-batch fermentation.     

The integrated development of network complexity modulates the diverse evolutionary mutation rates of individual proteins

The rate of evolution-related mutation varies widely among proteins while the unity of the organism implies an integrated evolution of its protein network. Focusing on the yeast interactome, we monitored the structural impact of amino acid substitution on yeast proteins with reported structure. The impact of mutation in creating or deleting structural markers for interactivity varies across proteins and modulates the evolutionary rates, yielding a unified kinetic law of accumulation of connectivities consistent with an integrated evolution of the interactome.     

Improvement of xylanase production in solid-state fermentation by alkali tolerant Aspergillus versicolor MKU3

AIMS: To optimize the media components for xylanase production by Aspergillus versicolor MKU3 in solid-state fermentation (SSF). METHODS AND RESULTS: Medium optimization was carried out using De Moe's fractional factorial design with seven components. Maximum production of xylanase 3249.9 U g(-1) was obtained in SSF with an optimized medium containing (g l(-1)): NaNO(3), 20; K(2)HPO(4), 20; MgSO(4), 10; FeSO(4), 0.001; KCl, 1; peptone, 10 and yeast extract, 10. Four components namely NaNO(3), MgSO(4), peptone and K(2)HPO(4) significantly increased the xylanase production by A. versicolor MKU3. CONCLUSIONS: Fractional factorial design was used to optimize the seven components in the fermentation medium for SSF. The optimized media increased xylanase production by 3.4-fold. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus versicolor MKU3 produced maximum xylanase after two steps of media optimization under alkaline condition. This medium will be significant value for xylanase production in SSF.