Repeatability of a running heat tolerance test

At present there is no standardised heat tolerance test (HTT) procedure adopting a running mode of exercise. Current HTTs may misdiagnose a runner's susceptibility to a hyperthermic state due to differences in exercise intensity. The current study aimed to establish the repeatability of a practical running test to evaluate individual's ability to tolerate exercise heat stress. Sixteen (8M, 8F) participants performed the running HTT (RHTT) (30 min, 9 km h⁻¹, 2% elevation) on two separate occasions in a hot environment (40 °C and 40% relative humidity). There were no differences in peak rectal temperature (RHTT1: 38.82±0.47 °C, RHTT2: 38.86±0.49 °C, Intra-class correlation coefficient (ICC)=0.93, typical error of measure (TEM)=0.13 °C), peak skin temperature (RHTT1: 38.12±0.45, RHTT2: 38.11±0.45 °C, ICC=0.79, TEM=0.30 °C), peak heart rate (RHTT1: 182±15 beats min⁻¹, RHTT2: 183±15 beats min⁻¹, ICC=0.99, TEM=2 beats min⁻¹), nor sweat rate (1721±675 g h⁻¹, 1716±745 g h⁻¹, ICC=0.95, TEM=162 g h⁻¹) between RHTT1 and RHTT2 (p>0.05). Results demonstrate good agreement, strong correlations and small differences between repeated trials, and the TEM values suggest low within-participant variability. The RHTT was effective in differentiating between individuals physiological responses; supporting a heat tolerance continuum. The findings suggest the RHTT is a repeatable measure of physiological strain in the heat and may be used to assess the effectiveness of acute and chronic heat alleviating procedures.     

Contemporary carbon accumulation in a boreal oligotrophic minerogenic mire – a significant sink after accounting for all C-fluxes

Based on theories of mire development and responses to a changing climate, the current role of mires as a net carbon sink has been questioned. A rigorous evaluation of the current net C-exchange in mires requires measurements of all relevant fluxes. Estimates of annual total carbon budgets in mires are still very limited. Here, we present a full carbon budget over 2 years for a boreal minerogenic oligotrophic mire in northern Sweden (64°11′N, 19°33′E). Data on the following fluxes were collected: land–atmosphere CO₂ exchange (continuous Eddy covariance measurements) and CH₄ exchange (static chambers during the snow free period); TOC (total organic carbon) in precipitation; loss of TOC, dissolved inorganic carbon (DIC) and CH₄ through stream water runoff (continuous discharge measurements and regular C-concentration measurements). The mire constituted a net sink of 27±3.4 (±SD) g C m⁻² yr⁻¹ during 2004 and 20±3.4 g C m⁻² yr⁻¹ during 2005. This could be partitioned into an annual surface–atmosphere CO₂ net uptake of 55±1.9 g C m⁻² yr⁻¹ during 2004 and 48±1.6 g C m⁻² yr⁻¹ during 2005. The annual NEE was further separated into a net uptake season, with an uptake of 92 g C m⁻² yr⁻¹ during 2004 and 86 g C m⁻² yr⁻¹ during 2005, and a net loss season with a loss of 37 g C m⁻² yr⁻¹ during 2004 and 38 g C m⁻² yr⁻¹ during 2005. Of the annual net CO₂-C uptake, 37% and 31% was lost through runoff (with runoff TOC>DIC≫CH₄) and 16% and 29% through methane emission during 2004 and 2005, respectively. This mire is still a significant C-sink, with carbon accumulation rates comparable to the long-term Holocene C-accumulation, and higher than the C-accumulation during the late Holocene in the region.     

Shifts in thermoregulatory strategy during ontogeny in harp seals (Pagophilus groenlandicus)

Heat balance can be difficult for young and/or small animals in polar regions because environmental conditions in combination with small body size or physiological immaturity can increase heat loss. We investigated how thermoregulatory patterns change with ontogeny in 5 age classes of harp seal (Pagophilus groenlandicus) from birth to post-molt to further understand the timing of thermoregulatory development in relation to their potential vulnerability to ongoing fluctuations in the extent and stability of Arctic pack ice. We measured changes in the amount, conductivity, and resistance of the seal pups׳ insulative layers (blubber and fur), the potential for endogenous heat-generation by shivering (muscle enzyme activity), and nonshivering thermogenesis (NST; brown adipose tissue (BAT) uncoupling protein 1 (UCP1) expression and mitochondrial density). There was no significant difference in blubber conductivity among age classes, though the amount of blubber insulation significantly increased from birth to weaning. Pelage conductivity was low (0.12±0.01 W m⁻¹ °C⁻¹) except in 9-day old pups (0.40±0.08 W m⁻¹ °C⁻¹); the significantly higher conductivity may signal the beginning of the molt, and this age group may be the most vulnerable to early water entry. Citrate synthase activity significantly increased (49.68±3.26 to 75.08±3.52 μmol min⁻¹ g wet weight⁻¹) in the muscle; however it is unlikely that increasing a single enzyme greatly impacts heat generation. BAT of younger pups contained UCP1, though expression and mitochondrial density quickly declined, and the ability of pups to produce heat via NST was lost by weaning. While total thermal resistance did not differ, neonatal and early nursing animals gained the majority of their thermal resistance from lanugo (82.5±0.03%); however, lanugo is not insulative when wet, and NST may be important to maintain euthermia and dry the coat if early immersion in water occurs. By late nursing, blubber seems sufficient as insulation (75.87±0.01% of resistance after 4 weeks), but high conductivity of fur may be responsible for retention of UCP1 expression. Weaned animals rely on blubber insulation, and no longer need NST, as wetted fur is no longer a threat to euthermia.     

Cryopreservation of Babesia caballi cultures

Babesia caballi cultures were cryopreserved with a solution of 10% (w/v) polyvinylpyrrolidone 40 as cryoprotectant. Samples were cooled at rates of 1, 10, 30 and 100°C min⁻¹ using a programmable freezer. Additionally, a styrofoam box designed to cool samples at an approximate rate of 10°C min⁻¹ when placed in a −80°C freezer was used. Samples were stored in liquid nitrogen, thawed rapidly and inoculated into cultures. Although, a high loss of infectivity was observed after cryopreservation, cultures could be initiated reliably from cryo-stabilates frozen at a rate of 10 and 30°C min⁻¹ or frozen with the styrofoam box.     

Characterization of receptors for α2-macroglobulin-trypsin complex in rat hepatocytes

High-affinity receptors for α₂-macroglobulin-trypsin complex were demonstrated in rat hepatocytes at 4°C. The dissociation rate constant for the labelled complex was very small at low receptor occupancies, approx. 4·10⁻⁴ min⁻¹. Dissociation was biphasic at high receptor occupancies with a rate constant for the rapid phase of about 2·10⁻² min⁻¹. At near-equilibrium, half of the receptors were saturated at a complex concentration of 150 pM, and the Scatchard plot was concave upwards. Thus, the binding shows complex kinetics with the probable involvement of negative cooperativity. Binding of the labelled complex was not influenced by galactose, mannose, mannose phosphate or fucoidin, whereas it was abolished in the absence of extracellular Ca²⁺ and inhibited by bacitracin. Approx. 70% of the labelled complex bound at 4°C was rapidly internalized (k_int about 3·10⁻¹ min⁻¹) after being warmed to 37°C. Radioactivity released from the cells at 37°C comprised intact labelled complex and iodide. The complex was initially released at a rapid rate (k₋₁ about 1·10⁻¹ min⁻¹) from about 25% of the cell-bound pool. This probably represents dissociation from the receptors. A slow phase of release followed, so that half of the bound pool was finally released as intact complex. Iodide release followed a sigmoidal curve after a 20 min lag period. Thus, specific high-affinity receptors mediate the internalization and eventual degradation of α₂-macroglobulin-proteinase complex into hepatocytes.     

Solubilization,characterization and photoaffinity labeling of the mitochondrial dihydropyridine receptor from bovine adrenal medulla

• 1.1. The mitochondrial dihydropyridine receptor was solubilized with Chaps at a detergent/ protein ratio of 2.5, during 45 min at 4°C.
• 2.2. From the rate constants of association (8.10 ± 0.25 × 10⁴ M⁻¹ min⁻¹) and dissociation (0.022 ± 0.001 min⁻¹ a K_d of 275 nM was calculated, while from saturation experiments a K_d of 270 ± 30 nM and a density of receptors of 106 ± 9 pmol/mg protein was obtained.
• 3.4. The solubilized receptors are heat-resistant, sensitive to the trypsin and to the reduction of disulfide bonds.
• 4.5. In native membranes, a polypeptide of 50 kDa was specifically photolabelled with [³H]Azidopine.

     

Production enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Halogeometricum borinquense,characterization of the bioplastic and desalination of the bioreactor effluent

Polyhydroxyalkanoates (PHAs) are a replacement of conventional single-use plastics. Bioprocess conditions of the extreme halophilic archaeon Halogeometricum borinquense strain RM-G1 were selected resulting in the synthesis of 66.80 ± 1.69 % PHA (of cell dry mass) in 72 h using glycerol and tryptone as carbon and nitrogen sources respectively, yielding volumetric productivity of 0.206 ± 0.006 gL⁻¹ h⁻¹ in a repeated batch process in a small-scale bioreactor where 20 % of the production medium was used as the inoculum for the subsequent batch. The purified PHA was characterized as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with 10.21 mol% 3-hydroxyvalerate content possessing glass transition temperature -12.6 °C, degradation temperature 285 °C, number average molecular weight 156,899 Da, weight average molecular weight 288,723 Da, polydispersity index 1.8 and melting temperatures 139.1 °C and 152.5 °C. Maximum (21.7 ± 0.6 L m^-2 h⁻¹) and average (17.2 ± 0.6 L m^-2 h⁻¹) flux values were their respective highest and crystallization time was its least (3.0 ± 0.16 h) when ΔT was 90 °C and polytetrafluoroethylene membrane was applied for desalination of the bioreactor effluent by Direct Contact Membrane Distillation. While using polyvinylidene fluoride membrane, maximum 25.5 ± 0.5 L m^-2 h⁻¹ and average 18.6 ± 0.2 L m^-2 h⁻¹ fluxes were obtained and crystallization time decreased (3.25 ± 0.16 h) even when ΔT was lowered by 20 °C.     

High throughput covalent immobilization process for improvement of shelf-life,operational cycles,relative activity in organic media and enzymatic kinetics of urease and its application for urea removal from water samples

High throughput covalent urease immobilization was performed through the amide bond formation between the urease and the amino-functional MNPs. The enzyme’s performances, including shelf-life, reusability, enzymatic kinetics, and the enzyme relative activity in organic media was improved. At optimal conditions, the immobilization efficiency was calculated about 95.0% with keeping 94.7% of the urease initial specific activity. The optimal pH for maximum activity of the free and immobilized urease was calculated as 7.0 at 37.0 °C and 8.0 at 60.0 °C, respectively. The kinetics studies showed the K_m of 26.0 mM and 8.0 mM and the V_max of 5.31 μmol mg⁻¹ min⁻¹ and 3.93 μmol mg⁻¹ min⁻¹ for the free and immobilized urease, respectively. The ratio K_cat/K_m as a measure of catalytic efficiency and enzyme specificity was calculated as 0.09 mg mL⁻¹ min⁻¹ and 0.22 mg mL⁻¹ min⁻¹ for the free and immobilized urease, respectively, indicating an improvement in the enzymatic kinetics. The shelf-life and operational studies of immobilized urease indicated that approximately 97.7% and 88.5% of its initial activity was retained after 40 days and 17 operational cycles, respectively. The immobilized urease was utilized to urea removal from water samples with an efficiency between 91.5–95.0%.     

Heart rate as an indicator of stress during the critical swimming speed test of farmed Atlantic salmon (Salmo salar L.)

A swim tunnel is to fish as a treadmill is to humans, and is a device used for indirect measuring of the metabolic rate. This study aims to explore the fish stress (if any) during the critical swimming test routines (fish handling, confinement, and swimming) using heart rate (f_H, heartbeat per minute) bio-loggers in farmed Atlantic salmon (Salmo salar L.). In addition, the recovery dynamics of exercised fish using f_H were explored for 48 h post swim tests. Continuous f_H data were acquired following the surgical implantation and throughout the trials, such as during fish handling, swim tests (critical swimming speed, U_crit), and 48 h post swim tests. After 3 weeks of surgical recovery, f_H stabilized at 46.20 ± 1.26 beats min⁻¹, equalizing a ~38% reduction in f_H recorded post-surgical tachycardia (74.13 ± 1.44 beats min⁻¹). Interestingly, f_H was elevated by ~200% compared to baseline levels not only due to the U_crit (92.04 ± 0.23 beats min⁻¹) but also due to fish handling and confinement in the swim tunnel, which was 66% above the baseline levels (77.48 ± 0.34 beats min⁻¹), suggesting fish stress. Moreover, significantly higher plasma cortisol levels (199.56 ± 77.17 ng mL⁻¹) corresponding to a ~300% increase compared to baseline levels (47.92 ± 27.70 ng mL⁻¹) were identified after U_crit, predicting post-swim test stress (physiological exhaustion). These findings reinforce the importance of fish acclimation in the swim tunnel prior to the swimming tests. However, f_H dropped over the course of the 48-h post-swim test, but remained comparatively higher than the basal levels, suggesting fish should be given at least 48 h to recover from handling stress for better fish welfare. This study further explored the influence of fish tagging on U_crit, which resulted in reduced swimming capabilities of tagged fish (1.95 ± 0.37 body lengths s⁻¹) compared to untagged fish (2.54 ± 0.42 body length s⁻¹), although this was not significant (p = 0.06), and therefore future tagging studies are warranted.     

Heart rates of Atlantic salmon Salmo salar during a critical swim speed test and subsequent recovery

In this study, heart rate (HR) bio-loggers were implanted in the abdominal cavity of 12 post-smolt Atlantic salmon Salmo salar weighing 1024 ± 31 g and acclimated to 12°C sea water. One week after the surgical procedure, a critical swim speed (U_crit) test was performed on tagged and untagged conspecifics, whereafter tagged fish were maintained in their holding tanks for another week. The U_crit was statistically similar between tagged and untagged fish (2.67 ± 0.04 and 2.74 ± 0.05 body lengths s⁻¹, respectively) showing that the bio-logger did not compromise the swimming performance. In the pre-swim week, a diurnal cycle was apparent with HR peaking at 65 beats min⁻¹ during the day and approaching 40 beats min⁻¹ at night. In the U_crit test, HR increased approximately exponentially with swimming speed until a plateau was reached at the final speed before fatigue with a maximum of 85.2 ± 0.7 beats min⁻¹. During subsequent recovery tagged fish could be divided into a surviving group (N = 8) and a moribund group (N = 4). In surviving fish HR had fully recovered to pre-swim levels after 24 h, including reestablishment of a diurnal HR cycle. In moribund fish HR never recovered and remained elevated at c. 80 beats min⁻¹ for 4 days, whereafter they started dying. We did not identify a proximal cause of death in moribund fish, but possible explanations are discussed. Tail beat frequency (TBF) was also measured and showed a more consistent response to increased swimming speeds. As such, when exploring correlations between HR, TBF and metabolic rates at different swimming speeds, TBF provides better predictions. On the contrary, HR measurements in free swimming fish over extended periods of time are useful for other purposes such as assessing the accumulative burden of various stressors and recovery trajectories from exhaustive exercise.     

Improving hydrogen production in microbial electrolysis cells through hydraulic connection with thermoelectric generators

Using alternative power sources to drive hydrogen production in microbial electrolysis cells (MECs) is important to implementation of MEC technology. Herein, thermoelectric generators (TEG) were to power MECs using simulated waste heat. With the MEC anolyte as a cold source for TEG, current generation of the MEC increased to 2.46 ± 0.06 mA and hydrogen production reached 0.14 m³ m⁻³ d^-1, higher than those of the TEG-MEC system without hydraulic connection (1.16 ± 0.07 mA and 0.07 ± 0.01 m³ m⁻³ d^-1). A high recirculation rate of 30 mL min^-1 doubled both current generation and hydrogen production with 10 mL min^-1, benefited from a stronger cooling effect that increased the TEG voltage output. However, the optimal recirculation rate was determined as 20 mL min^-1 because of comparable performance but potentially less energy requirement. Reducing anolyte hydraulic retention time to 4 h has increased hydrogen production to 0.25 ± 0.05 m³ m⁻³ d^-1 but decreased organic removal efficiency to 69 ± 2%. Adding three more TEG units that captured more heat energy further enhanced hydrogen production to 0.36 m³ m⁻³ d^-1. Those results have demonstrated a successful integration of TEG with MEC through both electrical and hydraulic connections for simultaneous wastewater treatment and energy recovery.     

Digital heart rate monitor derived from the arterial pressure pulse

This monitor displays heart rate without the need for electrical contact with the experimental animal. The device uses a quartz pressure transducer connected to an arterial catheter and has a full scale accuracy of ± 3 beats min⁻¹; its range is 0–400 beats min⁻¹. There is an output voltage proportional to heart rate and internal calibration facilities are provided at 60 and 360 beats min⁻¹.     

Ventilation and oxygen consumption in the hagfish, Myxine glutinosa L.

Ventilation was measured directly in the hagfish, Myxine glutinosa L., by means of an electro-magnetic blood flowmeter. Ventilatory flow and frequency increased from 0.86 ± 0.27 ml·min^?, and 18.2 ± 5.1·min^?, respectively, at 7°C to 1.70 ± 0.20 ml·min^?, and 70.1 ± 9.5·min^? at 15 ·C.Standard oxygen consumption,V?O₂, was measured in non-buried hagfish. V?O₂ was 0.57 ± 0.17μl O₂·g^?1·min^?1 at 7°C, and 0.85 ± 0.12μl O₂·g^?1·min^?1 at 15°C.     

Metabolic rates in harvestmen (Arachnida, Opiliones): the influence of running activity

Abstract. Metabolic rates of adult Lophopilio palpinalis (Herbst, 1799) (Arachnida, Opiliones, Phalangioidea) and Paranemastoma quadripunctatum (Perty, 1833) (Arachnida, Opiliones, Troguloidea) are measured during rest and activity. Carbon dioxide release during rest is continuous in both species. Mean values at 20 °C are 4.2 µL min⁻¹ g⁻¹ for the males of P. quadripunctatum, 4.1 µL min⁻¹ g⁻¹ for the males of L. palpinalis and 4.7 µL min⁻¹ g⁻¹ for the females of L. palpinalis, thus being significantly higher in the egg-producing females. In L. palpinalis, respiratory quotient at rest is 0.84. Spontaneous walking activity with speeds of 15–30 cm min⁻¹ raises the metabolic rate by up to three-fold in both species. Lophopilio palpinalis is made to undertake constant running on a treadmill with speeds of 60, 72 and 96 cm min⁻¹. Enforced activity causes the animals to raise their metabolic rates by up to five-fold above resting rates. Animals reach a steady state of CO₂ release on the treadmill and show a fast t_1/2 on-response, indicating aerobic exercise. The minimum cost of locomotion is determined to be 2.5 × 10⁻³ J cm⁻¹ g⁻¹, thus fitting the predicted values for terrestrial locomotion.     

Effects of temperature on metabolic rate and lower dissolved oxygen tolerance of juvenile speckled peacock bass Cichla temensis

The speckled peacock bass Cichla temensis is a popular sport and food fish that generates substantial angling tourism and utilitarian harvest within its range. Its popularity and value make this species important for management and a potential aquaculture candidate for both fisheries enhancement and food fish production. However, little is known of optimal physiochemical conditions in natural habitats, which also are important for the development of hatchery protocols for handling, spawning and grow-out. Speckled peacock bass have been documented to have high sensitivity to extreme temperatures, but the metabolic underpinnings have not been evaluated. In this study, the effects of temperature (25, 30 and 35°C) on the standard metabolic rate (SMR) and lower dissolved oxygen tolerance (LDOT) of juvenile speckled peacock bass (mean ± standard error total length 153 ± 2 mm and wet weight 39.09 ± 1.37 g) were evaluated using intermittent respirometers after an acclimation period of 2 weeks. Speckled peacock bass had the highest SMR at 35°C (345.56 ± 19.89 mgO₂ kg⁻¹ h⁻¹), followed by 30°C (208.16 ± 12.45 mgO₂ kg⁻¹ h⁻¹) and 25°C (144.09 ± 10.43 mgO₂ kg⁻¹ h⁻¹). Correspondingly, the Q₁₀, or rate of increase in aerobic metabolic rate (MO₂) relative to 10°C, for 30–35°C was also greater (2.76) than from 25 to 30°C (2.08). Similarly, speckled peacock bass were the most sensitive to hypoxia at the warmest temperature, with an LDOT at pO₂ of 90 mmHg (4.13 mg l⁻¹) at 35°C compared to pO₂ values of 45 mmHg (2.22 mg l⁻¹) and 30 mmHg (1.61 mg l⁻¹) at 30 and 25°C, respectively. These results indicate that speckled peacock bass are sensitive to temperatures near 35°C, therefore we recommend managing and rearing this species at 25–30°C.     

Immobilized glucose oxidase on different supports for biotransformation removal of glucose from oligosaccharide mixtures

Four immobilized forms of glucose oxidase (GOD) were used for biotransformation removal of glucose from its mixture with dextran oligosaccharides. GOD was biospecifically bound to Concanavalin A-bead cellulose (GOD-ConA-TBC) and covalently to triazine-bead cellulose (GOD-TBC). Eupergit C and Eupergit CM were used for preparation of other two forms of immobilized GOD: GOD-EupC and GOD-EupCM. GOD-ConA-TBC and GOD-EupC exhibited the best operational and storage stabilities. pH and temperature optima of these two immobilized enzyme forms were broadened and shifted to higher values (pH 7 and 35 °C) in comparison with those of free GOD. The decrease of V_max values after immobilization was observed, from 256.8 ± 7.0 μmol min⁻¹ mg_GOD⁻¹ for free enzyme to 63.8 ± 4.2 μmol min⁻¹ mg_GOD⁻¹ for GOD-ConA-TBC and 45 ± 2.7 μmol min⁻¹ mg_GOD⁻¹ for GOD-EupC, respectively. Depending on the immobilization mode, the immobilized GODs were able to decrease the glucose content in solution to 3.8–15.6% of its initial amount The best glucose conversion, was achieved by an action of GOD-EupCM on a mixture of 100 g dextran with 9 g of glucose (i.e. 98.7% removal of glucose).     

Kinetics of imidazole addition to the axial site of the iron(II) complex of 2, 3, 9,10-tetraphenyl-1,4, 8,11-tetraaza-1,3, 8,10-cyclotetradecatetraene in dimethyl sulfoxide. Evidence for a dissociative-interchange mechanism

The axial adduct formation of the iron(II) complex of 2,3,9,10-tetraphenyl-l,4,8,11-tetraaza-1,3,8,10-cyclotetradecatetraene (L) with imidazole in dimethyl sulfoxide has been investigated spectrophotometrically at various temperatures and pressures. In the presence of a large excess of imidazole the reaction with the two phases has been observed. The first faster reaction is the formation of the monoimidazole complex of FeL²⁺, and the second slower reaction corresponds to the formation of the bisimidazole complex. Activation parameters are as follows: for the first step with k₁ (25.0°C) = (6.8 ±0.2)×10⁵ mol⁻¹ kg s⁻¹, ΔH³₁ = 47.5 ± 4.9 kJ mol⁻¹, ΔS³₁ = 26±16 J K⁻¹ mol⁻¹, and ΔV³₁ (30.0°C) = 27.2±1.5 cm³ mol⁻¹; for the second step with k₂ (25.0°C) = 26.8±0.8 mol⁻¹ kg s⁻¹, ΔH³₂ = 91.6± 0.8 kJ mol⁻¹, ΔS³₂ = 90±3 J K⁻¹ mol⁻¹, and ΔV³₂ (35.0°C) = 21.8±0.9 cm³ mol⁻¹. The large positive activation volumes strongly indicate a dissociative character of the activation process.     

A 30% reduction in switchgrass rhizome reserves did not decrease biomass yield

A long-standing question in perennial grass breeding and physiology is whether yield improvement strategies could compromise winter survival. Since perennial grasses rely on stored carbohydrates for winter maintenance and spring regrowth, yield improvement strategies could reduce winter survival if they increase biomass and grain yields at the expense of carbon allocation to storage. Therefore, it is crucial to comprehend the dependence of regrowth on storage reserves. We experimentally depleted switchgrass (Panicum virgatum L.) rhizome reserves by storing rhizomes for 2 weeks at 5°C (control treatment) and 25°C (reserve-depleted treatment). During the storage period rhizome respiration was 5.3× higher at 25°C (0.010 μmol CO₂ g⁻¹ min⁻¹ at 5°C vs. 0.054 μmol CO₂ g⁻¹ min⁻¹ at 25°C; p < 0.0001) and the starch content was depleted by 30% by the end of storage. Surprisingly, reserve-depleted switchgrass had 60% larger leaf area (LA; LA_control = 149 cm² pot⁻¹ vs. LA_depleted = 239 cm² pot⁻¹; p = 0.013) and produced ~40% more aboveground biomass than control plants (9.46 g pot⁻¹ vs. 6.63 g pot⁻¹; p = 0.112). In addition, reserve-depleted switchgrass restored its rhizome starch reserves to pre-storage levels. Switchgrass showed a large plasticity among its source-sink components to buffer the imposed reserve depletion. It increased plant photosynthesis by increasing the photosynthetic leaf area while keeping photosynthesis constant on a leaf area basis and readjusted the timing and activity of sink organs. These results suggest that switchgrass, and potentially other perennial grasses, largely over-invest in storage reserves. Therefore, current breeding strategies in perennial grasses aimed to extend the aboveground growing season should not compromise crop persistence. Our study also has implications on long-term yield dynamics as it highlights sink limitations as potential driver of the yield decline commonly observed in perennial grasses 5+ years after cultivation.     

A high pressure liquid chromatography-based assay for glutathione-S-transferase class distinction assay

In order to expedite the process of classification of the members of the family of glutathione-S-transferases (GSTs) high performance liquid chromatography with photodiode array detection (HPLC-PDA) was used as a means for measuring enzymatic activity. The GST chosen for the development of the HPLC-PDA technique was from equine liver (E-GST). The characterizing substrates, ethacrynic acid (EA) and bromosulfophthalein (BSP), along with previously gathered characterization data allowed for the distinction of α, μ or π-class enzymes. In an initial characterization of the previously unclassified E-GST it was determined that the enzyme was of the π-class with specific activities of 0.062, ± 0.0015 μmol min^− 1 mg^− 1 and 0.0019, ± 0.00064 μmol min^− 1 mg^− 1 for EA and BSP, respectively. Finally, the activity of the E-GST with the EA and BSP substrates, was measured by HPLC-PDA, and was found to be 0.027, ± 0.003 μmol min^− 1 mg^− 1 and 0.002, ± 0.0005 μmol min^− 1 mg^− 1, respectively. While the HPLC-PDA data do not mirror the spectrophotometric results quantitatively the overall response by the E-GST was the same. In general, the E-GSTs were shown to belong to the π-class when characterized by HPLC-PDA due to an EA specific activity greater than 0.01 μmol min^− 1 mg^− 1 and a negligible BSP activity (≤ 0.002 μmol min^− 1 mg^− 1).     

The immunomodulation of inducible nitric oxide in scallop Chlamys farreri

Nitric oxide (NO) is an important signalling molecule which plays an indispensable role in immunity of all vertebrates and invertebrates. In the present study, the immunomodulation of inducible NO in scallop Chlamys farreri was examined by monitoring the alterations of haemocyte behaviours and related immune molecules in response to the stimulations of LPS and/or with S-Methylisothiourea Sulphate (SMT), an inhibitor of inducible NO synthase (NOS). The total activity of NOS and NO concentration in the haemolymph of scallop C. farreri increased significantly at 3, 6 and 12 h after LPS stimulation respectively, whereas their increases were fully repressed when scallops were treated in the collaborating of LPS and SMT. Meanwhile, some cellular and humoral immune parameters were determined after the stimulation of LPS and SMT to investigate the role of inducible NO in innate immunity of scallop. After LPS stimulation, the highest levels of haemocytes apoptosis and phagocytosis were observed at 24 h (38.5 ± 2.5%, P < 0.01) and 12 h (38.6 ± 0.2%, P < 0.01), respectively, and the reactive oxygen species (ROS) level (5.88 ± 0.90%, P < 0.01) of haemocytes and anti-bacterial activity of haemolymph (10.0 ± 2.2%, P < 0.01) all elevated dramatically at 12 h. Although the activity of lysozyme and phenoloxidase (PO) in haemolymph both declined at 48 h (93.0 ± 6.3 U mgprot^?1, 0.40 ± 0.06 U mgprot^?1, P < 0.01), superoxide dismutase (SOD) activity and GSH concentration both increased to the highest level at 24 h post treatment (99.2 ± 8.1 U mgprot^?1, 93.0 ± 6.3 nmol mgprot^?1, P < 0.01). After the collaborating treatment of LPS and SMT, the apoptosis index increased much higher from 48 h, while the increase of haemocytes phagocytosis, ROS level and haemolymph anti-bacteria activities were suppressed completely at 12 h. The declines of lysozyme and PO activity in haemolymph were reversed at 48 h, and the rise of SOD activity and GSH concentration started earlier from 3 h. These results indicated clearly that NO could participate in the scallop immunity and play a crucial role in the modulation of immune response including haemocytes apoptosis and phagocytosis, anti-bacterial activity and redox homeostasis in the haemolymph of scallop.