Engineering volatile thiol formation in yeast.

AIMS: Volatile thiols are very potent aroma molecules that contribute to the aroma of many beverages. The characteristic thiols of certain wine varieties such as Sauvignon blanc are partly released during the yeast-based fermentation from plant-synthesized glutathione- or cysteine-conjugated and dipeptic precursors present in the must. In this work, we aimed at the construction and characterization of yeast strains with the ability to synthesize volatile thiols from respective precursors. METHODS AND RESULTS: Besides genome integration of the Escherichia coli gene tnaA, which encodes an enzyme with high ß-lyase activity, a glutathione synthetase and glutathione-S-transferases were overexpressed. Up to 8.9 µg L-1 3-mercaptohexan-1-ol could be formed with the strain from externally added trans-2-hexen-1-ol. Well-characterized thiols such as 2-methyl-2-butanethiol, 3-mercapto-3-methylbutan-1-ol, and 8-mercapto-p-menthan-3-one, as well as several so far undescribed thiol compounds could be synthesized. CONCLUSION: Volatile thiols could be produced by feeding alcohol, alkenol, aldehyde, or ketone precursors like trans-2-hexenal, trans-2-hexen-1-ol, cis-2-hexen-1-ol, 3-methyl-2-buten-1-ol, 3-buten-2-one, and pulegone to the optimized yeast cells.

Investigation of non-Saccharomyces yeasts with intracellular ß-glycosidase activity for wine aroma modification.

Since high proportions of aroma-relevant molecules in plant-derived juices are present in glycosylated forms, the introduction of glycosidase activity during processing is an important tool to modify the aroma composition of the product. During winemaking, the addition of ß-glycosidase enzyme or microorganisms with ß-glycosidase activity is an established technology. However, low stability under acidic conditions and low selectivity for hydrolysis of different glycosides are still drawbacks, which limit application possibilities. Here, we report the identification and characterization of non-Saccharomyces yeast strains with relatively high ß-glycosidase activity in their cultures. We found strong indications for intracellular localization of the enzymes, which is in line with the pH robustness found in experiments with whole cells. Furthermore, we compared the selectivity of aroma compound release from glycoside mixtures using whole cells or cell extracts. The results showed strong differences for the released aroma patterns, which indicates the transport of glycosides and intracellular hydrolysis. Our work demonstrates the application potential of yeasts with intracellular ß-glycosidase activities as catalysts with high pH robustness and selective aroma release properties. PRACTICAL APPLICATION: The yeast strains identified and characterized within this work can be applied in wine processing but also in other processes to release aroma molecules from their glycosylated precursors provided by the plants. The strains show relatively high activity of the relevant enzyme, ß-glycosidase, also at low pH, which is essential in many processes. In contrast to most other approaches, the enzyme is inside the cells, which can lead to a specific release of certain aroma compounds.

Biomechanical Model-Based Development of an Active Occupational Upper-Limb Exoskeleton to Support Healthcare Workers in the Surgery Waiting Room.

Occupational ergonomics in healthcare is an increasing challenge we have to handle in the near future. Physical assistive systems, so-called exoskeletons, are promising solutions to prevent work-related musculoskeletal disorders (WMSDs). Manual handling like pushing, pulling, holding and lifting during healthcare activities require practical and biomechanical effective assistive devices. In this article, a musculoskeletal-model-based development of an assistive exoskeleton is described for manual patient transfer in the surgery waiting room. For that purpose, kinematic data collected with an experimental set-up reproducing real patient transfer conditions are first used to define the kinetic boundary conditions for the model-based development approach. Model-based analysis reveals significant relief potential in the lower back and shoulder area of the musculoskeletal apparatus. This is corroborated by subjective feedback collected during measurements with real surgery assistants. A shoulder-arm exoskeleton design is then proposed, optimized and evaluated within the same simulation framework. The presented results illustrate the potential for the proposed design to reduce significantly joint compressions and muscle activities in the shoulder complex in the considered patient transfer scenarios.

Investigation of the Durability of a Diaphragm for a Total Artificial Heart.

One of the most critical components regarding the durability of the ReinHeart total artificial heart (TAH) is its biocompatible diaphragm, which separates the drive unit from the ventricles. Hence, a durability tester was designed to investigate its required 5-year lifetime. The aim of this study was to prove the validity of accelerated testing of the polyurethane diaphragm. The durability tester allows simultaneous testing of 12 diaphragms and mimics physiological conditions. To accelerate the time of testing, it operates with an increased speed at a frequency of 8 Hz. To prove the correctness of this acceleration, a servo-hydraulic testing machine was used to study the effect of different frequencies and their corresponding loads. Thereby the viscoelastic behavior of the polyurethane was investigated. Additionally, high-speed video measurements were performed. The force against frequency and the high-speed video measurements showed constant behavior. In the range of 1-10 Hz, the maximum resulting forces varied by 3%, and the diaphragm movement was identical. Frequencies below 10 Hz allow a valid statement of the diaphragm's mechanical durability. Viscoelasticity of the polyurethane in the considered frequency-range is negligible. The accelerated durability test is applicable to polyurethane diaphragms, and the results are applicable to TAH use. The reliability of the diaphragm for a lifetime of 5 years was found to be 80% with a confidence of 62%.

Molecular characterization and virus neutralization patterns of severe, non-epizootic forms of feline calicivirus infections resembling virulent systemic disease in cats in Switzerland and in Liechtenstein.

Feline calicivirus (FCV) infections are associated with oral ulceration, chronic stomatitis and a limping syndrome. Epizootic outbreaks of virulent systemic disease (VSD) have been reported in the USA and Europe. Here, the molecular characterization and neutralization patterns of FCV isolates from cases of severe, non-epizootic infection associated with skin ulceration and edema are presented. Samples from eleven symptomatic cats, four in-contact cats and 27 cats with no contact with symptomatic cats were collected and tested for FCV, feline herpesvirus-1 (FHV-1), feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV). Phylogenetic analyses based on the capsid (VP1) gene of FCV and virus neutralization with antisera raised against four FCV vaccine strains were performed. Nine kittens and two adult cats in two shelters and two veterinary clinics in four geographically distinct locations in Switzerland and Liechtenstein were affected. The cats showed fever, tongue and skin ulceration, head and paw edema, and occasionally jaundice, generalized edema and dyspnea. All symptomatic cats tested FCV-positive but were negative for FHV-1, FeLV and FIV, with the exception of one FIV-positive kitten. All kittens of one litter and both adult cats died. The disease did not spread to cats in the environment. Cats in the environment displayed phylogenetically distinct, but related, FCV strains. Virus neutralization patterns suggested that some cases might have been potentially prevented by vaccination with the optimal vaccine strain. In conclusion, clinicians should be aware of severe, non-epizootic forms of FCV infections with initial clinical presentations similar to VSD.

Design and Evaluation of a Fully Implantable Control Unit for Blood Pumps.

As the number of donor hearts is limited while more and more patients suffer from end stage biventricular heart failure, Total Artificial Hearts become a promising alternative to conventional treatment. While pneumatic devices sufficiently supply the patients with blood flow, the patient's quality of life is limited by the percutaneous pressure lines and the size of the external control unit. This paper describes the development of the control unit of the ReinHeart, a fully implantable Total Artificial Heart. General requirements for any implantable control unit are defined from a technical and medical point of view: necessity of a Transcutaneous Energy Transmission, autonomous operation, safety, geometry, and efficiency. Based on the requirements, a prototype is designed; it incorporates a LiFePo4 battery pack with charger, a rectifier for transcutaneous energy transmission, the motor's driver electronics, and a microcontroller which monitors and controls all functions. In validation tests, the control unit demonstrated a stable operation on TET and battery supply and a safe switching from one supply to the other. The overall mean efficiency is 14% on TET and 22% on battery supply. The control unit is suitable for chronic animal trials of the ReinHeart.

Mock circulation loop to investigate hemolysis in a pulsatile total artificial heart.

Hemocompatibility of blood pumps is a crucial parameter that has to be ensured prior to in vivo testing. In contrast to rotary blood pumps, a standard for testing a pulsatile total artificial heart (TAH) has not yet been established. Therefore, a new mock circulation loop was designed to investigate hemolysis in the left ventricle of the ReinHeart TAH. Its main features are a high hemocompatibility, physiological conditions, a low priming volume, and the conduction of blood through a closed tubing system. The mock circulation loop consists of a noninvasive pressure chamber, an aortic compliance chamber, and an atrium directly connected to the ventricle. As a control pump, the clinically approved Medos-HIA ventricular assist device (VAD) was used. The pumps were operated at 120 beats per minute with an aortic pressure of 120 to 80 mm Hg and a mean atrial pressure of 10 mm Hg, generating an output flow of about 5 L/min. Heparinized porcine blood was used. A series of six identical tests were performed. A test method was established that is comparable to ASTM F 1841, which is standard practice for the assessment of hemolysis in continuous-flow blood pumps. The average normalized index of hemolysis (NIH) values of the VAD and the ReinHeart TAH were 0.018 g/100 L and 0.03 g/100 L, respectively. The standard deviation of the NIH was 0.0033 for the VAD and 0.0034 for the TAH. Furthermore, a single test with a BPX-80 Bio-Pump was performed to verify that the hemolysis induced by the mock circulation loop was negligible. The performed tests showed a good reproducibility and statistical significance. The mock circulation loop and test protocol developed in this study are valid methods to investigate the hemolysis induced by a pulsatile blood pump.

Numerical washout study of a pulsatile total artificial heart.

PURPOSE: For blood pumps with long term indication, blood stagnation can result in excessive thromboembolic risks for patients. This study numerically investigates the washout performance of the left pump chamber of a pulsatile total artificial heart (TAH) as well as the sensitivity of the rotational orientation of the inlet bileaflet mechanical heart valve (MHV) on blood stagnation. METHODS: To quantitatively evaluate the washout efficiency, a fluid-structure interaction (FSI) simulation of the artificial heart pumping process was combined with a blood washout model. Four geometries with different orientations (0°, 45°, 90° and 135°) of the inlet valve were compared with respect to washout performance. RESULTS: The calculated flow field showed a high level of agreement with particle image velocimetry (PIV) measurements. Almost complete washout was achievable after three ejection phases. Remains of old blood in relation to the chamber volume was below 0.6% for all configurations and were mainly detected opposite to the inlet and outlet port at the square edge where the membrane and the pump chamber are connected. Only a small variation in the washout efficiency and the general flow field was observed. An orientation of 0° showed minor advantages with respect to blood stagnation and recirculation. CONCLUSIONS: Bileaflet MHVs were demonstrated to be only slightly sensitive to rotation regarding the washout performance of the TAH. The proposed numerical washout model proved to be an adequate tool to quantitatively compare different configurations and designs of the artificial organ regarding the potential for blood stagnation where experimental measurements are limited.