Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE

The Retinal Pigment Epithelium (RPE) forms the primary site of pathology in several blinding retinopathies. RPE cultures are being continuously refined so that dynamic disease processes in this important monolayer can be faithfully studied outside the eye over longer periods. The RPE substrate, which mimics the supportive Bruch’s membrane (BrM), plays a key role in determining how well in-vitro cultures recapitulate native RPE cells. Here, we evaluate how two different types of BrM substrates; (1) a commercially-available polyester transwell membrane, and (2) a novel electrospun scaffold developed in our laboratory, could support the generation of realistic RPE tissues in culture. Our findings reveal that both substrates were capable of supporting long-lasting RPE monolayers with structural and functional specialisations of in-situ RPE cells. These cultures were used to study autofluorescence and barrier formation, as well as activities such as outer-segment internalisation/trafficking and directional secretion of key proteins; the impairment of which underlies retinal disease. Hence, both substrates fulfilled important criteria for generating authentic in-vitro cultures and act as powerful tools to study RPE pathophysiology. However, RPE grown on electrospun scaffolds may be better suited to studying complex RPE-BrM interactions such as the formation of drusen-like deposits associated with early retinal disease.     

A cold-adapted reptile becomes a more effective thermoregulator in a thermally challenging environment

Thermoregulation is of great importance for the survival and fitness of ectotherms as physiological functions are optimized within a narrow range of body temperature (T _b). The precision with which reptiles thermoregulate has been proposed to be related to the thermal quality of their environments. Although a number of studies have looked at the effect of thermal constraints imposed by diel, seasonal and altitudinal variation on thermoregulatory strategies, few have addressed this question in a laboratory setting. We conducted a laboratory experiment to test whether tuatara, Sphenodon punctatus (order Rhynchocephalia), a cold-adapted reptile endemic to New Zealand, modify their thermoregulatory behaviour in response to different thermal environments. We provided tuatara with three thermal treatments: high-quality habitat [preferred T _b (T _sel) could be reached for 8 h/day], medium-quality habitat (T _sel available for 5 h/day) and low-quality habitat (T _sel available for 3 h/day). All groups maintained body mass, but tuatara in the low-quality habitat thermoregulated more accurately and tended to maintain higher T _bs than tuatara in the high-quality habitat. This study thus provides experimental evidence that reptiles are capable of adjusting their thermoregulatory behaviour in response to different thermal constraints. This result also has implications for the conservation of tuatara. A proposed translocation from their current habitat to a higher latitudinal range within New Zealand (similar to the shift from our 8 h/day to our 5 h/day regime) is unlikely to induce thermoconformity; rather, tuatara will probably engage in more effective thermoregulatory behaviour.     

Effect of carbohydrate intake on net muscle protein synthesis during recovery from resistance exercise.

The purpose of this study was to determine the effect of ingestion of 100 g of carbohydrates on net muscle protein balance (protein synthesis minus protein breakdown) after resistance exercise. Two groups of eight subjects performed a resistance exercise bout (10 sets of 8 repetitions of leg presses at 80% of 1-repetition maximum) before they rested in bed for 4 h. One group (CHO) received a drink consisting of 100 g of carbohydrates 1 h postexercise. The other group (Pla) received a noncaloric placebo drink. Leg amino acid metabolism was determined by infusion of 2H5- or 13C6-labeled phenylalanine, sampling from femoral artery and vein, and muscle biopsies from vastus lateralis. Drink intake did not affect arterial insulin concentration in Pla, whereas insulin increased several times after the drink in CHO (P < 0.05 vs. Pla). Arterial phenylalanine concentration fell slightly after the drink in CHO. Net muscle protein balance between synthesis and breakdown did not change in Pla, whereas it improved in CHO from -17 +/- 3 nmol.ml(-1).100 ml leg(-1) before drink to an average of -4 +/- 4 and 0 +/- 3 nmol.ml(-1).100 ml leg(-1) during the second and third hour after the drink, respectively (P < 0.05 vs. Pla during last hour). The improved net balance in CHO was due primarily to a progressive decrease in muscle protein breakdown. We conclude that ingestion of carbohydrates improved net leg protein balance after resistance exercise. However, the effect was minor and delayed compared with the previously reported effect of ingestion of amino acids.     

Thermal dependence of locomotor performance in two cool-temperate lizards

Temperate-zone ectotherms experience varying or very low ambient temperatures and may have difficulty in attaining preferred body temperatures. Thus, adaptations to reduce the thermal dependence of physiological processes may be present. We measured the optimal temperature range for sprint speed and compared it with the selected body temperatures (T _sel) of two sympatric, cool-temperate lizards: the diurnal skink Oligosoma maccanni and the primarily nocturnal gecko Woodworthia (previously Hoplodactylus) “Otago/Southland”. We also investigated whether time-of-day influenced sprint speed. Contrary to results for other reptiles, we found that time-of-day did not influence speed in either species. For each species, the optimal temperature range for sprinting and T _sel overlapped, supporting the ‘thermal coadaptation’ hypothesis. However, the optimal range of temperatures for speed is not always attainable during activity by either species, which have limited opportunities to attain T _sel in the field. The thermal sensitivity of sprint speed in these two species does not appear to have evolved to fully match their current thermal environment. More data on cold-adapted species are needed to fully understand physiological adaptation in ectotherms.     

In vitro indices of tissue adherence in Staphylococcus intermedius

In vitro indices of adherence showed that strains of Staphylococcus intermedius from lesions of canine pyoderma differed from strains isolated from normal carrier sites in that a significantly greater proportion of pyoderma strains adhered to extracellular matrix proteins whilst fewer adhered to polystyrene. Slime production and a hydrophobicity index did not differ between the groups. This suggests that exposure of extracellular matrix proteins due to underlying disease may result in the selection of a narrower spectrum of strains from amongst those at carrier sites.     

Recovery of labeled CO2 from acetate in severely burned children

The purpose of this study was to determine the fractional recovery rate of labeled CO(2) in the breath of severely burned children. This information is needed to perform tracer studies of substrate oxidation using carbon-labeled fatty acids. Nine children, ages 4-14 yr with massive burns participated in the study. All experiments were performed 7 days post burn after an overnight fast. A primed (60 micromol/kg), constant (2.0 micromol.kg(-1).min(-1)) infusion of [1,2-(13)C]acetate was given during a 4-h basal period and during a 4-h hyperinsulinemic euglycemic clamp. A priming dose (150 micromol/kg) of NaH(13)CO(3) was given at the beginning of the study. Breath samples were collected every 10 min during the last 40 min of each period. Indirect calorimetry was performed during the last 30 min of each period. The isotopic enrichment of (13)CO(2) was determined by isotope ratio-mass spectrometry, and total CO(2) excretion was measured by indirect calorimetry. The fractional recovery of acetate label was 0.89 +/- 0.05 and 0.88 +/- 0.04 during the basal state and clamp, respectively. We conclude that the fractional recovery of labeled acetate in severely burned children is approximately three times the recovery of a nonburned adult and similar to the value in exercising adults. The high recovery rate reflects the rapid turnover of the TCA cycle in burned children relative to the rate of exchange reactions. Minimal correction of expired CO(2) data is needed in this circumstance to quantify fatty acid oxidation using (13)C-labeled fatty acids.     

Reproductive cycles of male and female tuatara (Sphenodon punctatus) on Stephens Island, New Zealand

Cycles in gonadal activity and plasma sex steroid concentrations were investigated in wild female and male tuatara on Stephens Island, New Zealand. Females nest once every four years on average. Vitellogenesis is spread over the first three years, and mating, ovulation and nesting occur in the fourth. Oviducal eggs are carried for6–8 months before nesting. Although the length of this ovarian cycle is unparalleled among oviparous reptiles, the associated cycles in plasma concentrations of sex steroids are similar to those in other reptiles. Mean plasma concentrations of oestradiol increase during vitellogenesis, peak at mating, fall rapidly before or around ovulation, are low during most of gravidity, and rise slightly during late gravidity-nesting. Plasma concentrations of testosterone show a similarcycle. Mean plasma concentrations of progesterone are low during vitellogenesis and peak around ovulation. This periovulatory surge falls within1–2 months, and mean concentrations are low during the final 5–6 months of gravidity. Male tuatara show an annual, pre-nuptial reproductive cycle. Mean plasma concentrations of testosterone are low during winter, rise during spring, peak during midsummer-early autumn, when pre-nuptial displays and mating occur, and fall in mid autumn. Limited histological data indicate that spermiogenesis occurs during midsummer-early autumn, and also support a previous study suggesting that there may be no period of complete testicular and epididymal regression. In comparison with the pre-nuptial cycles of other temperate-zone reptiles, the cycle in male tuatara shows a more prolonged duration of testicular activity and of rising or elevated plasma testosterone concentrations. The prolonged reproductive cycles of male and female tuatara may be adaptations to a temperate environment with cool summers and cool, but not freezing, winters.