Predicting small endotherm body temperatures from scalp temperatures

We used 2-d to 3-d-old mallard ducklings (Anas platyrhynchos) to test whether remote thermographic measurement of scalp temperature could be used to estimate core (cloacal) temperature from a distance. The scalp was exposed by trimming down from an area of ⩽1 cm², and surface temperature measured to ±0.12 °C with a radiometric thermal imager. For wind speeds of up to 2.5 m/s, we found cloacal temperature could be estimated to within 1 °C by using a regression model incorporating only scalp temperature and ambient temperature. The inexpensive method of dyeing the scalp black and painting it with temperature-sensitive cholesteric liquid crystal paint is more difficult and provides less accuracy, but appears to be a useful means for monitoring change in body temperature. These methods allow body temperatures of small or young endotherms to be monitored from distances of up to several meters without surgery or encumbering wires, minimizing stress that may alter behavior patterns and physiological parameters.     

Evaluation of a method to measure long term cortisol levels

IntroductionElevated levels of cortisol are known to induce various symptoms and diseases, e.g. abdominal obesity, type 2 diabetes, osteoporosis and cardiovascular disease. Measuring serum, saliva and urine cortisol is limited to one time point. Measurement of cortisol in scalp hair is a recently developed method to measure long term cortisol levels. The aim of this study was to investigate whether hair cortisol is a feasible parameter to measure cortisol exposure.ExperimentalWe collected hair samples of 195 healthy individuals, 9 hypercortisolemic and one hypocortisolemic patient and measured hair cortisol levels. Cortisol was extracted from scalp hair using methanol and cortisol levels were measured using a salivary ELISA kit. Measurement of waist and hip circumferences and blood pressure was performed in 46 healthy subjects.ResultsWe found a positive correlation between hair cortisol and both waist circumference (r = 0.392, p = 0.007) and waist-to-hip ratio (WHR) (r = 0.425, p = 0.003). No correlations were found between hair cortisol levels and BMI, blood pressure or age. There was no decline in cortisol levels in six consecutive hair segments. Hair cortisol levels were elevated in patients with known hypercortisolism (p < 0.0001).ConclusionsHair cortisol was positively correlated with WHR, suggesting that hair cortisol reflects cortisol exposure at tissue level, which was also supported by elevated hair cortisol levels in hypercortisolemic patients and concordance between hair cortisol levels and clinical disease course. Cortisol levels in hair are slightly influenced by hair treatment but not by natural hair colour, use of hair products, gender or age.     

Exceptional thermal tolerance and water resistance in the mite Paratarsotomus macropalpis (Erythracaridae) challenge prevailing explanations of physiological limits

Physiological performance and tolerance limits in metazoans have been widely studied and have informed our understanding of processes such as extreme heat and cold tolerance, and resistance to water loss. Because of scaling considerations, very small arthropods with extreme microclimatic niches provide promising extremophiles for testing predictive physiological models. Corollaries of small size include rapid heating and cooling (small thermal time constants) and high mass-specific metabolic and water exchange rates. This study examined thermal tolerance and water loss in the erythracarid mite Paratarsotomus macropalpis (Banks, 1916), a species that forages on the ground surface of the coastal sage scrub habitat of Southern California, USA. Unlike most surface-active diurnal arthropods, P. macropalpis remains active during the hottest parts of the day in midsummer. We measured water-loss gravimetrically and estimated the critical thermal maximum (CT_max) by exposing animals to a given temperature for 1 h and then increasing temperature sequentially. The standardized water flux of 4.4 ng h⁻¹ cm⁻² Pa⁻¹, averaged for temperatures between 22 and 40 °C, is among the lowest values reported in the literature. The CT_max of 59.4 °C is, to our knowledge, the highest metazoan value reported for chronic (1-h) exposure, and closely matches maximum field substrate temperatures during animal activity. The extraordinary physiological performance seen in P. macropalpis likely reflects extreme selection resulting from its small size and resultant high mass-specific water loss rate and low thermal time-constant. Nevertheless, the high water resistance attained with a very thin lipid barrier, and the mite’s exceptional thermal tolerance, challenge existing theories seeking to explain physiological limits.     

Thermal environment affects hair coat development of Norway rats

Newborn rats have limited homeothermic capabilities and little insulation. Nevertheless, rats flourish in diverse thermal environments. We hypothesized that the environmental temperature influences the development of their hair coat insulation. Rats were housed from birth (0 d) in either a cool (17 °C), moderate (25 °C), or warm (33 °C) environment. Measures were recorded from young juveniles (22 d), old juveniles (43 d), and young adults (85 d). Cool-housed rats were found to develop greater body and coat mass and a higher coat mass to body mass ratio than warm-reared rats. These results indicate that thermal environment affects the growth of the hair coat insulation of developing rats.     

BMI and levels of zinc,copper in hair,serum and urine of Turkish male patients with androgenetic alopecia

ObjectiveMale pattern androgenetic alopecia is characterized by progressive hair loss from the scalp. It is known that imbalances of some trace elements play a role in the pathomechanism of many forms of alopecia. The aim of this study was to evaluate the levels of zinc and copper in hair, serum and urine samples of Turkish males with male pattern androgenetic alopecia and to compare with healthy controls.Material and methods116 males with male pattern androgenetic alopecia and 100 controls were involved in this study.ResultsLevels of zinc and copper in hair were decreased significantly in the patients (p < 0.05), although zinc and copper levels of serum and urine were not different between patients and controls (p > 0.05). Body mass index of patients were higher than control group. In addition, in the group with body mass index of 25 and lower zinc level in hair and urine, copper level in serum were significantly higher (p < 0.05). Body mass index was negatively correlated with hair zinc levels.ConclusionWe thought that decreased zinc and copper levels in hair may play a role in the etiology of male pattern androgenetic alopecia. In addition, obesity by making changes in the balance of the trace elements in hair, serum and urine may play a role in male pattern androgenetic alopecia. Hence, assessing the levels of trace elements in hair of male pattern androgenetic alopecia patients may be more valuable compared to serum and urine for treatment planning.     

Effect of rearing temperature on growth and thermal tolerance of Schizothorax (Racoma) kozlovi larvae and juveniles

Effect of rearing temperature on growth and thermal tolerance of Schizothorax (Racoma) kozlovi Nikolsky larvae and juveniles was investigated. The fish (start at 12 d post hatch) were reared for nearly 6 months at five constant temperatures of 10, 14, 18, 22 and 26 °C. Then juvenile fish being acclimated at three temperatures of 14, 18 and 22 °C were chosen to determine their critical thermal maximum (CTMax) and lethal thermal maximum (LTMax) by using the dynamic method. Growth rate of S. kozlovi larvae and juveniles was significantly influenced by temperature and fish size, exhibiting an increase with increased rearing temperature, but a decline with increased fish size. A significant ontogenetic variation in the optimal temperatures for maximum growth were estimated to be 24.7 °C and 20.6 °C for larvae and juveniles of S. kozlovi, respectively. The results also demonstrated that acclimation temperature had marked effects on their CTMax and LTMax, which ranged from 32.86 °C to 34.54 °C and from 33.79 °C to 34.80 °C, respectively. It is suggested that rearing temperature must never rise above 32 °C for its successful aquaculture. Significant temperature effects on the growth rate and thermal tolerance both exhibit a plasticity pattern. Determination of critical heat tolerance and optima temperature for maximum growth of S. kozlovi is of ecological significance in the conservation and aquaculture of this species.     

Investigation of temperature-dependent viscoelastic properties of thermal lesions in ex vivo animal liver tissue

The viscoelastic characteristics of thermal lesions in ex vivo animal liver are investigated in this paper. Characterization of the moduli of thermal lesions prepared at several temperatures will provide additional information for the elastographic monitoring of radio frequency ablation of hepatic tumors. In this study, the frequency-dependent complex modulus of thermal lesions prepared at temperatures ranging from 60 to 90 °C over a frequency range from 0.1 to 50 Hz are presented. Lesions were prepared using either radio frequency ablation or double immersion boiling. It was found that both the magnitude and phase of the modulus increase with frequency, a behavior that has been noted in the literature. A new result reported shows that the modulus dependence on temperature reveals a local maximum around 70–75 °C corresponding to the temperature at which tissue has released most of its water content. The modulus values at temperatures higher than 70 °C continued to increase, but the extent of increase depend on animal species and other factors.     

Thermal tolerance and preference of exploited turbinid snails near their range limit in a global warming hotspot

Predicted global climate change has prompted numerous studies of thermal tolerances of marine species. The upper thermal tolerance is unknown for most marine species, but will determine their vulnerability to ocean warming. Gastropods in the family Turbinidae are widely harvested for human consumption. To investigate the responses of turbinid snails to future conditions we determined critical thermal maxima (CTMax) and preferred temperatures of Turbo militaris and Lunella undulata from the tropical-temperate overlap region of northern New South Wales, on the Australian east coast. CTMax were determined at two warming rates: 1 °C/30 min and 1 °C/12 h. The number of snails that lost attachment to the tank wall was recorded at each temperature increment. At the faster rate, T. militaris had a significantly higher CTMax (34.0 °C) than L. undulata (32.2 °C). At the slower rate the mean of both species was lower and there was no significant difference between them (29.4 °C for T. militaris and 29.6 °C for L. undulata). This is consistent with differences in thermal inertia possibly allowing animals to tolerate short periods at higher temperatures than is possible during longer exposure times, but other mechanisms are not discounted. The thermoregulatory behaviour of the turban snails was determined in a horizontal thermal gradient. Both species actively sought out particular temperatures along the gradient, suggesting that behavioural responses may be important in ameliorating short-term temperature changes. The preferred temperatures of both species were higher at night (24.0 °C and 26.0 °C) than during the day (22.0 °C and 23.9 °C). As the snails approached their preferred temperature, net hourly displacement decreased. Preferred temperatures were within the average seasonal seawater temperature range in this region. However, with future predicted water temperature trends, the species could experience increased periods of thermal stress, possibly exceeding CTMax and potentially leading to range contractions.     

Trace elements content and hormonal profiles in women with androgenetic alopecia

It is well-known that some trace element imbalances play a significant role in the pathomechanism of many forms of alopecia. Androgenetic alopecia, however, is a specific local sensitivity of hair follicle receptors to androgens. In a clinical and laboratory study, 153 women with androgenetic alopecia (AGA) and 32 control women were examined. In AGA patients telogen hair and vellus hair (miniaturization, D < 30 μm) significantly differed in frontal and parietal hair comparison with occipital area (20 ± 0.9% vs. 12 ± 0.5% and 33 ± 0.9% vs. 12 ± 0.6% respectively). In the AGA group levels of androstenedione and dihydrotestosterone were higher than in the control group. Hair elemental content, analyzed by ICP-MS, demonstrated a lowered Cu and Zn content in the frontal area in comparison to the occipital area. It is important to note, that the AGA patients with elevated levels of androstenedione and dihydrotestosterone presented an increased Cu content and decreased Mn, Se, Zn contents in the occipital area of scalp. The occipital level of Cu positively correlated with the concentration of free testosterone in the serum. A negative correlation between the Zn content in the occipital area and the dehydroepiandrosterone level in the blood was found. Unfortunately, a routine treatment course of AGA patients, including topical inhibitor of 5-alpha-reductase and minoxidil, had no effect on the Cu hair content in occipital and frontal areas. However, there were positive changes in the morphological structure and other trace element contents. These data led us to hypothesize a key role of Cu metabolism disturbances in the AGA onset, development of AGA, and potential pharmaceutical targets for the treatment of AGA.     

Injury due to extravasation of thiopental and propofol: Risks/effects of local cooling/warming in rats

Inadvertent leakage of medications with vesicant properties can cause severe necrosis in tissue, which can have devastating long-term consequences. The aim of this study was to evaluate the extent of extravasation injury induced by thiopental and propofol, and the effects of cooling or warming of local tissue on extravasation injury at macroscopic and histopathologic levels. Rats were administered intradermally thiopental (2.5 mg/100 µL) or propofol (1.0 mg/100 µL). Rats were assigned randomly to three groups: control (no treatment), cooling and warming. Local cooling (18–20 °C) or warming (40–42 °C) was applied for 3 h immediately after agent injection. Lesion sizes (erythema, induration, ulceration, necrosis) were monitored after agent injection. Histopathology was evaluated in skin biopsies taken 24 h after agent injection. Thiopental injection induced severe skin injury with necrosis. Peak lesions developed within 24 h and healed gradually 18–27 days after extravasation. Propofol induced inflammation but no ulceration, and lesions healed within 1–2 days. Local cooling reduced thiopental- and propofol-induced extravasation injuries but warming strongly exacerbated the skin lesions (e.g., degeneration, necrosis) induced by extravasation of thiopental and propofol. Thiopental can be classified as a “vesicant” that causes tissue necrosis and propofol can be classified as an “irritant”. Local cooling protects (at least in part) against skin disorders induced by thiopental and propofol, whereas warming is harmful.     

Skin temperature changes in humans induced by local peripheral cooling

Local peripheral cooling (immerson of legs up to the knees into 12°C water) increased heart rate and blood pressure by 10–20% within the first 3–10 min of cooling. During further cooling heart rate remained elevated, while systolic and diastolic blood pressures decreased to the control value. Data on heart rate indicate a permanent activation of the sympathetic nervous system during local cooling.Skin temperatures (measured topically by thermosensors) decreased on some non-cooled areas of the body (fingers, palms and thighs) immediately after the start of local cooling. On the other hand, skin temperatures on chest and forehead were not influenced. During cooling skin temperatures on thighs remained low, but skin temperatures on fingers tended to increase. Changes in skin temperatures on non-cooled areas of the body indicate that a permanent and generalized activation of the sympathetic nervous system occurs during local cooling.Cold induced cycles of vasodilation (CIVD) were observed on fingers, palms and forearms during local cooling. Minute cycles in skin temperatures were observed on forehead, thighs and chest. Minute cycles coincided with those in the heart rate, indicating a permanent, generalized but discontinuous control of vasomotion by the sympathetic nervous system during local cooling.Infrared thermographic recordings from different body areas indicated that local peripheral cooling lowered skin temperatures in all areas of the body within 5 min. Distant areas of the body (extremities) and pectoral muscles showed greater hypothermia than abdominal areas and head. After 10 min of cooling average skin temperatures in all areas of the body returned to the original level and further fluctuated at approximately 10–15 min intervals.Data indicate that during local cooling skin blood flow in all areas of the body surface permanently fluctuates forming a mosaic of dynamic changes in skin temperatures. Since tympanic temperature increases, while skin temperature decreases immediately after the start of the local cooling, it appears that the initial vasoconstrictor response is being controlled independently of the central temperature input.     

Thermal stability of starch degrading enzymes of teff (Eragrostis tef) malt during isothermal mashing

Thermal stability of starch degrading enzymes varies from one source to another. This research was aimed to study thermal stability of starch degrading enzymes of teff malt. Isothermal mashing at temperatures ranging between 40 and 75 °C with sampling in 15 min interval for a total of 90 min was conducted. The study showed that deactivation rate constants of alpha- and beta-amylases ranged from 0.0003 to 0.0409 min^?1, and 0.002 to 0.032 min^?1, respectively. Rate of deactivation of limit dextrinase was not significant at temperatures lower than 60 °C but showed high deactivation at higher temperatures with rate constants ranging from 0.02 to 0.1 min^?1. The thermal deactivation energies of alpha-amylase, beta-amylase, and limit dextrinase were found to be 148, 82, and 144 kJ/mol, respectively. The present findings have significant applications in commercial processes where determination of the upper temperature limits for these enzymes is required.     

Effects of rapid temperature fluctuations prior to breeding on reproductive efficiency in replacement gilts

Rapidly cooling pigs after heat stress (HS) results in a pathophysiological condition, and because rapid temperature fluctuations may be associated with reduced reproductive success in sows, it lends itself to the hypothesis that these conditions may be linked. Objectives were to determine the effects of rapid cooling on thermal response and future reproductive success in pigs. Thirty-six replacement gilts (137.8±0.9 kg BW) were estrus synchronized and then 14.1±0.4 d after estrus confirmation, pigs were exposed to thermoneutral conditions (TN; n=12; 19.7±0.9°C) for 6 h, or HS (36.3±0.5°C) for 3 h, followed by 3 h of rapid cooling (HSRC; n=12; immediate TN exposure and water dousing) or gradual cooling (HSGC; n=12; gradual decrease to TN conditions) repeated over 2 d. Vaginal (T_V) and gastrointestinal tract temperatures (T_GI) were obtained every 15 min, and blood was collected on d 1 and d 2 during the HS and recovery periods at 180 and 60 min, respectively. Pigs were bred 8.3±0.8 d after thermal treatments over 2 d. Reproductive tracts were collected and total fetus number and viability were recorded 28.0±0.8 d after insemination. HS increased T_V and T_GI (P=0.01; 0.98 °C) in HSRC and HSGC compared to TN pigs. During recovery, T_V was reduced from 15 to 105 min (P=0.01; 0.33 °C) in HSRC compared to HSGC pigs, but no overall differences in T_GI were detected (P<0.05; 39.67 °C). Rapid cooling increased (P<0.05) TNFα compared to HSGC and TN pigs during recovery-d 1 (55.2%), HS-d 2 (35.1%), and recovery-d 2 (64.9%). Viable fetuses tended to be reduced (P=0.08; 10.5%) and moribund fetuses tended to be increased (P=0.09; 159.3%) in HSRC compared to HSGC and TN pigs. In summary, rapid cooling prior to breeding may contribute to reduced fetal viability and reproductive success in pigs.     

The effects of increased constant incubation temperature and cumulative acute heat shock exposures on morphology and survival of Lake Whitefish (Coregonus clupeaformis) embryos

Increasing incubation temperatures, caused by global climate change or thermal effluent from industrial processes, may influence embryonic development of fish. This study investigates the cumulative effects of increased incubation temperature and repeated heat shocks on developing Lake Whitefish (Coregonus clupeaformis) embryos. We studied the effects of three constant incubation temperatures (2 °C, 5 °C or 8 °C water) and weekly, 1-h heat shocks (+3 °C) on hatching time, survival and morphology of embryos, as these endpoints may be particularly susceptible to temperature changes. The constant temperatures represent the predicted magnitude of elevated water temperatures from climate change and industrial thermal plumes. Time to the pre-hatch stage decreased as constant incubation temperature increased (148 d at 2 °C, 92 d at 5 °C, 50 d at 8 °C), but weekly heat shocks did not affect time to hatch. Mean survival rates and embryo morphometrics were compared at specific developmental time-points (blastopore, eyed, fin flutter and pre-hatch) across all treatments. Constant incubation temperatures or +3 °C heat-shock exposures did not significantly alter cumulative survival percentage (~50% cumulative survival to pre-hatch stage). Constant warm incubation temperatures did result in differences in morphology in pre-hatch stage embryos. 8 °C and 5 °C embryos were significantly smaller and had larger yolks than 2 °C embryos, but heat-shocked embryos did not differ from their respective constant temperature treatment groups. Elevated incubation temperatures may adversely alter Lake Whitefish embryo size at hatch, but weekly 1-h heat shocks did not affect size or survival at hatch. These results suggest that intermittent bouts of warm water effluent (e.g., variable industrial emissions) are less likely to negatively affect Lake Whitefish embryonic development than warmer constant incubation temperatures that may occur due to climate change.     

Breaking the temperature-size rule: Thermal effects on growth,development and fecundity of a crustacean from temporary waters

The temperature-size rule (TSR) is a well-established phenomenon to describe the growth response of ectotherms to temperature by which individuals maintained at low temperatures grow more slowly, but attain a larger size upon maturity. Although there are adaptive and non-adaptive theories about the plasticity of body size in response to temperature, these cannot be applied to all ectotherms, and little is known about the changes in growth and development rates through ontogeny. The ostracod species Heterocypris bosniaca, an inhabitant of freshwater temporary ponds, was used to examine the growth and development rates of its nine growth stages and female fecundity at four different temperatures (15 °C, 20 °C, 25 °C and 30 °C). The development rate of this species accelerates with increasing temperature, reaching a maximum value at 25 °C. The growth factor has a reverse-TSR in younger instars, and the typical TSR is followed only in the last two moults, resulting in non-monotonic response of adult size to temperature. Fecundity (total offspring per female) was not directly related to adult size and was generally higher at lower temperatures. Our results agree with recent research showing that the TSR may vary during ontogeny, and may not be a general trend in ostracod species from temporary waters. Indeed, adult carapace size seems to follow the pattern of a thermal reaction norm, probably influenced by the reduction of oxygen bioavailability at low temperature and the drastic increase in metabolic demand at the upper extreme of the thermal gradient.     

Explicit formula of finite difference method to estimate human peripheral tissue temperatures during exposure to severe cold stress

During cold exposure, peripheral tissues undergo vasoconstriction to minimize heat loss to preserve the maintenance of a normal core temperature. However, vasoconstricted tissues exposed to cold temperatures are susceptible to freezing and frostbite-related tissue damage. Therefore, it is imperative to establish a mathematical model for the estimation of tissue necrosis due to cold stress. To this end, an explicit formula of finite difference method has been used to obtain the solution of Pennes' bio-heat equation with appropriate boundary conditions to estimate the temperature profiles of dermal and subdermal layers when exposed to severe cold temperatures. The discrete values of nodal temperature were calculated at the interfaces of skin and subcutaneous tissues with respect to the atmospheric temperatures of 25 °C, 20 °C, 15 °C, 5 °C, −5 °C and −10 °C. The results obtained were used to identify the scenarios under which various degrees of frostbite occur on the surface of skin as well as the dermal and subdermal areas. The explicit formula of finite difference method proposed in this model provides more accurate predictions as compared to other numerical methods. This model of predicting tissue temperatures provides researchers with a more accurate prediction of peripheral tissue temperature and, hence, the susceptibility to frostbite during severe cold exposure.     

Determination of heat transfer coefficients in plastic French straws plunged in liquid nitrogen

The knowledge of the thermodynamic process during the cooling of reproductive biological systems is important to assess and optimize the cryopreservation procedures. The time–temperature curve of a sample immersed in liquid nitrogen enables the calculation of cooling rates and helps to determine whether it is vitrified or undergoes phase change transition. When dealing with cryogenic liquids, the temperature difference between the solid and the sample is high enough to cause boiling of the liquid, and the sample can undergo different regimes such as film and/or nucleate pool boiling.In the present work, the surface heat transfer coefficients (h) for plastic French straws plunged in liquid nitrogen were determined using the measurement of time–temperature curves. When straws filled with ice were used the cooling curve showed an abrupt slope change which was attributed to the transition of film into nucleate pool boiling regime. The h value that fitted each stage of the cooling process was calculated using a numerical finite element program that solves the heat transfer partial differential equation under transient conditions. In the cooling process corresponding to film boiling regime, the h that best fitted experimental results was h = 148.12 ± 5.4 W/m² K and for nucleate-boiling h = 1355 ± 51 W/m² K. These values were further validated by predicting the time–temperature curve for French straws filled with a biological fluid system (bovine semen-extender) which undergoes freezing. Good agreement was obtained between the experimental and predicted temperature profiles, further confirming the accuracy of the h values previously determined for the ice-filled straw. These coefficients were corroborated using literature correlations.The determination of the boiling regimes that govern the cooling process when plunging straws in liquid nitrogen constitutes an important issue when trying to optimize cryopreservation procedures. Furthermore, this information can lead to improvements in the design of cooling devices in the cryobiology field.     

Model-based cap thickness and peak cap stress prediction for carotid MRI

A rupture-prone carotid plaque can potentially be identified by calculating the peak cap stress (PCS). For these calculations, plaque geometry from MRI is often used. Unfortunately, MRI is hampered by a low resolution, leading to an overestimation of cap thickness and an underestimation of PCS. We developed a model to reconstruct the cap based on plaque geometry to better predict cap thickness and PCS.We used histological stained plaques from 34 patients. These plaques were segmented and served as the ground truth. Sections of these plaques contained 93 necrotic cores with a cap thickness <0.62 mm which were used to generate a geometry-based model. The histological data was used to simulate in vivo MRI images, which were manually delineated by three experienced MRI readers. Caps below the MRI resolution (n = 31) were (digitally removed and) reconstructed according to the geometry-based model. Cap thickness and PCS were determined for the ground truth, readers, and reconstructed geometries.Cap thickness was 0.07 mm for the ground truth, 0.23 mm for the readers, and 0.12 mm for the reconstructed geometries. The model predicts cap thickness significantly better than the readers. PCS was 464 kPa for the ground truth, 262 kPa for the readers and 384 kPa for the reconstructed geometries. The model did not predict the PCS significantly better than the readers.The geometry-based model provided a significant improvement for cap thickness estimation and can potentially help in rupture-risk prediction, solely based on cap thickness. Estimation of PCS estimation did not improve, probably due to the complex shape of the plaques.     

Impact of cofactor-binding loop mutations on thermotolerance and activity of E. coli transketolase

Improvement of thermostability in engineered enzymes can allow biocatalysis on substrates with poor aqueous solubility. Denaturation of the cofactor-binding loops of Escherichia coli transketolase (TK) was previously linked to the loss of enzyme activity under conditions of high pH or urea. Incubation at temperatures just below the thermal melting transition, above which the protein aggregates, was also found to anneal the enzyme to give an increased specific activity. The potential role of cofactor-binding loop instability in this process remained unclear. In this work, the two cofactor-binding loops (residues 185–192 and 382–392) were progressively mutated towards the equivalent sequence from the thermostable Thermus thermophilus TK and variants assessed for their impact on both thermostability and activity. Cofactor-binding loop 2 variants had detrimental effects on specific activity at elevated temperatures, whereas the H192P mutation in cofactor-binding loop 1 resulted in a two-fold improved stability to inactivation at elevated temperatures, and increased the critical onset temperature for aggregation. The specific activity of H192P was 3-fold and 19-fold higher than that for wild-type at 60 °C and 65 °C respectively, and also remained 2.7-4 fold higher after re-cooling from pre-incubations at either 55 °C or 60 °C for 1 h. Interestingly, H192P was also 2-times more active than wild-type TK at 25 °C. Optimal activity was achieved at 60 °C for H192P compared to 55 °C for wild type. These results show that cofactor-binding loop 1, plays a pivotal role in partial denaturation and aggregation at elevated temperatures. Furthermore, a single rigidifying mutation within this loop can significantly improve the enzyme specific activity, as well as the stability to thermal denaturation and aggregation, to give an increased temperature optimum for activity.     

The effect of acclimation temperature on thermal activity thresholds in polar terrestrial invertebrates

In the Maritime Antarctic and High Arctic, soil microhabitat temperatures throughout the year typically range between ?10 and +5 °C. However, on occasion, they can exceed 20 °C, and these instances are likely to increase and intensify as a result of climate warming. Remaining active under both cool and warm conditions is therefore important for polar terrestrial invertebrates if they are to forage, reproduce and maximise their fitness. In the current study, lower and upper thermal activity thresholds were investigated in the polar Collembola, Megaphorura arctica and Cryptopygus antarcticus, and the mite, Alaskozetes antarcticus. Specifically, the effect of acclimation on these traits was explored. Sub-zero activity was exhibited in all three species, at temperatures as low as ?4.6 °C in A. antarcticus. At high temperatures, all three species had capacity for activity above 30 °C and were most active at 25 °C. This indicates a comparable spread of temperatures across which activity can occur to that seen in temperate and tropical species, but with the activity window shifted towards lower temperatures. In all three species following one month acclimation at ?2 °C, chill coma (=the temperature at which movement and activity cease) and the critical thermal minimum (=low temperature at which coordination is no longer shown) occurred at lower temperatures than for individuals maintained at +4 °C (except for the CTmin of M. arctica). Individuals acclimated at +9 °C conversely showed little change in their chill coma or CTmin. A similar trend was demonstrated for the heat coma and critical thermal maximum (CTmax) of all species. Following one month at ?2 °C, the heat coma and CTmax were reduced as compared with +4 °C reared individuals, whereas the heat coma and CTmax of individuals acclimated at +9 °C showed little adjustment. The data obtained suggest these invertebrates are able to take maximum advantage of the short growing season and have some capacity, in spite of limited plasticity at high temperatures, to cope with climate change.