Processing of the electroencephalogram in cardiac surgery

The objective of this investigation was to find a method to automatically detect several types of abnormal EEG patterns which occurred during cardiac surgery. The EEGs were analyzed by means of several EEG processing and pattern recognition methods. It was found that a good classification into normal and abnormal EEG patterns was generally obtained if only two EEG features were used. The results of this investigation are of importance for implementing into a computer-based monitoring system for use during cardiac surgery.     

Quantitative Physiology of Saccharomyces cerevisiae at Near-Zero Specific Growth Rates

Growth at near-zero specific growth rates is a largely unexplored area of yeast physiology. To investigate the physiology of Saccharomyces cerevisiae under these conditions, the effluent removal pipe of anaerobic, glucose-limited chemostat culture (dilution rate, 0.025 h⁻¹) was fitted with a 0.22-μm-pore-size polypropylene filter unit. This setup enabled prolonged cultivation with complete cell retention. After 22 days of cultivation, specific growth rates had decreased below 0.001 h⁻¹ (doubling time of >700 h). Over this period, viability of the retentostat cultures decreased to ca. 80%. The viable biomass concentration in the retentostats could be accurately predicted by a maintenance coefficient of 0.50 mmol of glucose g⁻¹ of biomass h⁻¹ calculated from anaerobic, glucose-limited chemostat cultures grown at dilution rates of 0.025 to 0.20 h⁻¹. This indicated that, in contrast to the situation in several prokaryotes, maintenance energy requirements in S. cerevisiae do not substantially change at near-zero specific growth rates. After 22 days of retentostat cultivation, glucose metabolism was predominantly geared toward alcoholic fermentation to meet maintenance energy requirements. The strict correlation between glycerol production and biomass formation observed at higher specific growth rates was not maintained at the near-zero growth rates reached in the retentostat cultures. In addition to glycerol, the organic acids acetate, d-lactate, and succinate were produced at low rates during prolonged retentostat cultivation. This study identifies robustness and by-product formation as key issues in attempts to uncouple growth and product formation in S. cerevisiae.Laboratory studies on microorganisms are often performed in batch cultures. During the initial phase of batch cultivation, all nutrients are usually present in excess. As a consequence, the initial specific growth rate, μ, of the microorganism in such cultures equals the maximum specific growth rate, μ_max. In natural environments, the specific growth rate of microorganisms is likely to be constrained by the limited availability of one or more growth-limiting nutrients, resulting in specific growth rates far below μ_max (8, 24). In chemostat cultures fed with a medium containing a single growth-limiting nutrient, the dilution rate determines the specific growth rate. Chemostat cultivation therefore offers the possibility to study microbial physiology at carefully controlled, submaximal specific growth rates and to investigate the effect of specific growth rate on cellular physiology (20). Chemostat cultivation of the yeast Saccharomyces cerevisiae has demonstrated strong effects of specific growth rate on biomass composition (26, 51), product formation (5, 37), and cell size (23). Moreover, during energy-limited growth at low specific growth rates, a relatively large fraction of the energy substrate has to be dissimilated for maintenance-related processes such as maintenance of chemi-osmotic gradients and turnover of cellular components (34). Not surprisingly, recent genome-wide studies have shown strong effects of specific growth rate on levels of mRNAs and proteins (9, 14, 38).In chemostat studies on S. cerevisiae, the steady-state specific growth rate is usually between 0.03 h⁻¹ and 0.40 h⁻¹. While this range is relevant for many industrial applications, there are several incentives to study growth of this yeast at even lower specific growth rates. In many natural environments, growth at a μ of 0.03 h⁻¹, corresponding to a doubling time of 23.1 h, probably still represents extremely fast growth. Furthermore, in industrial applications, S. cerevisiae and other microorganisms can be considered as self-replicating catalysts, and, unless biomass is the desired product, growth can be considered as undesirable by-product formation leading to nonproductive substrate consumption. This problem is further augmented when the excess yeast biomass cannot be valorized because it is genetically modified or has been used for the production of compounds that are not compatible with use as, for example, cattle feed. A third incentive for exploring the physiology of S. cerevisiae at near-zero growth rates is related to the increasing interest in this yeast as a systems biology model for human cells (16, 27, 33). At near-zero growth rates, the age of individual yeast cells becomes much higher than can be achieved in conventional batch or chemostat cultures. Studies on extremely slow growth of S. cerevisiae under defined conditions may therefore provide an interesting model for ageing of human cells.Retentostat cultivation, first proposed by Herbert (18), is a modification of chemostat cultivation that has been specifically designed to study microbial physiology at near-zero specific growth rates. In a retentostat, sometimes referred to as recycling fermentor or recyclostat, the growth-limiting energy substrate is fed at a constant rate, and biomass is retained in the fermentor by an internal filter probe connected to the effluent line or by an external filter module. Prolonged retentostat cultivation should, in theory, result in a situation where the specific growth rate becomes zero and where the specific rate of substrate consumption equals the maintenance energy requirement. This situation is fundamentally different from starvation, which involves deterioration of physiological processes, and from resting states typified by spores, which have little or no metabolic activity. Retentostat cultivation has been applied to several bacterial systems including Escherichia coli (11), Paracoccus denitrificans, and Bacillus licheniformis (49) and the autotrophs Nitrosomonas europaea and Nitrobacter winogradskyi (46, 47). These studies demonstrated that the physiology of these prokaryotes at extremely low specific growth rates could not be accurately predicted by a simple extrapolation of results obtained at higher specific growth rates. In particular, near-zero specific growth rates coincided with increased levels of ppGpp (2), which induces the stringent response, a regulatory program that diverts cellular resources from growth to amino acid biosynthesis (10, 21). Furthermore, it was concluded that extremely slow growth led to a reduction of the maintenance energy requirement of prokaryotes. A recent quantitative analysis on cell retention cultures of S. cerevisiae was performed under severely nitrogen-limited growth conditions and used incomplete cell retention (7), which precluded a quantitative comparison with maintenance energy requirements calculated from energy-limited chemostat cultures.The goal of the present study was to quantitatively analyze the physiology of S. cerevisiae at extremely low specific growth rates in glucose-limited retentostat cultures. To this end, an internal filter probe was introduced in the effluent line of standard laboratory chemostat fermentors and used in long-term cultivation runs with complete cell retention. Anaerobic conditions were chosen to facilitate quantification of catabolic fluxes and growth energetics.     

Inhibition of type 2A secretory phospholipase A2 reduces death of cardiomyocytes in acute myocardial infarction

During acute myocardial infarction (AMI), ischemia leads to necrotic areas surrounded by border zones of reversibly damaged cardiomyocytes, showing membrane flip-flop. During reperfusion type IIA secretory phopholipase A₂ (sPLA₂-IIA) induces direct cell-toxicity and facilitates binding of other inflammatory mediators on these cardiomyocytes. Therefore, we hypothesized that the specific sPLA₂-IIA-inhibitor PX-18 would reduce cardiomyocyte death and infarct size in vivo. Wistar rats were treated with PX-18 starting minutes after reperfusion, and at day 1 and 2 post AMI. After 28 days hearts were analyzed. Furthermore, the effect of PX-18 on membrane flip-flop and apoptosis was investigated in vitro. PX-18 significantly inhibited sPLA₂-IIA activity and reduced infarct size (reduction 73 ± 9%, P < 0.05), compared to the vehicle-treated group, without impairing wound healing. In vitro, PX-18 significantly reduced reversible membrane flip-flop and apoptosis in cardiomyocytes. However, no sPLA₂-IIA activity could be detected, suggesting that PX-18 also exerted a protective effect independent of sPLA₂-IIA. In conclusion, PX-18 is a potent therapeutic to reduce infarct size by inhibiting sPLA₂-IIA, and possibly also by inhibiting apoptosis of cardiomyocytes in a sPLA₂-IIA independent manner. A. van Dijk and P. A. J. Krijnen have contributed equally to the study.     

Key Process Conditions for Production of C4 Dicarboxylic Acids in Bioreactor Batch Cultures of an Engineered Saccharomyces cerevisiae Strain

A recent effort to improve malic acid production by Saccharomyces cerevisiae by means of metabolic engineering resulted in a strain that produced up to 59 g liter⁻¹ of malate at a yield of 0.42 mol (mol glucose)⁻¹ in calcium carbonate-buffered shake flask cultures. With shake flasks, process parameters that are important for scaling up this process cannot be controlled independently. In this study, growth and product formation by the engineered strain were studied in bioreactors in order to separately analyze the effects of pH, calcium, and carbon dioxide and oxygen availability. A near-neutral pH, which in shake flasks was achieved by adding CaCO₃, was required for efficient C₄ dicarboxylic acid production. Increased calcium concentrations, a side effect of CaCO₃ dissolution, had a small positive effect on malate formation. Carbon dioxide enrichment of the sparging gas (up to 15% [vol/vol]) improved production of both malate and succinate. At higher concentrations, succinate titers further increased, reaching 0.29 mol (mol glucose)⁻¹, whereas malate formation strongly decreased. Although fully aerobic conditions could be achieved, it was found that moderate oxygen limitation benefitted malate production. In conclusion, malic acid production with the engineered S. cerevisiae strain could be successfully transferred from shake flasks to 1-liter batch bioreactors by simultaneous optimization of four process parameters (pH and concentrations of CO₂, calcium, and O₂). Under optimized conditions, a malate yield of 0.48 ± 0.01 mol (mol glucose)⁻¹ was obtained in bioreactors, a 19% increase over yields in shake flask experiments.In recent years, biologically produced 1,4-dicarboxylic acids (succinate, malate, and fumarate) have attracted great interest as more sustainable replacements for oil-derived commodity chemicals, such as maleic anhydride (50). Malate is currently mainly produced via petrochemical routes for use in food and beverages (18). Development of a biotechnological production process started in the early 1960s with the investigation of the natural malate producer Aspergillus flavus (2). Although process improvements eventually resulted in high product yields and productivities (6), the potential production of aflatoxins (20) prevented the use of this filamentous fungus in industry. Other investigated natural malate-producing fungi (listed in reference 51) produced insufficient malate for industrial use. With the rational design options of metabolic engineering, microorganisms that do not naturally produce large amounts of malic acid may also be considered as production platforms. Wild-type Saccharomyces cerevisiae strains produce little if any malate but would be an interesting starting point for the construction of an efficient malate producer. This yeast has a relatively high tolerance to organic acids and low pH, and due to its role as a model organism in research, a well-developed metabolic engineering toolbox is available. In addition, wild-type S. cerevisiae strains have GRAS (Generally Regarded As Safe) status, so that modified strains are more likely to be allowed in the production of food-grade malic acid.One of the main challenges in the development of an organic acid-producing strain of S. cerevisiae has been the elimination of ethanol formation, which in wild-type strains occurs even under aerobic conditions when glucose concentrations are high (45). Deletion of the pyruvate decarboxylase-encoding genes was found to prevent ethanolic fermentation (17). After evolutionary engineering to remove the growth defects usually associated with pyruvate decarboxylase-negative S. cerevisiae strains, a strain was obtained that produced large amounts of pyruvate, a direct precursor to malate, when grown on glucose (42). Subsequent overexpression of the anaplerotic enzyme pyruvate carboxylase, a cytosolically relocalized malate dehydrogenase and a heterologous malate transporter from Schizosaccharomyces pombe led to a strain that produced significant amounts of malate (51). Cultivation in calcium carbonate (CaCO₃)-buffered shake flasks resulted in malate titers of up to 59 g liter⁻¹ at a yield of 0.42 mol (mol glucose)⁻¹.There are many differences between cultivation in shake flasks and cultivation in (laboratory or industrial) bioreactors. As shake flask cultures lack online pH monitoring and control, there is often significant pH variation over time. The pH is of particular importance. If the yeast can be persuaded to produce organic acids at lower pH values, this reduces the need for active neutralization and thereby reduces by-product formation such as gypsum. However, thermodynamic constraints on acid export, as well as increased stress levels from (undissociated) acid and the low pH, often limit the ability of the microorganisms to produce acids at low pH (32, 43). For this reason, the poorly soluble compound CaCO₃ has traditionally been used to maintain a near-neutral pH in malic acid-producing microbial cultures (6, 29, 51). Adding CaCO₃ also gives increased concentrations of bicarbonate (and thereby CO₂), a substrate for pyruvate carboxylase in the carboxylation of pyruvate (a C₃ carbon molecule) to oxaloacetate (C₄ carbon), as well as calcium. Calcium is known to be involved in cellular signaling pathways (22, 26, 33, 46) and to influence pyruvate carboxylase activity (21, 24). Finally, oxygen transfer rates in shake flasks are often poor compared to those in stirred (laboratory) bioreactors. The formation of significant concentrations (25 g liter⁻¹) of glycerol, a well-known redox sink in S. cerevisiae (41), in shake flask cultures of the engineered malate-producing strain (51) was a strong indication of oxygen limitation.Initial experiments in aerobic, pH-controlled bioreactor cultures of the malate- and succinate-producing Saccharomyces cerevisiae strain RWB525 yielded only low concentrations of these C₄ dicarboxylic acids. The goal of the present study was to identify process parameters that explain the different production levels in shake flask and bioreactor cultures. To this end, we analyzed, both separately and in combination, the impact of culture pH and concentrations of calcium, carbon dioxide, and oxygen on the production of malate and succinate.     

Elimination of Glycerol Production in Anaerobic Cultures of a Saccharomyces cerevisiae Strain Engineered To Use Acetic Acid as an Electron Acceptor

In anaerobic cultures of wild-type Saccharomyces cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. Consequently, glycerol is a major by-product during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in industrial biotechnology. The present study investigates the possibility of completely eliminating glycerol production by engineering S. cerevisiae such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Consistent with earlier studies, deletion of the two genes encoding NAD-dependent glycerol-3-phosphate dehydrogenase (GPD1 and GPD2) led to elimination of glycerol production and an inability to grow anaerobically. However, when the E. coli mhpF gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10; acetaldehyde + NAD⁺ + coenzyme A ↔ acetyl coenzyme A + NADH + H⁺), was expressed in the gpd1Δ gpd2Δ strain, anaerobic growth was restored by supplementation with 2.0 g liter⁻¹ acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production, and partially convert acetate, which is a well-known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address the kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g., osmotolerance).Bioethanol production by Saccharomyces cerevisiae is currently, by volume, the single largest fermentation process in industrial biotechnology. A global research effort is under way to expand the substrate range of S. cerevisiae to include lignocellulosic hydrolysates of nonfood feedstocks (e.g., energy crops and agricultural residues) and to increase productivity, robustness, and product yield (for reviews see references 20 and 35). A major challenge relating to the stoichiometry of yeast-based ethanol production is that substantial amounts of glycerol are invariably formed as a by-product (24). It has been estimated that, in typical industrial ethanol processes, up to 4% of the sugar feedstock is converted into glycerol (24). Although glycerol also serves as a compatible solute at high extracellular osmolarity (10), glycerol production under anaerobic conditions is primarily linked to redox metabolism (34).During anaerobic growth of S. cerevisiae, sugar dissimilation occurs via alcoholic fermentation. In this process, the NADH formed in the glycolytic glyceraldehyde-3-phosphate dehydrogenase reaction is reoxidized by converting acetaldehyde, formed by decarboxylation of pyruvate to ethanol via NAD⁺-dependent alcohol dehydrogenase. The fixed stoichiometry of this redox-neutral dissimilatory pathway causes problems when a net reduction of NAD⁺ to NADH occurs elsewhere in the metabolism. Such a net production of NADH occurs in assimilation when yeast biomass is synthesized from glucose and ammonia (34). Under anaerobic conditions, NADH reoxidation in S. cerevisiae is strictly dependent on reduction of sugar to glycerol (34). Glycerol formation is initiated by reduction of the glycolytic intermediate dihydroxyacetone phosphate to glycerol-3-phosphate, a reaction catalyzed by NAD⁺-dependent glycerol-3-phosphate dehydrogenase. Subsequently, the glycerol-3-phosphate formed in this reaction is hydrolyzed by glycerol-3-phosphatase to yield glycerol and inorganic phosphate.The importance of glycerol production for fermentative growth of yeasts was already observed in the 1960s during studies of non-Saccharomyces yeasts that exhibit a so-called “Custers effect.” In such yeast species, which are naturally unable to produce glycerol, fermentative growth on glucose is possible only in the presence of an external electron acceptor that can be reduced via an NADH-dependent reaction (e.g., the reduction of acetoin to butanediol via NAD⁺-dependent butanediol dehydrogenase) (29). It was later shown that gpd1Δ gpd2Δ strains of S. cerevisiae, which are also unable to produce glycerol, are similarly unable to grow under anaerobic conditions unless provided with acetoin as an external electron acceptor (8).In view of its large economic significance, several metabolic engineering strategies have been explored to reduce or eliminate glycerol production in anaerobic cultures of S. cerevisiae. Nissen et al. (25) changed the cofactor specificity of glutamate dehydrogenase, the major ammonia-fixing enzyme of S. cerevisiae, thereby increasing NADH consumption in biosynthesis. This approach significantly reduced glycerol production in anaerobic cultures grown with ammonia as the nitrogen source. Attempts to further reduce glycerol production by expression of a heterologous transhydrogenase, with the aim to convert NADH and NADP⁺ into NAD⁺ and NADPH, were unsuccessful (24) because intracellular concentrations of these pyridine nucleotide cofactor couples favor the reverse reaction (23).The goal of the present study was to investigate whether the engineering of a linear pathway for the NADH-dependent reduction of acetic acid to ethanol can replace glycerol formation as a redox sink in anaerobic, glucose-grown cultures of S. cerevisiae and thus provide a stoichiometric basis for elimination of glycerol production during industrial ethanol production. Significant amounts of acetic acid are released upon hydrolysis of lignocellulosic biomass, and, in fact, acetic acid is studied as an inhibitor of yeast metabolism in lignocellulosic hydrolysates (5, 7, 26). The S. cerevisiae genome already contains genes encoding acetyl coenzyme A (acetyl-CoA) synthetase (32) and NAD⁺-dependent alcohol dehydrogenases (ADH1-5 [12]). To complete the linear pathway for acetic acid reduction, we expressed an NAD⁺-dependent, acetylating acetaldehyde dehydrogenase (EC 1.2.1.10) from Escherichia coli into a gpd1Δ gpd2Δ strain of S. cerevisiae. This enzyme, encoded by the E. coli mhpF gene (15), catalyzes the reaction acetaldehyde + NAD⁺ + coenzyme A ↔ acetyl coenzyme A + NADH + H⁺. Growth and product formation of the engineered strain were then compared in the presence and absence of acetic acid and compared to those of a congenic reference strain.     

Soil Type-Dependent Responses to Phenanthrene as Revealed by Determining the Diversity and Abundance of Polycyclic Aromatic Hydrocarbon Ring-Hydroxylating Dioxygenase Genes by Using a Novel PCR Detection System

A novel PCR primer system that targets a wide range of polycyclic aromatic hydrocarbon ring-hydroxylating dioxygenase (PAH-RHD_α) genes of both Gram-positive and Gram-negative bacteria was developed and used to study their abundance and diversity in two different soils in response to phenanthrene spiking. The specificities and target ranges of the primers predicted in silico were confirmed experimentally by cloning and sequencing of PAH-RHD_α gene amplicons from soil DNA. Cloning and sequencing showed the dominance of phnAc genes in the contaminated Luvisol. In contrast, high diversity of PAH-RHD_α genes of Gram-positive and Gram-negative bacteria was observed in the phenanthrene-spiked Cambisol. Quantitative real-time PCR based on the same primers revealed that 63 days after phenanthrene spiking, PAH-RHD_α genes were 1 order of magnitude more abundant in the Luvisol than in the Cambisol, while they were not detected in both control soils. In conclusion, sequence analysis of the amplicons obtained confirmed the specificity of the novel primer system and revealed a soil type-dependent response of PAH-RHD_α gene-carrying soil bacteria to phenanthrene spiking.Polycyclic aromatic hydrocarbons (PAHs) are hydrophobic compounds composed of two or more fused aromatic rings. Although PAHs are ubiquitous in the environment (from natural oil seeps, brush fires, and plant derivatives), anthropogenic activities, such as disposal of coal-processing waste, mining accidents, petroleum wastes, and vehicle exhaust, have drastically increased their occurrence in the environment. The fate of PAHs in soil is of great interest due to their potential for bioaccumulation, persistence, transport, and toxicity. Microbe-driven aerobic degradation of PAHs is well documented (15-17). The diversity of PAH-degrading genes in soils is assumed to be huge, but the extent of diversity and how it is influenced by different soil types or their history and type of pollution are not yet fully explored. Knowledge of the genes coding for dioxygenase enzymes that catalyze the primary step of PAH degradation by incorporating molecular oxygen into the aromatic nucleus is an essential prerequisite to unraveling the contributions of microbial population networks to transformation, assimilation, and degradation of organic chemicals in soil. Recently, the complete genomes of several PAH-degrading bacteria became available and allowed new insights into degradative pathways (6, 18, 36). Organic pollutants also serve as nutrients for those microbes that have the appropriate genetic makeup to utilize them, resulting in their increased metabolic activity and abundance (4, 14). In the last decade, impressive progress was seen in techniques that allow cultivation-independent analysis of microbial communities and thus overcome the most severe limitations in studying microbial communities in natural habitats, namely, that only a rather small portion of microbes are accessible to standard cultivation conditions (1, 29). For more than a decade, cultivation-independent approaches have also been employed to unravel the responses of microbial communities in soils and sediments to PAH pollution. In all these studies, PCR amplification of PAH-degrading gene fragments from nucleic acids directly extracted from environmental samples was used to explore the abundance and diversity of PAH ring-hydroxylating dioxygenase (PAH-RHD_α) genes (4, 8, 9, 13, 14, 22, 34, 37). Despite the known biases of PCR amplification from mixed templates, these techniques allow highly sensitive and specific detection even from minute amounts of nucleic acids. In order to select suitable primer systems, previously published primer systems were analyzed for their ranges of target sequences. The existing primer systems were found to have limitations, as they often target only a rather narrow range of sequences, e.g., nahAc- or phnAc-type sequences (21, 34) or only PAH-RHD_α genes from Gram-negative bacteria (3, 13). In other studies, two-primer systems were used to target PAH-RHD_α genes of both Gram-positive and Gram-negative bacteria (4, 37). Only one primer system targeting the Rieske gene fragment was described that amplified a small fragment from PAH-RHD_α genes from both Gram-negative and Gram-positive bacteria (24). However, the amplicon size was only 78 bp and the primer might also target genes coding for dioxygenases that attack nonpolar aromatic compounds, such as benzene, toluene, and xylene. Therefore, this work aimed to design an improved primer system that targets PAH-RHD_α genes from both Gram-positive and Gram-negative bacteria and provides larger amplicon sizes. The novel primer system was tested in silico and validated by sequencing cloned PAH-RHD_α genes amplified from total-community (TC) DNA and was used in endpoint and quantitative real-time PCR (qPCR) formats. The primer system was also applied to study the responses of soil microbial communities in two different soils (a Cambisol and a Luvisol representing typical arable soils in Central Europe with different texture compositions) to artificial phenanthrene pollution.