Interleukin-10 (IL-10) is an important anti-inflammatory cytokine increasing with exercise. It is stated in the literature that the increase of IL-10 after exercise may be related to muscle damage. Creatine kinase (CK) is a dimeric protein whose plasma levels are increasing especially in muscle pathologies. At the same time, it is known that it increases relating to the muscle damage caused by exercise. In this study planned, it shall be researched there is IL-10 and CK values before and immediately after the exercise according to Bruce protocol. Moreover, the changes in white blood cells shall also be examined. Those who are convenient for the test were elected from 30 healthy voluntary males in the same age group who do sports amateurly and who don’t do sport. The subjects were applied to exercise test on a treadmill according to Bruce protocol, and the blood samples were taken into heparinize tubes before and after the exercise. Evaluations were made in biochemistry and microbiology laboratories. While IL-10 levels did not change in sedentaries after the exercise (P>0,05), it decreased in sportsmen (P<0,05). CK values increased in both of the groups after the (P<0,01). No regressional relation was found between IL-10 and CK values in both of the groups. The Bruce protocol we applied increased CK and leukocyte levels in sportsmen but decreased plasma IL -10 level significantly.

Summary The thermal challenge associated with cold acclimation (CA) and hibernation requires effective cardio-respiratory function over a large range of temperatures. We examined the impact of acute cooling in a cold-naive hibernator to quantify the presumed improvement in cardio-respiratory dysfunction triggered by CA, and estimate the role of the autonomic nervous system in optimising cardiac and respiratory function. Golden hamsters (Mesocricetus auratus) were held at a 12h:12h light/dark (L:D) photoperiod and room temperature (21°C euthermic control) or exposed to simulated onset of winter in an environmental chamber, by progression to 1h:23h L:D and 4°C over 4weeks. In vivo acute cooling (core temperature Tb=25°C) in euthermic controls led to a hypotension (P<0.05), bradycardia (P<0.05) but preserved cardiac output (NS). CA induced a hypertension at normothermia (Tb=37°C) but on cooling led to decreases in diastolic pressure below euthermic controls (P<0.05) and a decrease in cardiac output (P<0.05), despite an increase in left ventricular conductance (P<0.05). Power Spectral Analysis of heart rate variability suggested a decline in vagal tone on cooling euthermic hamsters (P<0.05 at Tb=25°C). Following CA, vagal tone was increased at Tb=37°C (P<0.05), but declined more quickly on cooling (Tb=25°C) to preserve vagal tone at levels similar to euthermic controls at Tb=37°C. For the isolated heart, CA led to concentric hypertrophy (P<0.05) with decreased end-diastolic volume (P<0.05) but with no change in intrinsic heart rate at either T=37°C or T=25°C (NS). Mechanical impairment was noted at T=37°C following CA, with peak developed pressure decreased by 50% (P<0.05) and peak rate-pressure product decreased by 65% (P<0.05); this difference was preserved at T=25°C. For euthermic hearts, coronary flow showed thermal sensitivity, decreasing 65% on cooling (T=25°C; P<0.05). By contrast, CA hearts had low coronary flow (P<0.05 compared to euthermic control) but with a loss of thermal sensitivity (NS). Together, these observations suggest that CA induced a functional impairment in the myocardium that limits performance of the cardiovascular system at euthermia, despite increased autonomic input to preserve cardiac function. On acute cooling this autonomic control was lost and cardiac performance declined further than for cold-naive hamsters, suggesting CA may compromise elements of cardiovascular function to facilitate preservation of those more critical for subsequent rewarming.

SUMMARY The consequences of acute hypothermia include impaired cardiovascular performance, ultimately leading to circulatory collapse. We examined the extent to which this results from intrinsic limitations to cardiac performance or physiological dysregulation/autonomic imbalance, and whether chronic cold exposure could ameliorate the impaired function. Wistar rats were held at a 12 h:12 h light:dark (L:D) photoperiod and room temperature (21°C; euthermic controls), or exposed to a simulated onset of winter in an environmental chamber by progressive acclimation to 1 h:23 h L:D and 4°C over 4 weeks. In vivo, acute cold exposure (core temperature, Tb=25°C) resulted in hypotension (approximately –20%) due to low cardiac output (approximately –30%) accompanying a bradycardia (approximately –50%). Cold acclimation (CA) induced only partial compensation for this challenge, including increased coronary flow at Tb=37°C (but not at Tb=25°C), maintenance of ventricular capillarity and altered sympathovagal balance (increased low:high frequency in power spectral analysis, PSA), suggesting physiological responses alone were insufficient to maintain cardiovascular performance. However, PSA showed maintenance of cardiorespiratory coupling on acute cold exposure in both groups. Ex vivo cardiac performance revealed no change in intrinsic heart rate, but a mechanical impairment of cardiac function at low temperatures following CA. While CA involved an increased capacity for β-oxidation, there was a paradoxical reduction in developed pressure as a result of adrenergic down-regulation. These data suggest that integrated plasticity is the key to cardiovascular accommodation of chronic exposure to a cold environment, but with the potential for improvement by intervention, for example with agents such as non-catecholamine inotropes.

Vascular endothelial growth factor (VEGF) is a cytokine that promotes endothelial cell proliferation, leucocyte chemotaxis and expression of adhesion molecules and is a major mediator of vascular permeability. It has been demonstrated that VEGF directly activates neutrophils and it could promote acute recruitment of leucocytes. It is known that neutrophils are the major cell population involved in acute inflammation in familial Mediterranean fever (FMF) and the role of VEGF in these cells may be crucial. The aim of this study was to investigate whether the 936 C/T functional polymorphism of the VEGF gene is associated with susceptibility to FMF and its relationship with the main clinical features of the disease. Polymerase chain reaction-restriction fragment length polymorphism technique was used to determine 936 C/T polymorphism within the VEGF gene in 75 patients with FMF and 122 non-related healthy controls. Genotype and allele frequencies of the VEGF 936 C/T polymorphism between patients with FMF and healthy control groups were not significantly different (OR = 0.74, 95% CI = 0.40-1.37, P = 0.335 for CT genotype; OR = 1.11, 95% CI = 0.67-1.83, P = 0.700, for T allele). Although VEGF 936 TT genotype was found to be more frequent in patients with FMF than in healthy controls (6.7% vs. 1.6%, respectively), the difference was not significant (OR = 4.28, 95% CI = 0.81-22.67, P = 0.108). No associations were found between the studied polymorphism and either the clinical features such as arthritis, abdominal pain, pleuritis, myalgia, arthralgia and erysipelas-like erythema of the disease or the four common studied exon 10 mutations (M694V, M680I, V726A, M694I) of the Mediterranean fever gene. Present results suggest that VEGF gene 936 C/T polymorphism does not seem to be associated with susceptibility to FMF and its clinical manifestations.

The cardiovascular and ventilatory responses of the Wistar rat (a non-hibernator) and the Syrian hamster (a hibernator) to acute and chronic cold exposure were investigated. The acute lowering of core temperature (T(c) = 22 degrees C, hypothermia) compared with normothermia (T(c) = 37 degrees C) and hyperthermia (T(c) = 40 degrees C) was used to examine the underlying differences in the extent of cold adaptation. In euthermic rats, acutely induced hypothermia resulted in a pronounced reduction in heart rate (f(H) reduced by 55%; P < 0.01), a modest but significant elevation of mean arterial blood pressure (mABP increased by 16%; P < 0.05), and a marked reduction in respiratory frequency (f(R) reduced by 64%; P < 0.01). All parameters returned to baseline values on returning T(c) to 37 degrees C, with a modest overshoot on acute hyperthermia. These data are consistent with the depressive effect of low temperature on biological rate functions and increased vagal tone in the cold, while matching f(R) to a lowered metabolic rate (MO(2)). Cold acclimation had little effect on this pattern of response, suggesting that any adaptive increase in thermogenesis is limited. Euthermic hamsters also showed a significant reduction in f(H) on acute cooling (74%; P < 0.01). In contrast to rats, hamsters developed a significant decrease in mABP (52%; P < 0.01) and maintained a relatively high f(R) (4%; n.s.). These data suggest a resetting of the baroreflex and relative hyperventilation, consistent with an elevated MO(2) associated with enhanced nonshivering thermogenesis. Cold acclimation had little effect on thermal sensitivity, though the response curves were displaced to produce a relative hypertension and tachycardia at a given T(c). These data suggest a reduced cardiorespiratory coupling in the hibernator compared with the non-hibernator.

Objectives: Normal muscle growth is accompanied by capillary proliferation, which usually lags behind the increase in muscle size, causing a decline in mean capillary density (CD). It is not known, however, how the capillary distribution is affected and what impact it has on the oxygenation of the muscle.Methods: The capillarization of soleus muscles of rats (64-425 g) was determined with the method of capillary domains. As well as quantifying CD, capillary to fiber ratio (C:F), and fiber size, this method provides a measure of the heterogeneity of capillary spacing. Capillary locations were used to mathematically model oxygenation levels within the muscle.Results: The increase in muscle mass was largely attributable to 5-fold increase in fiber size, accompanied by a more than 3-fold rise in C:F. The mismatch between rates of angiogenesis and muscle growth resulted in a decrease in CD. However, the heterogeneity of capillary spacing was unaffected (heterogeneity index log(R)SD: 0.091 +/- 0.013; mean +/- SD) as was muscle PO2, with modal values between 4 and 60 mmHg (0.5 and 8 kPa).Conclusions: Angiogenesis during normal muscle growth does not maintain CD, but with similar heterogeneity of capillary spacing it preserves the potential for adequate intramuscular oxygenation.

Control (normothermic) and cold-acclimated (environmental temperature gradually reduced from 20 to 5 degrees C for 4 weeks) groups of male rats and hamsters were compared to elucidate the nature of angiogenesis in oxidative and glycolytic muscles of these species during progressive cold exposure. Skeletal muscle capillarity and fibre cross-sectional area were measured in the tonic soleus (SOL) and phasic tibialis anterior (TA). Cryostat sections were stained for alkaline phosphatase (ALP) activity to identify all capillaries, and proliferating cell nuclear antigen (PCNA) to localise the site of cellular proliferation. Cold-induced angiogenesis, indicated by an increase in capillary to fibre ratio (C:F), occurred in SOL of rats (approximately 20 % increase, P < 0.05) but not hamsters (approximately 9.5 % increase, n.s.), and in TA of hamsters (approximately 22 % increase, P < 0.01) but not rats (approximately 1 % increase, n.s.). The change in C:F was highest in the glycolytic cortex region of TA where fibre size is larger than in the oxidative core. Capillary-specific cell proliferation (co-localised ALP and PCNA labelling) increased in parallel with C:F. The total PCNA label density within the interstitium was some 5-fold higher than that co-localised with capillaries, but where angiogenesis occurred the relative increase in capillary labelling was 2-fold greater than for other cells of the interstitium. These data suggest a significant role for endothelial cell proliferation in the angiogenic response, indicative of the sprouting form of angiogenesis. There was a tendency for fibre hypertrophy in both SOL and TA of rats, especially in the core region of TA (P < 0.01), such that capillary density (CD) and intramuscular diffusion distances (DD) were largely unchanged following cold exposure. In contrast, fibre size was maintained in hamsters, DD reduced and CD increased compared to control TA (P < 0.01). In conclusion, cold acclimation stimulated angiogenesis in muscle of hamsters more than in rats, possibly due to a higher metabolic rate in the smaller species. Angiogenesis was also seen in SOL of rat, where oxidative capacity and muscle activity is higher than the TA. Thus, a combination of oxidative capacity, muscle activity, and fibre size may determine the degree of angiogenesis in response to low environmental temperature.

The physiological, metabolic and anatomical adaptations of skeletal muscle to chronic cold exposure were investigated in Wistar rats (Rattus norvegicus), a species that defends core temperature, and Syrian hamsters (Mesocricetus auratus), which may adopt a lower set point under unfavourable conditions. Animals were exposed to a simulated onset of winter in an environmental chamber, progressively shortening photoperiod and reducing temperature from 12 h:12 h L:D and 22 degrees C to 1 h:23 h L:D and 5 degrees C over 4 weeks. The animals were left at 4 degrees C for a further 4 weeks to complete the process of cold-acclimation. M. tibialis anterior from control (euthermic) and cold-acclimated animals of similar mass showed a significant hyperactivity-induced hypertrophy in the rat, but a small disuse atrophy in the hamster. Little evidence was found for interconversion among fibre types in skeletal muscle on cold-acclimation, and only modest differences were seen in activity of oxidative or glycolytic enzymes in either species. However, adjustments in Type II fibre size paralleled the muscle hypertrophy in rat and atrophy in hamster. Cold-induced angiogenesis was present in the rat, averaging a 28 % increase in capillary-to-fibre ratio (C:F) but, as this was balanced by fibre hypertrophy across the whole muscle, there was no change in capillary density (CD). In contrast, the C:F was similar in both groups of hamsters, whereas CD rose by 33 % in line with fibre atrophy. Within distinct regions of the m. tibialis anterior, there was a correlation between angiogenesis and fibre size in rats, in which oxygen diffusion distance increased, but not in hamsters, in which there was a reduced oxygen diffusion distance. Consequently, the change in C:F was greatest (39 %) in the glycolytic cortex region of the m. tibialis anterior in rats. We conclude that non-hibernator and hibernator rodents improve peripheral oxygen transport following cold-acclimation by different mechanisms. In rats, an increase in fibre girth was accompanied by a true angiogenesis, while the improved apparent capillary supply in hamsters was due to smaller fibre diameters. These responses are consistent with the strategies of resisting and accommodating, respectively, an annual fall in environmental temperature.

The angiogenic response of glycolytic (tibialis anterior, TA) and oxidative (soleus, SOL) muscles of rats to progressive cooling (20 to 5degreesC) for 4 weeks and hypoxia (12%O-2) for 3 weeks was investigated. Consistent areas were investigated in each muscle, with the TA divided into core and cortex regions. Cold exposure increased capillary-to-fibre ratio (C/F) in SOL from 1.97 +/- 0.06 to 2.37 +/- 0.17 (P<0.05, ANOVA), but not in the TA. Capillary density (CD, mm(-2)) and fibre cross sectional area (FCSA, mum(-2)) did not change in either muscle. However, proliferating cell nuclear antigen (PCNA) density increased in SOL (52. +/- 9 vs. 88 +/- 8 mm(-1), P<0.05) and staining co-localised with capillaries also increased from 5.1 +/- 1.6 to 12.9 +/- 1.1 mm-2 (P<0.05) in cold acclimated rats. Hypoxia-induced angiogenesis also occurred in SOL (C/F 2.19 +/- 0.10 vs. 2.64 +/- 0.15, P<0.05), but not in the TA when averaged across the whole muscle. However, in the cortex region of TA angiogenesis was seen (1.45 +/- 0.02 vs. 1.83 +/- 0.07) where FCSA was largest. PCNA density increased in SOL and that co-localised with capillaries also increased in hypoxic rats. These results show that both cold acclimation and hypoxia stimulate angiogenesis in SOL. This may indicate that oxidative capacity and muscle activity levels are important factors for angiogenesis.

Skeletal muscle capillarity and fibre cross-sectional area were investigated within and between diaphragm (Diaph), extensor digitorum longus (EDL), soleus (SOL) and tibialis anterior (TA) muscles of control and chronic hypoxic (12 % O(2) for 6 weeks) adult male Wistar rats (final body mass approximately 355 g). Cryostat sections were stained for alkaline phosphatase activity to depict all capillaries, and for succinic dehydrogenase to demonstrate regional differences in oxidative capacity within the muscles. Hypoxia-induced angiogenesis occurred in all muscles (P < 0.01), with capillary-to-fibre ratio (C:F) being higher in the more active and oxidative muscles, Diaph (27 %) and SOL (26 %), than phasically active and glycolytic muscles, TA (21 %) and EDL (15 %). Diaph, SOL and EDL maintained fibre size, and hence showed an increased capillary density (CD) and reduced intramuscular diffusion distance (DD), whereas TA showed fibre hypertrophy and maintained CD and DD compared to control muscles. The extent of angiogenesis among different regions of muscle varied so as to suggest that muscle fibre size has an additional influence on capillary growth during chronic systemic hypoxia, which is progressive over an extended period of systemic hypoxia.

Alleviation of muscle ischaemia by improving capillary supply has proved difficult, possibly reflecting the inability to substantially increase blood flow. We reasoned that muscle overload, which induces angiogenesis in the absence of altered blood flow, may be an alternative to drug therapy. Male Wistar rats underwent unilateral ligation of the common iliac artery, with or without ipsilateral extirpation of the tibialis anterior muscle. Six weeks later ischaemic (I) extensor digitorum longus (EDL) had a 10% (P <0.05) decrease in relative muscle mass, while overloaded muscles (O) had undergone hypertrophy of 39% and 52% relative to contralateral (CL) and control (C) muscle masses, respectively (P < 0.01). Muscle atrophy was prevented by the combination of overload and ischaemia (O/I), with hypertrophy of 24% (vs. CL) and 35% (vs. C), respectively (P < 0.01). Changes in muscle fibre cross‐sectional area paralleled the changes in muscle mass, with means of 1898 ± 59, 1531 ± 90, 2253 ± 155 and 2292 ± 80 mm2 for C, I, O and O/I, respectively (P < 0.01 vs. C and I). Capillary to fibre ratio (C:F) was significantly increased in overloaded (2.58 ± 0.09) compared to contralateral (1.78 ± 0.04), control (1.61 ± 0.05) and ischaemic (1.73 ± 0.06) muscles (P < 0.001). A similar increase in C:F was seen in overloaded plus ischaemic muscle (2.59 ± 0.07) compared to contralateral (1.40 ± 0.01) and control or ischaemic values (P < 0.01). In both O and O/I muscle groups, C:F and capillary density (CD) increased most in the region of EDL where fibre size was largest, while hypertrophy of fibres was least in the same region for both groups. These data suggest that the microvascular deficit evident in chronic muscle ischaemia may be alleviated by angiogenesis that is induced by mechanical stimuli via chronic muscle overload.

We examined the differential change in body composition in response to a gradual reduction in both environmental temperature and photoperiod to mimic seasonal fluctuations in the wild (summer–winter transition), from ambient to 5°C and 1:23 light:dark for 8 weeks. In contrast to acute cold exposure used in previous studies, cold-acclimated rats showed an initial increase in growth rate relative to normothermic controls, possibly due to cold-stimulated hyperphagia. In hamsters, maintenance of growth rate during initial cold exposure reflects the intrinsic high oxidative capacity, while subsequent cessation of growth is consistent with the preparation for hibernation. Cold-induced atrophy of skeletal muscles coincided with increased capacity for non-shivering thermogenesis (NST) associated with a greater mass of brown adipose tissue (BAT). Cardiac hypertrophy may compensate for an increase in total peripheral resistance and/or work load of heart in both species (40% and 20%, respectively), while hypertrophy of lung (20% and 40%) and diaphragm muscle (7% and 40%) was consistent with increased ventilation associated with a cold-induced increase in basal metabolic rate. Gonadal atrophy in hamsters (160%) may be an energy saving strategy during the non-reproductive season, while maintenance of other endocrine (thyroid, adrenal, pineal) gland masses reflects the continued importance of hormonal regulation of homeostasis. The interspecific differences appear to accommodate the increased demands of shivering thermogenesis (skeletal muscle hypertrophy) or NST (BAT, diaphragm) in rats and hamsters, respectively. Those systems representing cardiovascular and metabolic control completed their adaptation quickly (within 4-week cold acclimation), while the respiratory and reproductive systems continued to respond to a further 4-week exposure. This differential time course may reflect the relative strength of selection pressure on these systems for the process of cold acclimation.

Whether chronic hypoxia causes angiogenesis in skeletal muscle is controversial. Male Wistar rats, 5--6 wk of age, were kept at constant 12% O(2) for 3 wk, and frozen sections of their postural soleus (SOL), phasic extensor digitorum longus (EDL), and tibialis anterior (TA) muscles were compared with those of normoxic controls. Capillary supply increased in SOL muscles [capillary-to-fiber ratio (C/F) = 2.55 +/- 0.09 hypoxia vs. 2.17 +/- 0.06 normoxia; capillary density (CD) = 942 +/- 14 hypoxia vs. 832 +/- 20 mm(-2) normoxia, P < 0.01] but not in EDL muscles (C/F = 1.44 +/- 0.04 hypoxia vs. 1.42 +/- 0.04 normoxia; CD = 876 +/- 52 hypoxia vs. 896 +/- 24 mm(-2) normoxia). The predominantly glycolytic cortex of TA muscles showed higher C/F after hypoxia (1.79 +/- 0.09 vs. 1.53 +/- 0.05 normoxia, P < 0.05), whereas the mainly oxidative TA core with smaller fibers showed no change in capillarity. The region of the SOL muscle with large-sized (mean fiber area 2,843 +/- 128 microm(2)) oxidative fibers (90% type I) had a higher C/F (by 30%) and CD (by 25%), whereas there was no angiogenesis in the region with sparse (76%) and smaller-sized (2,200 +/- 85 microm(2)) type I fibers. Thus systemic hypoxia differentially induces angiogenesis between and within hindlimb skeletal muscles, with fiber size contributing either directly (via a metabolic stimulus) or indirectly (via a mechanical stimulus) to the process.

Male rats and hamsters were exposed to a progressively lower air temperature and shorter photoperiod to simulate the onset of winter. Normothermic hamsters had a higher haematological oxygen carrying capacity (OCC) and coagulability (shorter prothrombin time and activated partial thromboplastin time) than rats. Cold acclimation significantly increased the OCC of rats, which parallels an increased metabolic rate, while no differences were observed in hamsters. Red cell transit time through filters was faster in the acclimated rats but not in hamsters, reflecting the lower mean cell volume due to a decreased rate of clearance from the circulation. Platelet counts were significantly lower in both cold-acclimated rats and hamsters, and there was a significant leucopenia in rats, which would reduce the degree of microvascular blockade. Whole blood viscosity, plasma viscosity, and serum osmolarity showed little change in either species. However, whole blood viscosity was significantly lower in cold-acclimated hamsters than control hamsters at the lowest shear rate tested (0.95 s(-1)). Interestingly, plasma viscosity and serum osmolarity were significantly lower in hamsters exposed to low temperatures for a shorter period (4 weeks), and may reflect the development of a reduced coagulability. These data suggest that blood composition in hamsters contributes to an innate tolerance of low temperatures, maintaining tissue perfusion under hypothermic conditions and aiding arousal from hibernation.

SUMMARY Tissues weighing from 0·006 to 3·0 g were dissected and put directly into 15 ml screw cap polypropylene tubes with a conical base (maximum of 3 g per tube). It proved unnecessary to mince tissue, even though smaller pieces may aid quicker digestion. Five millilitres of 2 M KOH in 99 % (IMS) ethanol with 0·5 % Tween‐80 was then added. Tissue digestion was usually completed in 2‐4 h using a dry heating block held at 60°C, with intermittent shaking. Samples were routinely processed using fresh tissues, although storage of frozen tissue (in the dark at ‐20°C) introduced no detectable error in BF estimation and tended to aid tissue maceration. After digestion was completed the tubes were centrifuged at 3000 r.p.m. (1500 g) for 15 min. The supernatant was carefully aspirated until < 500 μl was left, thereby reducing the possibility of accidental loss of microspheres. After 1 ml dH2O was added the tubes were quickly vortexed to prevent microsphere flocculation and aid resuspension of remaining pellets, while the subsequent addition of ethanoic Tween (100 % ethanol + 0·5 % Tween‐80) allowed complete sedimentation by centrifugation. Nine millilitres of ethanoic Tween‐80 was added, and the tubes were vortexed and spun at 1500 g for 15 min. The supernatant was aspirated as above (Step 3). Five millilitres of 100 mM phosphate buffer (pH 7·00) was added to neutralize the pellet as alkaline solutions quench fluorescence. Using aqueous solutions increased the possibility of microsphere loss by adhesion to the surface of tubes or aggregation, which could readily be seen by eye when tubes were examined under bright light, and hence the buffer was followed by addition of 4 ml absolute ethanol and further vortexing, before spinning (1500 g) for 20 min. The supernatant was aspirated, leaving up to 300 μl depending on the amount of tissue residue, and the remaining microspheres and pellet were quickly vortexed to ensure complete resuspension. The tubes were left to evaporate in an oven at 60°C and then briefly vortexed during this period to disperse more of the flocculent, until around 100 μl fluid remained. This improves solvent extraction of microspheres, which may be less efficient in a completely dry pellet. Two or three millilitres of solvent (di(ethylene glycol) ethyl ether acetate, 98 %; Aldrich Chemical Co., Poole, Dorset, UK) was added according to the expected fluorescence intensity, and vortexed several times over 3‐5 min. After 30 min the tubes were sonicated in a water bath for 5 min to complete dye extraction by the solvent. The tubes were kept as far as possible in the dark after the solvent was added to avoid photo‐bleaching of the fluorescent dyes. After sonication, the tubes were occasionally spun (1500 g) once more to sediment any undigested/undissolved material, though this was rarely necessary in our experiments. Readings should be completed within 1 h to avoid loss of signal intensity.