Protein thermostability above 100°C: A key role for ionic interactions

The discovery of hyperthermophilic microorganisms and the analysis of hyperthermostable enzymes has established the fact that multisubunit enzymes can survive for prolonged periods at temperatures above 100°C. We have carried out homology-based modeling and direct structure comparison on the hexameric glutamate dehydrogenases from the hyperthermophiles Pyrococcus furiosus and Thermococcus litoralis whose optimal growth temperatures are 100°C and 88°C, respectively, to determine key stabilizing features. These enzymes, which are 87% homologous, differ 16-fold in thermal stability at 104°C. We observed that an intersubunit ion-pair network was substantially reduced in the less stable enzyme from T. litoralis, and two residues were then altered to restore these interactions. The single mutations both had adverse effects on the thermostability of the protein. However, with both mutations in place, we observed a fourfold improvement of stability at 104°C over the wild-type enzyme. The catalytic properties of the enzymes were unaffected by the mutations. These results suggest that extensive ion-pair networks may provide a general strategy for manipulating enzyme thermostability of multisubunit enzymes. However, this study emphasizes the importance of the exact local environment of a residue in determining its effects on stability.

The'cave' mineral oxammite: a high resolution thermogravimetry and Raman spectroscopic study

The thermal decomposition of natural ammonium oxalate known as oxammite has been studied using a combination of high resolution thermogravimetry coupled to an evolved gas mass spectrometer and Raman spectroscopy coupled to a thermal stage. Three mass loss steps were found at 57, 175 and 188°C attributed to dehydration, ammonia evolution and carbon dioxide evolution respectively. Raman spectroscopy shows two bands at 3235 and 3030 cm-1 attributed to the OH stretching vibrations and three bands at 2995, 2900 and 2879 cm-1, attributed to the NH vibrational modes. The thermal degradation of oxammite may be followed by the loss of intensity of these bands. No intensity remains in the OH stretching bands at 100°C and the NH stretching bands show no intensity at 200°C. Multiple CO symmetric stretching bands are observed at 1473, 1454, 1447 and 1431cm-1, suggesting that the mineral oxammite is composed of a mixture of chemicals including ammonium oxalate dihydrate, ammonium oxalate monohydrate and anhydrous ammonium oxalate.

Volatile properties of particles emitted by compressed natural gas and diesel buses during steady state and transient driving modes

Volatile properties of particle emissions from four compressed natural gas (CNG) and four diesel buses were investigated under steady state and transient driving modes on a chassis dynamometer. The exhaust was diluted utilising a full-flow continuous volume sampling system and passed through a thermodenuder at controlled temperature. Particle number concentration and size distribution were measured with a condensation particle counter and a scanning mobility particle sizer, respectively. We show that, while almost all the particles emitted by the CNG buses were in the nanoparticle size range, at least 85% and 98% were removed at 100ºC and 250ºC, respectively. Closer analysis of the volatility of particles emitted during transient cycles showed that volatilisation began at around 40°C with the majority occurring by 80°C. Particles produced during hard acceleration from rest exhibited lower volatility than that produced during other times of the cycle. Based on our results and the observation of ash deposits on the walls of the tailpipes, we suggest that these non-volatile particles were composed mostly of ash from lubricating oil. Heating the diesel bus emissions to 100ºC removed ultrafine particle numbers by 69% to 82% when a nucleation mode was present and just 18% when it was not.

The effect of light intensity and temperature on photocatalytic water splitting

Photocatalytic water splitting is a process which could potentially lead to commercially viable solar hydrogen production. This thesis uses an engineering perspective to investigate the technology. The effect of light intensity and temperature on photocatalytic water splitting was examined to evaluate the prospect of using solar concentration to increase the feasibility of the process. P25 TiO2 films deposited on conducting glass were used as photocatalyst electrodes and coupled with platinum electrodes which were also deposited on conducting glass. These films were used to form a photocatalysis cell and illuminated with a Xenon arc lamp to simulate solar light at intensities up to 50 suns. They were also tested at temperatures between 20°C and 100°C. The reaction demonstrated a sub-linear relationship with intensity. Photocurrent was proportional to intensity with an exponential value of 0.627. Increasing temperature resulted in an exponential relationship. This proved to follow an Arrhenius relationship with an activation energy of 10.3 kJ mol-1 and a pre-exponential factor of approximately 8.7×103. These results then formed the basis of a mathematical model which extrapolated beyond the range of the experimental tests. This model shows that the loss of efficiency from performing the reaction under high light intensity is offset by the increased reaction rate and efficiency from the associated temperature increase. This is an important finding for photocatalytic water splitting. It will direct future research in system design and materials research and may provide an avenue for the commercialisation of this technology.

The formation of YBa2Cu3O7-x in melt-texture heat treatments

A study of the bulk formation of YBa2Cu3O7-x from the Y2BaCuO5 plus liquid regime reveals that phase formation occurs at appreciable rates below 950°C in air. This result has been observed for phase-pure YBa2Cu3O7-x starting material given two types of heat treatment: held at 1100°C and slow-cooled from 1030°C at 6°C/h or heat-treated isothermally. Differential thermal analysis, with a cooling rate of 10°C/min indicates that the degree of undercooling for the peritectic formation of YBa2Cu3O7-x is greater than 100°C. © 1994.

Oriented graphitic carbon films for hydrogen gas sensors

An oriented graphitic nanostructured carbon film has been employed as a conductometric hydrogen gas sensor. The carbon film was energetically deposited using a filtered cathodic vacuum arc with a -75 V bias applied to a stainless steel grid placed 1cm from the surface of the Si substrate. The substrate was heated to 400°C prior to deposition. Electron microscopy showed evidence that the film consisted largely of vertically oriented graphitic sheets and had a density of 2.06 g/cm3. 76% of the atoms were bonded in sp2 or graphitic configurations. A change in the device resistance of >; 1.5% was exhibited upon exposure to 1 % hydrogen gas (in synthetic, zero humidity air) at 100°C. The time for the sensor resistance to increase by 1.5 % under these conditions was approximately 60 s and the baseline (zero hydrogen exposure) resistance remained constant to within 0.01% during and after the hydrogen exposures.

Pt/Graphene Nano-sheet Based Hydrogen Gas Sensor

In this paper, we present gas sensing properties of Pt/graphene-like nano-sheets towards hydrogen gas. The graphene-like nano-sheets were produced via the reduction of spray-coated graphite oxide deposited on SiC substrates by hydrazine vapor. Structural and morphological characterizations of the graphene sheets were analyzed by scanning electron and atomic force microscopy. Current-voltage and dynamic responses of the sensors were investigated towards different concentrations of hydrogen gas in a synthetic air mixture at 100°C. A voltage shift of 100 mV was recorded at 1 mA reverse bias current.

The effects of cold air (-110°C) and water (8°C) cryotherapy on knee skin temperature

Exposure to cold air, whole body cryotherapy (WBC), is a novel treatment employed by athletes. In WBC individuals dressed in minimal clothing are exposed to a temperature below -100°C for 2-4 min. The use of WBC has been advocated as a treatment for various knee injuries. PURPOSE: To compare the effects of two modalities of cryotherapy, -110°C WBC and 8°C cold water immersion (CWI) on knee skin temperature (Tsk). METHODS: With ethical approval and written informed consent 10 healthy active male participants (26.5±4.9 yr, 183.5±6.0 cm, 90.7±19.9 kg, 26.8±5.0 kg/m2, 23.0±9.3% body fat (measured by DXA), 7.6 ± 2.0 mm patellar skin fold; mean±SD) were exposed to 4 min of CWI and WBC. The treatment order was randomised in a controlled crossover design, with a minimum of 7 days between treatments. During WBC participants stood in a chamber (-60±3°C) for 20 s before entering the main chamber (-110°C±3°C) where they remained for 3 min and 40 s. For CWI participants were seated in a tank filled with cold water (8±0.3°C) and immersed to the level of the sternum for 4 min. Right knee Tsk was assessed via non-contact, infrared thermal imaging. A quadrilateral region of interest was created using inert markers placed 5 cm above and below the most superior and inferior aspect of the patella. Tsk within this quadrilateral was recorded pre, immediately post and every 10 min thereafter for 60 min. Tsk changes were examined using a two-way (treatment x time) repeated measures analyses of variance. In addition, a paired sample t-test was used to compare baseline Tsk before both treatments. RESULTS: Knee Tsk was similar before treatment (WBC: 29.9±0.7°C, CWI: 29.6±0.9°C, p>0.05). There was a significant main effect for treatment (p<0.05) and time (p<0.001). Compared to baseline, Tsk was significantly reduced (p<0.05) immediately post and at 10, 20, 30, 40, 50 and 60 min after both cooling modalities. Knee Tsk was lower (p<0.05) immediately after WBC (19.0±0.9°C) compared to CWI (20.5±0.6°C). However, from 10 to 60 min post, knee Tsk was lower (p<0.05) following the CWI treatment. CONCLUSION: WBC elicited a greater decrease in knee Tsk compared to CWI immediately after treatment. However, both modalities display different recovery patterns and Tsk after CWI was significantly lower than WBC at 10, 20, 30, 40, 50 and 60 min after treatment.

Whole-body cryotherapy : empirical evidence and theoretical perspectives

Whole-body cryotherapy (WBC) involves short exposures to air temperatures below –100°C. WBC is increasingly accessible to athletes, and is purported to enhance recovery after exercise and facilitate rehabilitation postinjury. Our objective was to review the efficacy and effectiveness of WBC using empirical evidence from controlled trials. We found ten relevant reports; the majority were based on small numbers of active athletes aged less than 35 years. Although WBC produces a large temperature gradient for tissue cooling, the relatively poor thermal conductivity of air prevents significant subcutaneous and core body cooling. There is weak evidence from controlled studies that WBC enhances antioxidant capacity and parasympathetic reactivation, and alters inflammatory pathways relevant to sports recovery. A series of small randomized studies found WBC offers improvements in subjective recovery and muscle soreness following metabolic or mechanical overload, but little benefit towards functional recovery. There is evidence from one study only that WBC may assist rehabilitation for adhesive capsulitis of the shoulder. There were no adverse events associated with WBC; however, studies did not seem to undertake active surveillance of predefined adverse events. Until further research is available, athletes should remain cognizant that less expensive modes of cryotherapy, such as local ice-pack application or cold-water immersion, offer comparable physiological and clinical effects to WBC.

Microprocessor-based temperature indicator using a thermistor as the sensor

A novel microprocessor-based temperature indicator has been developed and described. This indicator provides a linear performance over a wide dynamic temperature range of 0-100°C with an accuracy of ±0-l°C. The interfacing module reduces computing time required by the microprocessor for solving the thermistor equation. Test results are given to support the theory.

A simple, wide-range, linear thermistor-based temperature indicator

A novel thermistor-based temperature indicator using an RC oscillator and an up/down counter has been developed and described. The indicator provides linear performance over a wide dynamic temperature range of 0-100°C. This indicator is free from the error due to lead resistances of the thermistor and gives a maximum error of ±0 · 1°C in the range 0-100°C. Test results are given to support the theory.

Development of new gas sensors based on oxidized galinstan

For the first time, we have fabricated and tested conductometric sensors based on oxidized liquid galinstan towards NO2 and NH3 gases at low operating temperatures. Galinstan based films on silicon substrates have been studied with two different loadings. Surface morphology of the films was investigated by means of field emission scanning electron microscopy (FESEM). The sensor with higher galinstan loading showed a better sensitivity, which can be attributed to a higher surface area, as confirmed by SEM. At 100°C, a detection limit as low as 1 and 20 ppm was measured for NO2 and NH3, respectively.

Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.