Adaptation of sea turtles to climate warming: Will phenological responses be sufficient to counteract changes in reproductive output?

Sea turtles are vulnerable to climate change since their reproductive output is influenced by incubating temperatures, with warmer temperatures causing lower hatching success and increased feminization of embryos. Their ability to cope with projected increases in ambient temperatures will depend on their capacity to adapt to shifts in climatic regimes. Here, we assessed the extent to which phenological shifts could mitigate impacts from increases in ambient temperatures (from 1.5 to 3°C in air temperatures and from 1.4 to 2.3°C in sea surface temperatures by 2100 at our sites) on four species of sea turtles, under a "middle of the road" scenario (SSP2-4.5). Sand temperatures at sea turtle nesting sites are projected to increase from 0.58 to 4.17°C by 2100 and expected shifts in nesting of 26-43 days earlier will not be sufficient to maintain current incubation temperatures at 7 (29%) of our sites, hatching success rates at 10 (42%) of our sites, with current trends in hatchling sex ratio being able to be maintained at half of the sites. We also calculated the phenological shifts that would be required (both backward for an earlier shift in nesting and forward for a later shift) to keep up with present-day incubation temperatures, hatching success rates, and sex ratios. The required shifts backward in nesting for incubation temperatures ranged from -20 to -191 days, whereas the required shifts forward ranged from +54 to +180 days. However, for half of the sites, no matter the shift the median incubation temperature will always be warmer than the 75th percentile of current ranges. Given that phenological shifts will not be able to ameliorate predicted changes in temperature, hatching success and sex ratio at most sites, turtles may need to use other adaptive responses and/or there is the need to enhance sea turtle resilience to climate warming.

Detecting intratumoral heterogeneity of EGFR activity by liposome-based in vivo transfection of a fluorescent biosensor.

Despite decades of research in the epidermal growth factor receptor (EGFR) signalling field, and many targeted anti-cancer drugs that have been tested clinically, the success rate for these agents in the clinic is low, particularly in terms of the improvement of overall survival. Intratumoral heterogeneity is proposed as a major mechanism underlying treatment failure of these molecule-targeted agents. Here we highlight the application of fluorescence lifetime microscopy (FLIM)-based biosensing to demonstrate intratumoral heterogeneity of EGFR activity. For sensing EGFR activity in cells, we used a genetically encoded CrkII-based biosensor which undergoes conformational changes upon tyrosine-221 phosphorylation by EGFR. We transfected this biosensor into EGFR-positive tumour cells using targeted lipopolyplexes bearing EGFR-binding peptides at their surfaces. In a murine model of basal-like breast cancer, we demonstrated a significant degree of intratumoral heterogeneity in EGFR activity, as well as the pharmacodynamic effect of a radionuclide-labeled EGFR inhibitor in situ. Furthermore, a significant correlation between high EGFR activity in tumour cells and macrophage-tumour cell proximity was found to in part account for the intratumoral heterogeneity in EGFR activity observed. The same effect of macrophage infiltrate on EGFR activation was also seen in a colorectal cancer xenograft. In contrast, a non-small cell lung cancer xenograft expressing a constitutively active EGFR conformational mutant exhibited macrophage proximity-independent EGFR activity. Our study validates the use of this methodology to monitor therapeutic response in terms of EGFR activity. In addition, we found iNOS gene induction in macrophages that are cultured in tumour cell-conditioned media as well as an iNOS activity-dependent increase in EGFR activity in tumour cells. These findings point towards an immune microenvironment-mediated regulation that gives rise to the observed intratumoral heterogeneity of EGFR signalling activity in tumour cells in vivo.

Heritable variation in heat shock gene expression: a potential mechanism for adaptation to thermal stress in embryos of sea turtles.

The capacity of species to respond adaptively to warming temperatures will be key to their survival in the Anthropocene. The embryos of egg-laying species such as sea turtles have limited behavioural means for avoiding high nest temperatures, and responses at the physiological level may be critical to coping with predicted global temperature increases. Using the loggerhead sea turtle (Caretta caretta) as a model, we used quantitative PCR to characterise variation in the expression response of heat-shock genes (hsp60, hsp70 and hsp90; molecular chaperones involved in cellular stress response) to an acute non-lethal heat shock. We show significant variation in gene expression at the clutch and population levels for some, but not all hsp genes. Using pedigree information, we estimated heritabilities of the expression response of hsp genes to heat shock and demonstrated both maternal and additive genetic effects. This is the first evidence that the heat-shock response is heritable in sea turtles and operates at the embryonic stage in any reptile. The presence of heritable variation in the expression of key thermotolerance genes is necessary for sea turtles to adapt at a molecular level to warming incubation environments.

Increased expression of Hsp70 and Hsp90 mRNA as biomarkers of thermal stress in loggerhead turtle embryos (Caretta Caretta).

The survival and viability of sea turtle embryos is dependent upon favourable nest temperatures throughout the incubation period. Consequently, future generations of sea turtles may be at risk from increasing nest temperatures due to climate change, but little is known about how embryos respond to heat stress. Heat shock genes are likely to be important in this process because they code for proteins that prevent cellular damage in response to environmental stressors. This study provides the first evidence of an expression response in the heat shock genes of embryos of loggerhead sea turtles (Caretta caretta) exposed to realistic and near-lethal temperatures (34°C and 36°C) for 1 or 3 hours. We investigated changes in Heat shock protein 60 (Hsp60), Hsp70, and Hsp90 mRNA in heart (n=24) and brain tissue (n=29) in response to heat stress. Under the most extreme treatment (36°C, 3h), Hsp70 increased mRNA expression by a factor of 38.8 in heart tissue and 15.7 in brain tissue, while Hsp90 mRNA expression increased by a factor of 98.3 in heart tissue and 14.7 in brain tissue. Hence, both Hsp70 and Hsp90 are useful biomarkers for assessing heat stress in the late-stage embryos of sea turtles. The method we developed can be used as a platform for future studies on variation in the thermotolerance response from the clutch to population scale, and can help us anticipate the resilience of reptile embryos to extreme heating events.

Modified hydra bioassay to evaluate the toxicity of multiple mycotoxins and predict the detoxification efficacy of a clay-based sorbent.

Food shortages and a lack of food supply regulation in developing countries often leads to chronic exposure of vulnerable populations to hazardous mixtures of mycotoxins, including aflatoxin B(1) (AFB(1)) and fumonisin B(1) (FB(1)). A refined calcium montmorillonite clay [i.e. uniform particle size NovaSil (UPSN)] has been reported to tightly bind these toxins, thereby decreasing bioavailability in humans and animals. Hence, our objectives in the present study were to examine the ability of UPSN to bind mixtures of AFB(1) and FB(1) at gastrointestinally relevant pH in vitro, and to utilize a rapid in vivo bioassay to evaluate AFB(1) and FB(1) toxicity and UPSN efficacy. Isothermal sorption data indicated tight AFB(1) binding to UPSN surfaces at both pH 2.0 and 6.5, but substantially more FB(1) bound at pH 2.0 than 6.5. Site-specific competition occurred between the toxins when exposed to UPSN in combination. Importantly, treatment with UPSN resulted in significant protection to mycotoxin-exposed hydra maintained at pH 6.9-7.0. Hydra were exposed to FB(1), AFB(1) and FB(1) /AFB(1) combinations with and without UPSN. A toxic response over 92 h was rated based on morphology and mortality. Hydra assay results indicated a minimum effective concentration (MEC) of 20 µg ml(-1) for AFB(1), whereas the MEC for FB(1) was not reached. The MEC for co-exposure was 400 µg ml(-1) FB(1) + 10 µg ml(-1) AFB(1). This study demonstrates that UPSN sorbs both mycotoxins tightly at physiologically relevant pH levels, resulting in decreased bioavailability, and that a modified hydra bioassay can be used as an initial screen in vivo to predict efficacy of toxin-binding agents.

Aflatoxins and fumonisins contamination of home-made food (weanimix) from cereal-legume blends for children.

BACKGROUND: Weanimix is an important food for children in Ghana. Mothers are trained to prepare homemade weanimix from beans, groundnuts and maize for their infants. Groundnuts and maize are prone to aflatoxin contamination while fumonisin contaminates maize. Aflatoxin, is produced by the Asperguillus fungi while fumonisin, is produced by Fusarium fungi. These mycotoxins occur in tropical areas worldwide due to favorable climate for their growth. OBJECTIVE: The objective of the study was to determine the levels of aflatoxin and fumonisin in homemade weanimix in the Ejura-Sekyedumase district in the Ashanti Region of Ghana. METHODS: Thirty six homemade weanimix samples (50g each) were collected from households. Aflatoxin and fumonisin were measured using a fluorometric procedure described by the Association of Official Analytical Chemist (AOAC official method 993.31, V1 series 4). RESULTS: Aflatoxin and fumonisin were detected in all 36 samples, range 7.9-500ppb. Fumonisin levels range: 0.74-11.0ppm). Thirty (83.3%) of the thirty six samples were over the action limit of 20ppb for aflatoxin with an overall mean of 145.2 ppb whiles 58.3% of the samples had fumonisins above the action limit of 4 ppm with an overall mean of 4.7 ppm. CONCLUSION: There were significant aflatoxin and fumonisin contamination of homemade weanimix. Children fed on this nutritional food were being exposed to unacceptable levels of aflatoxin and fumonisin. Therefore there is a critical need to educate mothers on the dangers of mycotoxin exposure and to develop strategies to eliminate exposure of children fed homemade weanimix to aflatoxin and fumonisin.

Calcium montmorillonite clay reduces urinary biomarkers of fumonisin B1 exposure in rats and humans.

Fumonisin B1 (FB1) is often a co-contaminant with aflatoxin (AF) in grains and may enhance AF's carcinogenicity by acting as a cancer promoter. Calcium montmorillonite (i.e. NovaSil, NS) is a possible dietary intervention to help decrease chronic aflatoxin exposure where populations are at risk. Previous studies show that an oral dose of NS clay was able to reduce AF exposure in a Ghanaian population. In vitro analyses from our laboratory indicated that FB1 (like aflatoxin) could also be sorbed onto the surfaces of NS. Hence, our objectives were to evaluate the efficacy of NS clay to reduce urinary FB1 in a rodent model and then in a human population highly exposed to AF. In the rodent model, male Fisher rats were randomly assigned to either FB1 control, FB1 + 2% NS or absolute control group. FB1 alone or with clay was given as a single dose by gavage. For the human trial, participants received NS (1.5 or 3 g day⁻¹) or placebo (1.5 g day⁻¹) for 3 months. Urines from weeks 8 and 10 were collected from the study participants for analysis. In rats, NS significantly reduced urinary FB1 biomarker by 20% in 24 h and 50% after 48 h compared to controls. In the humans, 56% of the urine samples analysed (n = 186) had detectable levels of FB1. Median urinary FB1 levels were significantly (p < 0.05) decreased by >90% in the high dose NS group (3 g day⁻¹) compared to the placebo. This work indicates that our study participants in Ghana were exposed to FB1 (in addition to AFs) from the diet. Moreover, earlier studies have shown conclusively that NS reduces the bioavailability of AF and the findings from this study suggest that NS clay also reduces the bioavailability FB1. This is important since AF is a proven dietary risk factor for hepatocellular carcinoma (HCC) in humans and FB1 is suspected to be a dietary risk factor for HCC and oesophageal cancer in humans.

Temperature-dependent sex determination and contemporary climate change.

Whether species that have persisted throughout historic climatic upheavals will survive contemporary climate change will depend on their ecological and physiological traits, their evolutionary potential, and potentially upon the resources that humans commit to prevent their extinction. For those species where temperatures influence sex determination, rapid global warming poses a unique risk of skewed sex ratios and demographic collapse. Here we review the specific mechanisms by which reptiles with temperature-dependent sex determination (TSD) may be imperilled at current rates of warming, and discuss the evidence for and against adaptation via behavioural or physiological means. We propose a scheme for ranking reptiles with TSD according to their vulnerability to rapid global warming, but note that critical data on the lability of the sex determining mechanism and on the heritability of behavioural and threshold traits are unavailable for most species. Nevertheless, we recommend a precautionary approach to management of reptiles identified as being at relatively high risk. In such cases, management should aim to neutralise directional sex ratio biases (e.g. by manipulating incubation temperatures or assisted migration) and promote adaptive processes, possibly by genetic supplementation of populations. These practices should aid species' persistence and buy time for research directed at more accurate prediction of species' vulnerability.

The energetics of endotrophic development in the frog Geocrinia vitellina (Anura: Myobatrachinae).

The energetics of endotrophic development, where the nutrition required to complete metamorphosis is provided solely by yolk, has seldom been quantified. The energy cost of development to metamorphosis of the endotrophic Australian frog Geocrinia vitellina was measured using bomb calorimetry and closed-system respirometry. Dry yolk had an energy density of 26.4 J x mg(-1), and an average 2.8-mm-diameter ovum contained 144 J. Incubation at 15 degrees C produced a froglet of 5.8 mm snout-vent length, containing 88 J in 87 d, with 11% of residual yolk in the gut, which is markedly less than the 50% recorded in another endotroph, Eleutherodactylus coqui. Geocrinia vitellina lost 56 J of metabolic energy during development to metamorphosis at 15 degrees C, and the total production efficiency was 61.0%. A review of published egg energy densities found a mean for amphibians of 25.1 kJ x g(-1), significantly lower than the mean of 27.1 kJ x g(-1) for reptiles. Moreover, available amphibian data suggest that endotrophic species have high yolk energy densities and low mass-specific rates of oxygen consumption relative to exotrophic species (with feeding larvae); consequently, large ovum size may not necessarily be prerequisite for endotrophic development.

Males call more from wetter nests: effects of substrate water potential on reproductive behaviours of terrestrial toadlets.

Laboratory studies of terrestrial-breeding frogs have demonstrated that wetter substrates produce fitter offspring but the relevance of substrate wetness to adult reproductive strategies is unknown. I hypothesized that male toadlets (Pseudophryne bibronii) would select wetter areas for nesting and would advertise wet nests strongly, and tested these predictions by manipulating water potentials at a breeding site. Males preferred to nest in the wettest areas, and called at greater rates on almost twice as many nights as males occupying drier nests. Overall, males that mated called on significantly more nights than unmated males. Hence, because males occupying wet nests called more, they also mated more and in 19 out of 20 cases, oviposition occurred in wet nests that were suitable for embryonic development. Males occupying drier nests may have risked dehydration by calling, and so were less able to signal to females. Hydration states therefore have the potential to influence the reproductive success of terrestrial male frogs.

Effects of temperature on energy cost and timing of embryonic and larval development of the terrestrially breeding moss frog, Bryobatrachus nimbus.

The Australian moss frog, Bryobatrachus nimbus, oviposits four to 16 large eggs in terrestrial nests constructed in moss or lichen in subalpine regions of southern Tasmania. Nidicolous larvae overwinter beneath snow, reaching metamorphosis without feeding after 395 d, the longest development time known for an endotrophic anuran. However, a few clutches develop more quickly and metamorphose before winter. This study examines the effect of temperature on development time and energy expenditure by measuring temperatures and developmental stages in field nests as well as rates of oxygen consumption (Vo2), developmental stage, body mass, and energy content in the laboratory at three relevant temperatures (5 degrees, 10 degrees, 15 degrees C). Eggs and larvae reared at 5 degrees C differentiated very slowly, and their development time far exceeded those in natural nests, but development times at 10 degrees and 15 degrees C averaged 277 and 149 d, respectively, and were shorter than field incubation times. Generally, respiration rates of aquatic hatchlings were low in comparison with other species but increased with larval age and jumped about 25% higher near metamorphosis when larvae were able to air breathe. The mean energy density was 26.0 J mg(-1) for the dry ova and 20.6 J mg(-1) for a dry gut-free froglet, and total production efficiency was 61.5%. We developed a model based on the relationships between incubation temperature and V&d2;o2 to estimate the respiratory cost of development to metamorphosis, the first such study for an amphibian. The cost was 177 J at 15 degrees C, 199 J at 10 degrees C, and at least 249 J at 5 degrees C, and we predicted that continual development at 5 degrees C would lead to premature yolk depletion because it equalled the 249 J contained in fresh ova. Continuously logged field-nest temperatures and interpolation of laboratory data provided estimates of development rates, Vo2, and respiratory energy costs in field nests. Development to metamorphosis required between 185 and 234 J when larvae overwintered, but completion of metamorphosis before winter saved 123 J. However, the advantage of emergence in warmer months, when conditions are suitable for feeding and growth, may offset the greater energy cost of overwintering.

Influence of environmental oxygen on development and hatching of aquatic eggs of the australian frog, Crinia georgiana.

The effect of oxygen partial pressure (Po(2)) on development and hatching was investigated in aquatic embryos of the myobatrachid frog, Crinia georgiana, in the field and in the laboratory. Eggs from 29 field nests experienced widely variable Po(2) but similar temperatures. Mean Po(2) in different nests ranged between 2.9 and 19.3 kPa (grand mean 12.9 kPa), and mean temperature ranged between 11.9 degrees and 16.8 degrees C (grand mean 13.7 degrees C). There was no detectable effect of Po(2) or temperature on development rate or hatching time in the field, except in one nest at 2.9 kPa where the embryos died, presumably in association with hypoxia. Laboratory eggs were incubated at 15 degrees C at a range of Po(2) between 2 and 25 kPa. Between 5 and 25 kPa, there was almost no effect of Po(2) on development rate to stage 26, but the embryos hatched progressively earlier-at earlier stages and lower gut-free body mass-at lower Po(2). At 2 kPa, development was severely delayed, growth of the embryo slowed, and morphological anomalies appeared. A high tolerance to low Po(2) may be an adaptation to embryonic development in the potentially hypoxic, aquatic environment.

Surgical face masks in modern operating rooms--a costly and unnecessary ritual?

Following the commissioning of a new suite of operating rooms air movement studies showed a flow of air away from the operating table towards the periphery of the room. Oral microbial flora dispersed by unmasked male and female volunteers standing one metre from the table failed to contaminate exposed settle plates placed on the table. The wearing of face masks by non-scrubbed staff working in an operating room with forced ventilation seems to be unnecessary.

Differential cytokeratin expression in normal, hyperplastic and malignant epithelial cells from human prostate.

Studies were undertaken to define the expression of cytokeratins in normal, hyperplastic and malignant epithelial cells from human prostate. Cytokeratin (CK) polypeptides, separated by two-dimensional electrophoresis, were identified by immunoblotting with CK-specific monoclonal antibodies. CK polypeptides 5, 7, 8, 15, 18 and 19 were identified in fresh normal and hyperplastic prostate. Expression of CK 15 has not been previously reported in human prostate. Analysis of central and peripheral zone tissues from human prostate did not reveal qualitative differences in CK expression between these areas. Epithelial cells harvested from fresh BPH tissue by percoll gradient centrifugation and propagated in vitro using selective culture techniques showed alterations in CK expression compared to intact human prostate. Specifically, CKs 6, 14, 16 and 17 were noted in cultured BPH epithelial cells but not fresh normal prostate or BPH tissue. Immunoblot analysis of the established prostate cancer cell lines PC3, DU145 and LNCAP showed expression of CKs 8 and 18 but not CKs 5, 7 and 15 which were observed in benign prostate. These studies further characterize CK expression in benign and malignant human prostate and provide insights which may be useful in differentiating normal, hyperplastic and malignant epithelial cells in the human prostate gland.

Whole body disinfection with chlorhexidine: is shower bathing more effective than bathing?

Whole body washing with chlorhexidine gluconate solution BP (equivalent to 4 per cent w/v chlorhexidine gluconate) achieved a variable reduction in total skin microbial colony counts whether applied in a standard bath or a shower bath. No significant differences between the two techniques were demonstrable.

Single dose metronidazole with and without cefuroxime in elective colorectal surgery.

Seventy patients undergoing elective colorectal surgery for malignant and inflammatory bowel disease were allocated randomly to one of two groups. Patients in group 1 were given, after induction of anaesthesia, a single dose of metronidazole 500 mg i.v. Patients in group 2 were given in addition a single dose of cefuroxime 1.5 g i.v. Of 34 patients in group 1, 4 (11.8 per cent) developed postoperative infection. Of 36 patients in group 2, 5 (13.6 per cent) developed postoperative infection. No significant difference was noted in the incidence of postoperative sepsis between the two treatment groups.

Reduction of skin bacteria in theatre air with comfortable, non-woven disposable clothing for operating-theatre staff.

Conventional loose-weave cotton operating garments were compared with clothing of a non-woven fabric to test their efficacy in reducing the dispersal of skin bacteria into theatre air. When men wore operating suits made of the non-woven fabric dispersal of skin bacteria was reduced by 72%. When all the operating-theatre staff wore suits and dresses of this fabric air bacterial counts during operating sessions were reduced by 55%; no reduction occurred when the fabric was worn by only the scrubbed team. The lowest levels of microbial contamination of the air in the operating theatre occurred when both the unscrubbed and scrubbed theatre staff wore clothes of non-woven fabric.