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Background
Minor changes in protein structure induced by small organic and inorganic molecules can result in significant metabolic effects. The effects can be even more profound if the molecular players are chemically active and present in the cell in considerable amounts. The aim of our study was to investigate effects of a nitric oxide donor (spermine NONOate), ATP and sodium/potassium environment on the dynamics of thermal unfolding of human hemoglobin (Hb). The effect of these molecules was examined by means of circular dichroism spectrometry (CD) in the temperature range between 25°C and 70°C. The alpha-helical content of buffered hemoglobin samples (0.1 mg/ml) was estimated via ellipticity change measurements at a heating rate of 1°C/min.
Results
Major results were:
1) spermine NONOate persistently decreased the hemoglobin unfolding temperature T u irrespectively of the Na + /K + environment,
2) ATP instead increased the unfolding temperature by 3°C in both sodium-based and potassium-based buffers and
3) mutual effects of ATP and NO were strongly influenced by particular buffer ionic compositions. Moreover, the presence of potassium facilitated a partial unfolding of alpha-helical structures even at room temperature.
Conclusion
The obtained data might shed more light on molecular mechanisms and biophysics involved in the regulation of protein activity by small solutes in the cell.
Cell spraying has become a feasible application method for cell therapy and tissue engineering approaches. Different devices have been used with varying success. Often, twin-fluid atomizers are used, which require a high gas velocity for optimal aerosolization characteristics. To decrease the amount and velocity of required air, a custom-made atomizer was designed based on the effervescent principle. Different designs were evaluated regarding spray characteristics and their influence on human adipose-derived mesenchymal stromal cells. The arithmetic mean diameters of the droplets were 15.4–33.5 µm with decreasing diameters for increasing gas-to-liquid ratios. The survival rate was >90% of the control for the lowest gas-to-liquid ratio. For higher ratios, cell survival decreased to approximately 50%. Further experiments were performed with the design, which had shown the highest survival rates. After seven days, no significant differences in metabolic activity were observed. The apoptosis rates were not influenced by aerosolization, while high gas-to-liquid ratios caused increased necrosis levels. Tri-lineage differentiation potential into adipocytes, chondrocytes, and osteoblasts was not negatively influenced by aerosolization. Thus, the effervescent aerosolization principle was proven suitable for cell applications requiring reduced amounts of supplied air. This is the first time an effervescent atomizer was used for cell processing.
A new method for improved autoclave loading within the restrictive framework of helicopter manufacturing is proposed. It is derived from experimental and numerical studies of the curing process and aims at optimizing tooling positions in the autoclave for fast and homogeneous heat-up. The mold positioning is based on two sets of information. The thermal properties of the molds, which can be determined via semi-empirical thermal simulation. The second information is a previously determined distribution of heat transfer coefficients inside the autoclave. Finally, an experimental proof of concept is performed to show a cycle time reduction of up to 31% using the proposed methodology.