Darcian permeability constant as indicator for shear stresses in regular scaffold systems for tissue engineering

The shear stresses in printed scaffold systems for tissue engineering depend on the flow properties and void volume in the scaffold. In this work, computational fluid dynamics (CFD) is used to simulate flow fields within porous scaffolds used for cell growth. From these models the shear stresses acting on the scaffold fibres are calculated. The results led to the conclusion that the Darcian (k 1) permeability constant is a good predictor for the shear stresses in scaffold systems for tissue engineering. This permeability constant is easy to calculate from the distance between and thickness of the fibres used in a 3D printed scaffold. As a consequence computational effort and specialists for CFD can be circumvented by using this permeability constant to predict the shear stresses. If the permeability constant is below a critical value, cell growth within the specific scaffold design may cause a significant increase in shear stress. Such a design should therefore be avoided when the shear stress experienced by the cells should remain in the same order of magnitud

Chemo-enzymatic peptide synthesis : bioprocess engineering aspects

Peptides, in particular oligopeptides, play an important role in the fields of health care, nutrition and cosmetics. Chemical synthesis is currently the most mature technique for the synthesis of peptides that range in length from 5 to 80 amino acids. Chemical synthesis is, however, expected to be more and more combined with enzyme-catalyzed synthesis, resulting in chemo-enzymatic approaches towards peptide synthesis. The racemization that hampers chemical synthesis can be prevented by forming the peptide bond enzymatically. In the work in this thesis the bioprocess engineering aspects of a specific chemo-enzymatic peptide synthesis route are studied. In this route, an N-protected, C‑terminally activated amino acid is coupled with a C-protected amino acid nucleophile. The coupling step is catalyzed by Alcalase. The initial idea was to also enzymatically catalyze the activation of the amino acid, which is used in the coupling step, and to carry out the coupling and activation steps in one pot. In the work of Chapter 2 lipase B from Candida antarctica (CalB) and Alcalase were used as a model system for such a one-pot chemo-enzymatic peptide synthesis, in order to investigate the (in)compatibility between the two enzymes. The rate of activity loss of native and immobilized CalB in the absence and presence of native and immobilized Alcalase was calculated from the rate of triacetin hydrolysis. It was shown that native Alcalase degrades native CalB under aqueous conditions. Immobilization of both or either CalB or Alcalase onto macroporous beads, however, effectively prevented hydrolysis of CalB by Alcalase. Due to the current impracticality of the enzyme-catalyzed activation step, the rest of the work in the thesis focuses on the Alcalase-catalyzed coupling step. The non-aqueous coupling in tetrahydrofuran (THF) of phenylalanine amide (Phe-NH2) and the carbamoylmethyl ester of phenylalanine (of which the amino group was benzyloxycarbonyl-protected, Z-Phe-OCam) was used as a model coupling reaction. In protease-catalyzed peptide synthesis the availability of water is essential, as a compromise must be made between on the one hand the overall enzymatic activity and, on the other hand, the rate of product synthesis. Water is essential for enzyme activity, but at the same time causes hydrolytic side reactions. In the work of Chapter 3 the model coupling reaction was catalyzed by cross-linked enzyme aggregates of Alcalase optimized for use in organic media (Alcalase CLEA-OM) at a range of water activity (aw) values, including the coupling in the presence of molecular sieves (i.e. at very low aw values). The rate of peptide synthesis could not be increased by increasing awvalueswithout significantly increasing the rate of hydrolysis, i.e. without significantly decreasing the synthesis / hydrolysis (S/H) ratio. Hydrolysis (in the present system, only the activated substrate, not the dipeptide product, may be hydrolyzed) was found to dominate above aw ≈0.2.To prevent hydrolysis, the presence of molecular sieves was found to be necessary. Nevertheless, the use of molecular sieves over longer periods of time should be carefully considered as they may dehydrate and thereby inactivate the enzyme in time. In the work of Chapter 4, besides CLEA-OM, also other Alcalase formulations were used to catalyze the model coupling reaction. The reaction was done in the presence of molecular sieves (i.e. under near-dry conditions). Hydration prior to drying (with anhydrous tert-butanol and anhydrous THF)of the Alcalase formulations resulted in a significant increase in rate of the subsequent dipeptide synthesis. Without such initial hydration, the enzymes seem to lack the water needed to maintain their catalytically active conformation. Repeated use in the presence of molecular sieves, without intermediate rehydration, led to inactivation of the enzyme. For three enzyme formulations this inactivation could be counteracted by intermediate rehydration. Inactivation of another enzyme formulation, Alcalase immobilized onto dicalite, was only partially reversible by hydration. Alcalase immobilized onto dicalite was found to be initially the most active in dipeptide synthesis. Nevertheless, due to its small particle size and its lack of operational stability, this formulation may not be the best choice for the synthesis of dipeptides in neat organic media on a large scale. The most promising enzyme formulation for this is Alcalase covalently immobilized onto macroporous acrylic beads (in this thesis abbreviated as Cov) due to its reasonable activity, its seemingly good operational stability, and its practical size and uniform spherical shape. If, for economic reasons, Cov should be reused repeatedly for dipeptide synthesis in organic media, its operational stability is important and thus its activity should not decrease significantly. The long-term stability and reuse of hydrated Cov in THF was investigated in the work of Chapter 5. Cov was incubated with and without molecular sieves (beads or powder) in anhydrous THF. After different incubation periods in THF, the enzyme activity was determined in an aqueous environment. In addition, Cov was repeatedly recycled in order to examine its reusability. The effect of reuse on the aqueous activity of Cov and on the Cov-catalyzed model coupling reaction in near-anhydrous THF was studied. Without molecular sieve beads, Cov hardly inactivated in THF. Nevertheless, when Cov was incubated with molecular sieve beads in THF in rotating reaction vials, Cov lost activity over time. Mechanical damage of Cov by the molecular sieve beads was found to be the main reason for the instability of Cov. In order to reuse Cov for the model coupling reaction in the presence of molecular sieves, it needs to be rehydrated in between the batches. Nevertheless, each intermediate rehydration step also caused a small but significant enzyme activity loss. In the work of Chapter 6, the coupling kinetics of the model coupling reaction, catalyzed by hydrated Cov, were investigated. Near-anhydrous conditions were maintained by a carefully chosenamount of molecular sieve powder (in contrast to molecular sieve beads, molecular sieve powder does not lead to mechanical damage of Cov). Kinetic characteristics were determined from reaction time courses up to full conversion at various initial concentrations of substrate and product. These progress curve data were fitted with different kinetic models to determine which of these models best approximates the kinetic properties of the immobilized Alcalase with respect to the coupling under study. It was found that the kinetics of the coupling can be described well with a two-substrate kinetic model with two inhibitory products. To reduce the effect of the product inhibition on Cov, a reactor should be designed in which at least glycolamide is selectively removed, as it was found to be the strongest inhibitor. In Chapter 4 it was shown that molecular sieves dehydrate and thereby reversibly inactivate the enzyme. In the work of Chapter 7 the effect of enzyme dehydration by molecular sieves on the Cov-catalyzed model coupling reaction was studied in detail. The dehydration kinetics of Cov by different amounts of molecular sieve powder were determined by incubating Cov with molecular sieve powder for different periods of time. Subsequently, the remaining coupling activity of Cov was measured. Dehydration-induced inactivation of Cov by molecular sieve powder seemed to occur in three phases: (1) an initial, rapid,major dehydration-induced inactivation that takes place during the first activity measurement (1 h), (2) a phase of first-order inactivation (20 h), and (3) a relatively low plateau phase in activity. These dehydration kinetics were incorporated into the reaction kinetics model described in Chapter 6. The resulting model was then used to fit progress curve data of the model coupling reaction in the presence of different amounts of molecular sieve powder. Using the estimated parameter values, the model was used to predict independent data sets and found to work well. The work of Chapter 8 is a case study about a process design for enzymatic peptide synthesis, which is based on the findings of the previous chapters. The choices with regard to Alcalase formulation, type of reactor, way to control the water content, and whether or not to recycle the enzyme, are discussed. In addition, an estimate is given for the reactor size, volumes of solvent, amounts of substrate, enzyme and molecular sieves, needed in order to produce a specific demand for peptides. We believe that this case study gives a good impression of the various choices that have to be made when designing a process for enzymatic peptide synthesis and the implications of these choices.</p

High power semiconductor switches in the 12 kV, 50 kA pulse generator of the SPS beam dump kicker system

Horizontal deflection of the beam in the dump kicker system of the CERN SPS accelerator is obtained with a series of fast pulsed magnets. The high current pulses of 50 kA per magnet are generated with capacitor discharge type generators which, combined with a resistive free-wheel diode circuit, deliver a critically damped half-sine current with a rise-time of 25 ms. Each generator consists of two 25 kA units, connected in parallel to a magnet via a low inductance transmission line

Pseudospark Switch Development for the LHC Extraction Kicker Pulse Generator

CERN, the European Laboratory for Particle Physics, has started construction of the Large Hadron Collider (LHC), a superconducting accelerator that will collide protons at a center of mass energy of 14 TeV from the year 2005 onwards. The kicker magnet pulse generators of the LHC beam extraction system require fast high power switches. One possible type is the pseudospark switch (PSS) which has several advantages for this application. A PSS fulfilling most of the requirements has been developed in the past years. Two outstanding problems, prefiring at high operating voltages and sudden current interruptions (quenching) at low voltage could be solved recently. Prefiring can be avoided for this special application by conditioning the switch at two times the nominal voltage after each power pulse. Quenching can be suppressed by choosing an appropriate electrode geometry and by mixing Krypton to the D2 gas atmosphere. One remaining problem, related to the required large dynamic voltage range (1.7 kV to 30 kV) is under active investigation: steps in forward voltage during conduction, occurring at low operation voltage at irregular time instants and causing a pulse to pulse jitter of the peak current. This paper presents results of electrical measurements concerning prefiring and quenching and explains how these problems have been solved. Furthermore the plans to cure the forward voltage step problem will be discussed

Added value of 18F-FDG-PET/CT and cardiac CTA in suspected transcatheter aortic valve endocarditis

Backgrounds: Transcatheter-implanted aortic valve infective endocarditis (TAVI-IE) is difficult to diagnose when relying on the Duke Criteria. Our aim was to assess the additional diagnostic value of 18F-fluorodeoxyglucose (18F-FDG) positron emission/computed tomography (PET/CT) and cardiac computed tomography angiography (CTA) in suspected TAVI-IE. Methods: A multicenter retrospective analysis was performed in all patients who underwent 18F-FDG-PET/CT and/or CTA with suspected TAVI-IE. Patients were first classified with Duke Criteria and after adding 18F-FDG-PET/CT and CTA, they were classified with European Society of Cardiology (ESC) criteria. The final diagnosis was determined by our Endocarditis Team based on ESC guideline recommendations. Results: Thirty patients with suspected TAVI-IE were included. 18F-FDG-PET/CT was performed in all patients and Cardiac CTA in 14/30. Using the Modified Duke Criteria, patients were classified as 3% rejected (1/30), 73% possible (22/30), and 23% definite (7/30) TAVI-IE. Adding 18F-FDG-PET/CT and CTA supported the reclassification of 10 of the 22 possible cases as “definite TAVI-IE” (5/22) or “rejected TAVI-IE” (5/22). This changed the final diagnosis to 20% rejected (6/30), 40% possible (12/30), and 40% definite (12/30) TAVI-IE. Conclusions: Addition of 18F-FDG-PET/CT and/or CTA changed the final diagnosis in 33% of patients and proved to be a valuable diagnostic tool in patients with suspected TAVI-IE

Phase transitions in biological membranes

Native membranes of biological cells display melting transitions of their lipids at a temperature of 10-20 degrees below body temperature. Such transitions can be observed in various bacterial cells, in nerves, in cancer cells, but also in lung surfactant. It seems as if the presence of transitions slightly below physiological temperature is a generic property of most cells. They are important because they influence many physical properties of the membranes. At the transition temperature, membranes display a larger permeability that is accompanied by ion-channel-like phenomena even in the complete absence of proteins. Membranes are softer, which implies that phenomena such as endocytosis and exocytosis are facilitated. Mechanical signal propagation phenomena related to nerve pulses are strongly enhanced. The position of transitions can be affected by changes in temperature, pressure, pH and salt concentration or by the presence of anesthetics. Thus, even at physiological temperature, these transitions are of relevance. There position and thereby the physical properties of the membrane can be controlled by changes in the intensive thermodynamic variables. Here, we review some of the experimental findings and the thermodynamics that describes the control of the membrane function.Comment: 23 pages, 15 figure

Cross-cultural adaptation of research instruments: language, setting, time and statistical considerations

<p>Abstract</p> <p>Background</p> <p>Research questionnaires are not always translated appropriately before they are used in new temporal, cultural or linguistic settings. The results based on such instruments may therefore not accurately reflect what they are supposed to measure. This paper aims to illustrate the process and required steps involved in the cross-cultural adaptation of a research instrument using the adaptation process of an attitudinal instrument as an example.</p> <p>Methods</p> <p>A questionnaire was needed for the implementation of a study in Norway 2007. There was no appropriate instruments available in Norwegian, thus an Australian-English instrument was cross-culturally adapted.</p> <p>Results</p> <p>The adaptation process included investigation of conceptual and item equivalence. Two forward and two back-translations were synthesized and compared by an expert committee. Thereafter the instrument was pretested and adjusted accordingly. The final questionnaire was administered to opioid maintenance treatment staff (n=140) and harm reduction staff (n=180). The overall response rate was 84%. The original instrument failed confirmatory analysis. Instead a new two-factor scale was identified and found valid in the new setting.</p> <p>Conclusions</p> <p>The failure of the original scale highlights the importance of adapting instruments to current research settings. It also emphasizes the importance of ensuring that concepts within an instrument are equal between the original and target language, time and context. If the described stages in the cross-cultural adaptation process had been omitted, the findings would have been misleading, even if presented with apparent precision. Thus, it is important to consider possible barriers when making a direct comparison between different nations, cultures and times.</p

Meta-omics approaches to understand and improve wastewater treatment systems

Biological treatment of wastewaters depends on microbial processes, usually carried out by mixed microbial communities. Environmental and operational factors can affect microorganisms and/or impact microbial community function, and this has repercussion in bioreactor performance. Novel high-throughput molecular methods (metagenomics, metatranscriptomics, metaproteomics, metabolomics) are providing detailed knowledge on the microorganisms governing wastewater treatment systems and on their metabolic capabilities. The genomes of uncultured microbes with key roles in wastewater treatment plants (WWTP), such as the polyphosphate-accumulating microorganism Candidatus Accumulibacter phosphatis, the nitrite oxidizer Candidatus Nitrospira defluvii or the anammox bacterium Candidatus Kuenenia stuttgartiensis are now available through metagenomic studies. Metagenomics allows to genetically characterize full-scale WWTP and provides information on the lifestyles and physiology of key microorganisms for wastewater treatment. Integrating metagenomic data of microorganisms with metatranscriptomic, metaproteomic and metabolomic information provides a better understanding of the microbial responses to perturbations or environmental variations. Data integration may allow the creation of predictive behavior models of wastewater ecosystems, which could help in an improved exploitation of microbial processes. This review discusses the impact of meta-omic approaches on the understanding of wastewater treatment processes, and the implications of these methods for the optimization and design of wastewater treatment bioreactors.Research was supported by the Spanish Ministry of Education and Science (Contract Project CTQ2007-64324 and CONSOLIDER-CSD 2007-00055) and the Regional Government of Castilla y Leon (Ref. VA038A07). Research of AJMS is supported by the European Research Council (Grant 323009

Складові компоненти мовної особистості в контексті міжкультурної комунікації

Стаття присвячена аналізу складових компонентів мовної особистості в контексті міжкультурної комунікації, їх взаємодії та функціонуванню з точки зору прагматичної спрямованості мовленнєвого впливу. Детально розглядаються три рівні структури мовної особистості (структурно-мовний, лінгвокогнітивний ті мотиваційний) із визначенням специфіки їхніх складових компонентів.Статья посвящена анализу составляющих компонентов языковой личности в контексте межкультурной коммуникаций, их взаимодействию и функционированию с точки зрения прагматической направленности речевого воздействия. Детально рассматриваются три уровня структуры языковой личности (структурно-языковой, лингвокогнитивный и мотивационный) с последующим определением специфики их составляющих компонентов.The article is dedicated to the linguistic personality constituent components' analysis in terms of cross-cultural communication, their interaction and functioning with the speech influence pragmatic orientation taken into consideration. The three levels of the linguistic personality (that is, structural linguistic, lingo cognitive and motivation ones) are under analysis with the following their constituent components specificity determinatio