The non-steroidal anti-inflammatory drug carprofen negatively impacts new bone formation and antibiotic efficacy in a rat model of orthopaedic-device-related infection.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for pain management during recovery from orthopaedic surgery. NSAID use is associated with increased risk of bone healing complications but it is currently unknown whether NSAIDs increase the risk of developing an orthopaedic-device-related infection (ODRI) and/or affects its response to antibiotic therapy. The present study aimed to determine if administration of the NSAID carprofen [a preferential cyclooxygenase-2 (COX-2) inhibitor] negatively affected Staphylococcus epidermidis (S. epidermidis) bone infection, or its subsequent treatment with antibiotics, in a rodent ODRI model. Sterile or S. epidermidis-contaminated screws (~ 1.5 x 106 CFU) were implanted into the proximal tibia of skeletally mature female Wistar rats, in the absence or presence of daily carprofen administration. A subset of infected animals received antibiotics (rifampicin plus cefazolin) from day 7 to 21, to determine if carprofen affected antibiotic efficacy. Bone changes were monitored using in vivo µCT scanning and histological analysis. The risk of developing an infection with carprofen administration was assessed in separate animals at day 9 using a screw contaminated with 10² CFU S. epidermidis. Quantitative bacteriological analysis assessed bacterial load at euthanasia. In the 28-day antibiotic treatment study, carprofen reduced osteolysis but markedly diminished reparative bone formation, although total bacterial load was not affected at euthanasia. Antibiotic efficacy was negatively affected by carprofen (carprofen: 8/8 infected; control: 2/9 infected). Finally, carprofen increased bacterial load and diminished bone formation following reduced S. epidermidis inoculum (10² CFU) at day 9. This study suggests that NSAIDs with COX-2 selectivity reduce antibiotic efficacy and diminish reparative responses to S. epidermidis ODRI.

[Palliative sedation : Development and consensus of a German language documentation template]. / Palliative Sedierung : Entwicklung und Konsentierung einer deutschsprachigen Dokumentationsvorlage.

BACKGROUND: Palliative sedation (PS) serves as a therapeutic option in cases of otherwise intractable suffering. As the use of sedative and hypnotic medication in many diverse situations is a core competency of anesthesiology, anesthesiologists are confronted with questions of sedative therapy at the end of life in institutions for specialized palliative care, in intensive care units and intermediate care wards. In recent years a number of guidelines have been published internationally but so far no official guidelines exist in Germany. The most recognized document is the European Association for Palliative Care (EAPC) framework on PS. This project aims to develop a German language template for the preparation, application, documentation and evaluation of PS according to the current frameworks, especially the EAPC framework on PS. METHODS: A first draft of the template was generated by the project team using the EAPC framework and individual templates of various institutions, which had been collected during a previous project. Professionals (n = 136) from inpatient and outpatient specialist palliative and hospice care were invited to assess all items of the draft regarding "relevance", "wording" and "feasibility" in an online Delphi survey (Unipark®, Questback, Cologne, Germany). After the second Delphi round an expert panel was asked to reflect the results and generate a final draft. Approval was granted if acceptance exceeded 75% of participants. RESULTS: The 3 rounds of the Delphi process were completed by 64, 46 and 41 participants, respectively. The Delphi process as well as the expert panel led to significant changes of the template. The indications for PS had to be clarified. The significance of documentation of vital parameters, such as oxygen saturation, blood pressure or respiratory rate during PS was intensively discussed. In many teams, predominantly hospice or outpatient palliative care teams, it seems to be difficult to measure these parameters or it is regarded as inappropriate in a palliative care setting. In contrast, the EAPC framework recommends monitoring of vital parameters in cases of intermittent or respite sedation. Finally, a solution was found to support documentation of additional data without the explicit mentioning of specific parameters. After the third Delphi round, all 16 items of the documentation template reached consensus with respect to relevance (82.9-100%), clarity of wording (80.5-100%), and feasibility in practice (78-100%). CONCLUSION: This article provides an empirically based, multiprofessional consented documentation template for PS. Core elements of the documentation of PS are the indications and the decision process towards PS. During the treatment, at least the level of sedation and the symptom burden have to be recorded. The documentation of vital signs during PS remains a highly disputed topic. The presented data suggest that especially in outpatient settings and in hospices measuring and documentation of vital parameters is uncommon and therefore is often regarded as not feasible. This template can help to support the medically and ethically sound use of PS and facilitate research. The template can be accessed at http://www.palliativmedizin.uk-erlangen.de/forschung/downloads/ .

Improving 2-phenylethanol and 6-pentyl-α-pyrone production with fungi by microparticle-enhanced cultivation (MPEC).

Trichoderma atroviride IMI 206040 synthesizes the coconut lactone 6-pentyl-α-pyrone (6-PAP) de novo and Aspergillus niger DSM 821 produces the rose-like flavour compound 2-phenylethanol (2-PE) from the precursor l-phenylalanine. Here, microparticles of different chemical composition and nominal particle diameter in the range 5-250 µm were added to shake-flask cultures of both fungi to investigate the particles' effect on product formation. Maximum 2-PE concentration increased by a factor of 1.3 to 1430 mg/l with the addition of 2% w/v talc (40 µm diameter). Maximum 6-PAP concentration increased by a factor of 2 to 40 mg/l with the addition of 2% w/v iron (II, III) oxide. The influence of ions leaching out of the particles was investigated by cultivating the fungi in leached particle medium. For the first time, the positive effect of the microparticle-enhanced cultivation (MPEC) technique on the microbial production of volatile metabolites, here flavour compounds from submerged fungal cultures, is demonstrated. The effect is strain- and particle-specific.

Hydrogen peroxide in inflammation: messenger, guide, and assassin.

Starting as a model for developmental genetics, embryology, and organogenesis, the zebrafish has become increasingly popular as a model organism for numerous areas of biology and biomedicine over the last decades. Within haematology, this includes studies on blood cell development and function and the intricate regulatory mechanisms within vertebrate immunity. Here, we review recent studies on the immediate mechanisms mounting an inflammatory response by in vivo analyses using the zebrafish. These recently revealed novel roles of the reactive oxygen species hydrogen peroxide that have changed our view on the initiation of a granulocytic inflammatory response.

Exploring metabolic pathways in genome-scale networks via generating flux modes.

MOTIVATION: The reconstruction of metabolic networks at the genome scale has allowed the analysis of metabolic pathways at an unprecedented level of complexity. Elementary flux modes (EFMs) are an appropriate concept for such analysis. However, their number grows in a combinatorial fashion as the size of the metabolic network increases, which renders the application of EFMs approach to large metabolic networks difficult. Novel methods are expected to deal with such complexity. RESULTS: In this article, we present a novel optimization-based method for determining a minimal generating set of EFMs, i.e. a convex basis. We show that a subset of elements of this convex basis can be effectively computed even in large metabolic networks. Our method was applied to examine the structure of pathways producing lysine in Escherichia coli. We obtained a more varied and informative set of pathways in comparison with existing methods. In addition, an alternative pathway to produce lysine was identified using a detour via propionyl-CoA, which shows the predictive power of our novel approach. AVAILABILITY: The source code in C++ is available upon request.

Morphology and rheology in filamentous cultivations.

Because of their metabolic diversity, high production capacity, secretion efficiency, and capability of carrying out posttranslational modifications, filamentous fungi are widely exploited as efficient cell factories in the production of metabolites, bioactive substances, and native or heterologous proteins, respectively. There is, however, a complex relationship between the morphology of these microorganisms, transport phenomena, the viscosity of the cultivation broth, and related productivity. The morphological characteristics vary between freely dispersed mycelia and distinct pellets of aggregated biomass, every growth form having a distinct influence on broth rheology. Hence, the advantages and disadvantages for mycelial or pellet cultivation have to be balanced out carefully. Because of the still inadequate understanding of the morphogenesis of filamentous microorganisms, fungal morphology is often a bottleneck of productivity in industrial production. To obtain an optimized production process, it is of great importance to gain a better understanding of the molecular and cell biology of these microorganisms as well as the relevant approaches in biochemical engineering. In this chapter, morphology and growth of filamentous fungi are described, with special attention given to specific problems as they arise from fungal growth forms; growth and mass transfer in fungal biopellets are discussed as an example. To emphasize the importance of the flow behavior of filamentous cultivation broths, an introduction to rheology is also given, reviewing important rheological models and recent studies concerning rheological parameters. Furthermore, current knowledge on morphology and productivity in relation to the environom is outlined in the last section of this review.

Volume measurement of cryogenic deuterium pellets by Bayesian analysis of single shadowgraphy images.

In situ commissioning of the Blower-gun injector for launching cryogenic deuterium pellets at ASDEX Upgrade tokamak was performed. This injector is designed for high repetitive launch of small pellets for edge localised modes pacing experiments. During the investigation the final injection geometry was simulated with pellets passing to the torus through a 5.5 m long guiding tube. For investigation of pellet quality at launch and after tube passage laser flash camera shadowgraphy diagnostic units before and after the tube were installed. As indicator of pellet quality we adopted the pellet mass represented by the volume of the main remaining pellet fragment. Since only two-dimensional (2D) shadow images were obtained, a reconstruction of the full three-dimensional pellet body had to be performed. For this the image was first converted into a 1-bit version prescribing an exact 2D contour. From this contour the expected value of the volume was calculated by Bayesian analysis taking into account the likely cylindrical shape of the pellet. Under appropriate injection conditions sound pellets with more than half of their nominal mass are detected after acceleration; the passage causes in average an additional loss of about 40% to the launched mass. Analyzing pellets arriving at tube exit allowed for deriving the injector's optimized operational conditions. For these more than 90% of the pellets were arriving with sound quality when operating in the frequency range 5-50 Hz.

Validation of a viral and bacterial inactivation step during the extraction and purification process of porcine collagen.

In the last few years, regulations for biomolecule production, and especially for extraction and purification of animal molecules such as collagen, have been reinforced to ensure the sanitary safety of the materials. To be authorized to market biomaterials based on collagen, manufacturers now have to prove that at least one step of their process is described in guidelines to inactivate prion, viruses, and bacteria. The present study focuses on the inactivation step performed during the extraction and purification of porcine type I atelocollagen. We chose to determine the reduction factor of a 1 M NaOH step on porcine parvovirus and four bacterial strains inactivation. During the extraction step, we deliberately inoculated the collagen suspension with the different microorganisms tested. Then, 1 M NaOH was added to the suspension for 1 hour at 20 degrees C. We demonstrated that this treatment totally inactivated S. aureus, P. aeruginosa, C. albicans and A. niger which are bacterial strains responsible of severe human pathology. The reduction factors reached more than 4 logs for B. cereus spores and 4 logs for the porcine parvovirus. are encouraging as those two microorganisms are known to be very resistant to inactivation.

Cryogenic pellet launcher adapted for controlling of tokamak plasma edge instabilities.

One of the main challenges posed recently on pellet launcher systems in fusion-oriented plasma physics is the control of the plasma edge region. Strong energy bursts ejected from the plasma due to edge localized modes (ELMs) can form a severe threat for in-vessel components but can be mitigated by sufficiently frequent triggering of the underlying instabilities using hydrogen isotope pellet injection. However, pellet injection systems developed mainly for the task of ELM control, keeping the unwanted pellet fueling minimized, are still missing. Here, we report on a novel system developed under the premise of its suitability for control and mitigation of plasma edge instabilities. The system is based on the blower gun principle and is capable of combining high repetition rates up to 143 Hz with low pellet velocities. Thus, the flexibility of the accessible injection geometry can be maximized and the pellet size kept low. As a result the new system allows for an enhancement in the tokamak operation as well as for more sophisticated experiments investigating the underlying physics of the plasma edge instabilities. This article reports on the design of the new system, its main operational characteristics as determined in extensive test bed runs, and also its first test at the tokamak experiment ASDEX Upgrade.

Mechanisms involved in A/J mouse lung tumorigenesis induced by inhalation of an environmental tobacco smoke surrogate.

Lung tumors have been reproducibly induced in A/J mice exposed to a surrogate for experimental environmental tobacco smoke (ETSS) in a 5-mo inhalation period followed by 4 mo without further exposure. In order to increase our mechanistic understanding of this model, male mice were whole-body exposed for 6 h/d, 5 d/wk to ETSS with a particulate matter concentration of 100 mg/m(3). Food restriction regimens were included to model or exceed the ETSS-related impairment of body weight development. Half of the mice were pretreated with a single ip injection of urethane to study the effect of the above treatments on lung tumor development induced by this substance. At 5 mo, the tumor response was statistically the same for all groups of non-pretreated mice; however, the expected urethane-induced lung tumorigenesis was significantly inhibited by approximately 25% by ETSS and food restriction. This inhibition was accompanied by a threefold increase in blood corticosterone as a common stress marker for both ETSS and food restriction. At 9 mo, in mice not pretreated, the lung tumor incidence and multiplicity were significantly increased by twofold in the ETSS group; in the urethane-treated groups, the same high tumor multiplicity was reached regardless of previous treatment. The predominant tumor type in all groups was bronchiolo-alveolar adenoma. There was no induction of a specific K-ras mutation pattern by ETSS exposure. These data suggest a stress-induced inhibition of lung tumorigenesis in this model, explaining the need for the posttreatment period.

Ecology and ecophysiology of tree stems: corticular and wood photosynthesis.

Below the outer peridermal or rhytidomal layers, most stems of woody plants possess greenish tissues. These chlorophyll-containing tissues (the chlorenchymes) within the stems are able to use the stem internal CO2 and the light penetrating the rhytidome to photoassimilate and produce sugars and starch. Although net photosynthetic uptake of CO2 is rarely found, stem internal re-fixation of CO2 in young twigs and branches may compensate for 60-90% of the potential respiratory carbon loss. Isolated chlorenchymal tissues reveal rather high rates of net photosynthesis (being up to 75% of the respective rates for leaf photosynthesis). Corticular photosynthesis is thus thought to be an effective mechanism for recapturing respiratory carbon dioxide before it diffuses out of the stem. Furthermore, chloroplasts of the proper wood or pith fraction also take part in stem internal photosynthesis. Although there has been no strong experimental evidence until now, we suggest that the oxygen evolved during wood or pith photosynthesis may play a decisive role in avoiding/reducing stem internal anaerobiosis.

Metabolic flux analysis using mass spectrometry.

Detailed knowledge on carbon flux distributions is crucial for the understanding and targeted optimization of cellular systems. Analytical methods to identify the topology of metabolic networks and to quantify fluxes through its different pathways are therefore in the core of metabolic engineering. An elegant approach for metabolic flux analysis is provided by tracer experiments. In such studies tracer substrates with stable isotopes such as 13C are applied and the labeling pattern of metabolites is subsequently measured. Detailed flux distributions can be obtained by a combination of tracer experiments and stoichiometric balancing. In recent years, mass spectrometry (MS) has emerged as an interesting method for labeling measurements in metabolic flux analysis and provided valuable insights into the cellular metabolism. The present review provides an overview on current experimental and modeling tools for metabolic flux analysis by MS. The application of MS for flux analysis is illustrated by examples from the literature for various biological systems, including bacteria, fungi, tissue cultures and in vivo studies in humans.

Influence of glucose, fructose and sucrose as carbon sources on kinetics and stoichiometry of lysine production by Corynebacterium glutamicum.

Batch cultivations of l-lysine-producing Corynebacterium glutamicum ATCC 21253 were carried out on the different carbon sources, glucose, sucrose and fructose. The time profiles of substrate and product concentrations were evaluated to compare kinetics and stoichiometry of lysine production. The lysine yield (mol C/mol C) on glucose was 8% higher than on sucrose and 30% higher than on fructose. The highest final biomass concentration of 5.0 g/l was obtained on glucose, whereas fructose and sucrose yielded 20% less biomass. Compared to glucose, fructose resulted in significantly higher respiration rates, a higher substrate uptake rate but a lower lysine production rate during the cultivation process. This was probably due to a higher tricarboxylic cycle activity combined with a lower activity of the pentose phosphate pathway. On sucrose, specific rates and yields differed significantly from those on fructose and glucose. Transport and metabolism of sucrose, therefore, are not a simple superposition of its building blocks, glucose and fructose.

Quantification of intracellular amino acids in batch cultures of Saccharomyces cerevisiae.

The dynamics of intracellular amino acid pools were determined in batch cultures of Saccharomyces cerevisiae. Immediate termination of metabolic activity was found to be necessary for accurate quantification of in vivo concentrations of intracellular amino acids, due to significant changes in most intracellular amino acid pools observed during extraction without an instantaneous stop of the metabolism. The method applied to batch-cultures of S. cerevisiae on glucose revealed complex dynamics in intracellular amino acid pools. The most drastic changes were observed during the diauxic shift and at the entry into the stationary phase. Even during phases of exponential growth on glucose and ethanol, cells showed significant variations in intracellular amino acid concentrations. The method presented can be used to investigate the physiology of yeast cultures, including industrially relevant batch and fed-batch processes.

MALDI-TOF MS for quantification of substrates and products in cultivations of Corynebacterium glutamicum.

The application of MALDI-TOF MS for the quantification of lysine, alanine, and glucose is described. The method is based on using stable isotopes as internal standards and allows fast, sensitive, and reproducible quantification of these compounds. It is demonstrated for aqueous standard solutions with concentrations of the analytes between 10 microM and 100 mM. The mean standard deviations from five replicates each were 4.3% (lysine), 3.7% (alanine), and 3.2% (glucose). In addition, sucrose could be measured by MALDI-TOF MS, but was not quantified due to lack of an internal standard. The method developed can be applied to quantify metabolites in cultivations of C. glutamicum ATCC 21253, without processing of the sample except a 1:5 dilution. Excellent agreement of the data with conventional techniques like HPLC or enzyme assay was found.

Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles.

The microtubule-binding protein tau has been implicated in the pathogenesis of Alzheimer's disease and related disorders. However, the mechanisms underlying tau-mediated neurotoxicity remain unclear. We created a genetic model of tau-related neurodegenerative disease by expressing wild-type and mutant forms of human tau in the fruit fly Drosophila melanogaster. Transgenic flies showed key features of the human disorders: adult onset, progressive neurodegeneration, early death, enhanced toxicity of mutant tau, accumulation of abnormal tau, and relative anatomic selectivity. However, neurodegeneration occurred without the neurofibrillary tangle formation that is seen in human disease and some rodent tauopathy models. This fly model may allow a genetic analysis of the cellular mechanisms underlying tau neurotoxicity.

Modeling and experimental design for metabolic flux analysis of lysine-producing Corynebacteria by mass spectrometry.

Experimental design of (13)C-tracer studies for metabolic flux analysis with mass spectrometric determination of labeling patterns was performed for the central metabolism of Corynebacterium glutamicum comprising various flux scenarios. Ratio measurement of mass isotopomer pools of Corynebacterium products lysine, alanine, and trehalose is sufficient to quantify the flux partitioning ratios (i) between glycolysis and pentose phosphate pathways (Phi(PPP)), (ii) between the split pathways in the lysine biosynthesis (Phi(DH)), (iii) at the pyruvate node (Phi(PC)), and reversibilities of (iv) glucose 6-phosphate isomerase (zeta(PGI)), (v) at the pyruvate node (zeta(PC/PEPCK)), and (vi) of transaldolase and transketolases in the PPP. Weighted sensitivities for flux parameters were derived from partial derivatives to quantitatively evaluate experimental approaches and predict precision for estimated flux parameters. Deviation of intensity ratios from ideal values of 1 was used as weighting function. Weighted flux sensitivities can be used to identify optimal type and degree of tracer labeling or potential intensity ratios to be measured. Experimental design for lysine-producing strain C. glutamicum MH 20-22B (Marx et al., Biotechnol. Bioeng. 49, 111-129, 1996) and various potential mutants with different alterations in the flux pattern showed that specific tracer labelings are optimal to quantify a certain flux parameter uninfluenced by the overall flux situation. Identified substrates of choice are [1-(13)C]glucose for the estimation of Phi(PPP) and zeta(PGI) and a 1 : 1 mixture of [U-(12)C/U-(13)C]glucose for the determination of zeta(PC/PEPCK). Phi(PC) can be quantified by feeding [4-(13)C]glucose or [U-(12)C/U-(13)C]glucose (1 : 1), whereas Phi(DH) is accessible via [4-(13)C]glucose. The sensitivity for the quantification of a certain flux parameter can be influenced by superposition through other flux parameters in the network, but substrate and measured mass isotopomers of choice remain the same. In special cases, reduced labeling degree of the tracer substrate can increase the precision of flux analysis. Enhanced precision and flux information can be achieved via multiply labeled substrates. The presented approach can be applied for effective experimental design of (13)C tracer studies for metabolic flux analysis. Intensity ratios of other products such as glutamate, valine, phenylalanine, and riboflavin also sensitively reflect flux parameters, which underlines the great potential of mass spectrometry for flux analysis.

Application of MALDI-TOF MS to lysine-producing Corynebacterium glutamicum: a novel approach for metabolic flux analysis.

In the present work, a novel comprehensive approach of (13)C-tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing Corynebacterium glutamicum during batch culture. MALDI-TOF MS methods established allow the direct quantification of labeling patterns of low molecular mass Corynebacterium products from 1 microL of diluted culture supernatant. A mathematical model of the central Corynebacterium metabolism was developed, that describes the carbon transfer through the network via matrix calculations in a generally applicable way and calculates steady state mass isotopomer distributions of the involved metabolites. The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI-TOF MS in tracer experiments either with 1-(13)C glucose or with mixtures of (13)C6/(12)C6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI-TOF MS sensitively reflecting the flux parameters of interest, the presented approach has an excellent potential to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.

Radon and thoron in cave dwellings (Yan'an, China).

222Rn and 220Rn concentrations were measured in cave dwellings and brick houses in the region of Yan'an (China) during summer 1997. The underground dwellings are built into Quaternary loess, and all investigated houses are founded on it. The median values of indoor 222Rn and 220Rn concentrations are 42 (n = 18) and 77 Bq m(-3) (n = 15) for brick houses and 92 (n = 23) and 215 (n = 17) Bq m(-3) for cave dwellings. To classify the dwellings in respect to their "cave-character," the fraction of walls having a direct contact to the loses is calculated for each dwelling. While the 222Rn concentrations are increasing with higher fractions, the 220Rn concentrations are not correlated with this fraction. On the other hand, due to the short half-life of 220Rn the distance from the measuring point to the walls is negatively correlated with the 220Rn concentration, while there is no correlation with the 222Rn concentration. Therefore, concentric isolines of 220Rn concentrations showing a strong gradient were detected in cave dwellings. An influence of the ventilation rate is distinct for 222Rn but weak for 220Rn. The effective dose rates for 222Rn and 220Rn and their progenies are calculated for brick houses (2.7 mSv y(-1)), cave dwellings (7.1 mSv y(-1)), and for traditional cave dwellings with a bed foundation built with loess (16.7 mSv y(-1)). These calculations are based on summer measurements only. It is expected that the true effective dose rates will be significantly higher.