Complementary use of stable isotopes and fatty acids for quantitative diet estimation of sympatric predators, the Antarctic pack-ice seals.

The quantitative use of stable isotopes (SIs) for trophic studies has seen a rapid growth whereas fatty acid (FA) studies remain mostly qualitative. We apply the Bayesian tool MixSIAR to both SI and FA data to estimate the diet of three sympatric predators: the crabeater (Lobodon carcinophaga), Weddell (Leptonychotes weddellii) and leopard seal (Hydrurga leptonyx). We used SI data of their vibrissae and FA data of their outer blubber to produce comparable diet estimates for the same individuals. Both SI and FA models predicted the same main diet components, although the predicted proportions differed. For the crabeater seal, both methods identified krill, Euphausia superba, as the main, and almost exclusive, food item, although the FA model estimated a slightly lower proportion, potentially due to the low lipid content of krill compared to the fish species used in the model. For the Weddell seal the FA model identified the fish Pleuragramma antarcticum as the most important prey, whereas the SI model was not able to distinguish among prey species, identifying a 'fish-squid' group as the main diet component. For the leopard seal, both models identified krill as the main contributor; however, the predicted proportions for the secondary sources differed. Although vibrissae and outer blubber may not represent the same timeframe, the use of MixSIAR with FA data provides diet estimates comparable to those obtained with SI data, thus, both approaches were complimentary. The use of both biotracers offers a feasible option to study diets of wild animals in a quantitative manner.

Revisiting the cost of carnivory in mammals.

Predator-prey relationships play a key role in the evolution and ecology of carnivores. An understanding of predator-prey relationships and how this differs across species and environments provides information on how carnivorous strategies have evolved and how they may change in response to environmental change. We aim to determine how mammals overcame the challenges of living within the marine environment; specifically, how this altered predator-prey body mass relationships relative to terrestrial mammals. Using predator and prey mass data collected from the literature, we applied phylogenetic piecewise regressions to investigate the relationship between predator and prey size across carnivorous mammals (51 terrestrial and 56 marine mammals). We demonstrate that carnivorous mammals have four broad dietary groups: small marine carnivores (< 11 000 kg) and small terrestrial carnivores (< 11 kg) feed on prey less than 5 kg and 2 kg, respectively. On average, large marine carnivores (> 11 000 kg) feed on prey equal to 0.01% of the carnivore's body size, compared to 45% or greater in large terrestrial carnivores (> 11 kg). We propose that differences in prey availability, and the relative ease of processing large prey in the terrestrial environment and small prey in marine environment, have led to the evolution of these novel foraging behaviours. Our results provide important insights into the selection pressures that may have been faced by early marine mammals and ultimately led to the evolution of a range of feeding strategies and predatory behaviours.

Macrophages as potential targets for zoledronic acid outside the skeleton-evidence from in vitro and in vivo models.

PURPOSE: Multiple cell types of the tumour microenvironment, including macrophages, contribute to the response to cancer therapy. The anti-resorptive agent zoledronic acid (ZOL) has anti-tumour effects in vitro and in vivo, but it is not known to what extent macrophages are affected by this agent. We have therefore investigated the effects of ZOL on macrophages using a combination of in vitro and in vivo models. METHODS: J774 macrophages were treated with ZOL in vitro, alone and in combination with doxorubicin (DOX), and the levels of apoptosis and necrosis determined. Uptake of zoledronic acid was assessed by detection of unprenylated Rap1a in J774 macrophages in vitro, in peritoneal macrophages and in macrophage populations isolated from subcutaneously implanted breast cancer xenografts following ZOL treatment in vivo. RESULTS: Exposure of J774 macrophages to 5 µM ZOL for 24 h caused a significant increase in the levels of uRap1A, and higher doses/longer exposure induced apoptotic cell death. DOX (10 nM/24 h) and ZOL (10 µM/4 h) given in sequence induced significantly increased levels of apoptotic cell death compared to single agents. Peritoneal macrophages and macrophage populations isolated from breast tumour xenografts had detectable levels of uRap1A 24 h following a single, clinically achievable dose of 100 µg/kg ZOL in vivo. CONCLUSION: We demonstrate that macrophages are sensitive to sequential administration of DOX and ZOL, and that both peritoneal and breast tumour associated macrophages rapidly take up ZOL in vivo. Our data support that macrophages may contribute to the anti-tumour effect of ZOL.

Toneburst-evoked auditory brainstem response in a leopard seal, Hydrurga leptonyx.

Toneburst-evoked auditory brainstem responses (ABRs) were recorded in a captive subadult male leopard seal. Three frequencies from 1 to 4 kHz were tested at sound levels from 68 to 122 dB peak equivalent sound pressure level (peSPL). Results illustrate brainstem activity within the 1-4 kHz range, with better hearing sensitivity at 4 kHz. As is seen in human ABR, only wave V is reliably identified at the lower stimulus intensities. Wave V is present down to levels of 82 dB peSPL in the right ear and 92 dB peSPL in the left ear at 4 kHz. Further investigations testing a wider frequency range on seals of various sex and age classes are required to conclusively report on the hearing range and sensitivity in this species.

Acoustic features involved in the neighbour-stranger vocal recognition process in male Australian fur seals.

Many territorial species have the ability to recognise neighbours from stranger individuals. If the neighbouring individual is assumed to pose less of a threat, the territorial individual responds less and avoids unnecessary confrontations with familiar individuals at established boundaries, thus avoiding the costly energy expenditure associated with fighting. Territorial male Australian fur seals respond more to strangers than to neighbouring males. The present study evaluated which acoustic features were important in the neighbour-stranger recognition process in male Australian fur seals. The results reveal that there was an increase in response strength or intensity from males when they heard more bark units, indicating the importance of repetition to detect a caller. However, lengthening and shortening the inter-unit spaces, (i.e. changing the rhythm of the call) did not appear to significantly affect an animal's response. In addition, the whole frequency spectrum was considered important to recognition with results suggesting that they may vary in their importance. A call containing the dominant and surrounding harmonics was considered important to a male's ability to recognise its neighbour. Furthermore, recognition occurs even with a partial bark, but males need to hear between 25 and 75% of each bark unit from neighbouring seals. Our study highlights which acoustic features induce stronger or weaker responses from territorial males, decoding the important features in neighbour-stranger recognition.

Determination of testosterone in saliva and blow of bottlenose dolphins (Tursiops truncatus) using liquid chromatography-mass spectrometry.

A rapid, accurate and reproducible assay utilising high performance liquid chromatography-mass spectrometry (LC-MS) has been developed and validated for determining testosterone concentrations in saliva and blow of bottlenose dolphins. Sample preparation used solid phase extraction with specific preconditioning of cartridges. Analytes were eluted with 100% acetonitrile, dried under nitrogen and stored at -80 degrees C. Samples were reconstituted in 60% acetonitrile for LC-MS analysis. Chromatographic separation was achieved with an Alltech Macrosphere C8 stainless steel analytical column (2.1 mm x 150 mm i.d., 5 microm particle size, 300 angstroms pore size) using a 55% mobile phase B isocratic method (mobile phase A = 0.5% acetic acid; mobile phase B = 0.5% acetic acid, 90% acetonitrile). Samples were analysed in SIM at m/z 289.20 (testosterone mw 288.40) and a positive ion ESI. The limit of quantification was 0.5 ng/ml with a limit of detection of 0.2 ng/ml. The concentration curve was linear from 0.5 to 50 ng/ml (y = 0.01x + 0.0045, r(2) = 0.959, r = 0.979, p < 0.001). The R.S.D.s of intra- and inter-batch precision were less than 15% for saliva and 11% blow. Recovery of the assay for saliva was 93.0 +/- 7.9% (50 ng/ml) and 91.5 +/- 3.72% (1 ng/ml), and for blow was 83.3 +/- 6.8% (50 ng/ml) and 85.8 +/- 4.6% (1 ng/ml). Recovery of the internal standard in saliva was 73.0 +/- 14.2% and in blow was 78.63 +/- 4.29. The described assay was used to determine the presence of endogenous testosterone in saliva (9.73-23 ng/ml, n = 10) and blow (14.71-86.20 ng/ml, n = 11) samples of captive bottlenose dolphins.

A new approach to the solution of the linear mixing model for a single isotope: application to the case of an opportunistic predator.

Mixing models are used to determine diets where the number of prey items are greater than one, however, the limitation of the linear mixing method is the lack of a unique solution when the number of potential sources is greater than the number (n) of isotopic signatures +1. Using the IsoSource program all possible combinations of each source contribution (0-100%) in preselected small increments can be examined and a range of values produced for each sample analysed. We propose the use of a Moore Penrose (M-P) pseudoinverse, which involves the inverse of a 2x2 matrix. This is easily generalized to the case of a single isotope with (p) prey sources and produces a specific solution. The Antarctic leopard seal (Hydrurga leptonyx) was used as a model species to test this method. This seal is an opportunistic predator, which preys on a wide range of species including seals, penguins, fish and krill. The M-P method was used to determine the contribution to diet from each of the four prey types based on blood and fur samples collected over three consecutive austral summers. The advantage of the M-P method was the production of a vector of fractions f for each predator isotopic value, allowing us to identify the relative variation in dietary proportions. Comparison of the calculated fractions from this method with 'means' from IsoSource allowed confidence in the new approach for the case of a single isotope, N.

Investigation of some commercially available spacer devices for the delivery of glucocorticoid steroids from a pMDI.

Five commercially available spacers were investigated to determine their influence on the percentage of drug retained in the spacer device, percentage fine particle fraction (FPF), percentage deposited in the induction port, mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD). Betamethasone valerate (BMV) and triamcinolone acetonide (TAA) were used as model drugs in the pressurized metered dose inhaler (pMDI) formulations containing the propellant HFA 134a. The BMV was dissolved in an ethanol/HFA 134a system, and the TAA was suspended in HFA 134a using ethanol as a dispersing agent. The metering chamber volume of the valve was either 50 microl or 150 microl. The spacer devices investigated included the ACE, Aerochamber, Azmacort, Easivent, and Ellipse spacers. Each spacer device was attached to an Andersen Cascade Impactor powered by a vacuum pump. Cascade impaction data were used to derive the percentage drug deposited in the induction port, MMAD, GSD, and FPF. The BMV particles emitted from the spacers were finer than the TAA particles because the dissolved drug precipitated as the cosolvent evaporated. The TAA particles had significantly larger MMADs because many undissolved drug particles were contained within each droplet following actuation. After evaporation of the liquid continuous phase, the suspended drug aggregated to form larger agglomerates than those particles precipitated from the BMV pMDI solution droplets. The addition of a spacer device lowered the MMAD to less than 4.7 microm for particles from both the BMV pMDI solution and the TAA pMDI suspension. The addition of a spacer device also lowered the percentage drug deposited in the induction port. The FPF was significantly increased when a spacer device was used. The MMAD significantly decreased when a spacer device was added for the two model drugs when using the 150-microl metering valves, but the difference was not statistically significant when the 50-microl valves were used (P < .05). The GSD was not influenced by the use of a spacer device. The use of a spacer device will enhance pMDI therapy by reducing the amount of drug deposited in the oropharyngeal region, which will lead to fewer instances of local and systemic side effects. In addition, the spacer devices investigated will allow a higher dose of drug to reach the deep lung, which may permit the use of lower dosage regimens with increased therapeutic efficacy.

Solution-based particle formation of pharmaceutical powders by supercritical or compressed fluid CO2 and cryogenic spray-freezing technologies.

Micronization is an important procedure used in the pharmaceutical industry to reduce the particle size of active pharmaceutical ingredients (APIs). The spray-drying and milling techniques presently used to micronize drug substances cannot be used to process thermolabile or physically unstable drug substances. Therefore, new micronization techniques, including particle precipitation with supercritical or compressed fluid CO2 and spray-freezing of drug solutions and suspensions into cryogenic gas to produce solid frozen microparticles, are currently being perfected for future use in the pharmaceutical industry. This review highlights the compressed gas and cryogenic liquid technologies being developed as potential solution-based particle formation technologies for drugs that cannot be processed by conventional micronization techniques.

Study of solubility of steroids in hydrofluoroalkane propellants.

The solubility of prednisone, hydrocortisone 21-acetate, hydrocortisone, dexamethasone, betamethasone 17-valerate, and danazol in hydrofluoroalkane (HFA) 134a and HFA 227 was determined at 5 degrees C and 25 degrees C. It was found that the solubility of steroid in HFA propellants was related to the melting point and the lipophilicity of the steroid. The solubility of the steroids in the binary system of HFA propellants and ethanol also was investigated in the study. Ethanol significantly increased the solubility of the steroids in HFA propellant. The magnitude of increase was related to the solubility of the corresponding steroid in ethanol alone.

A visual method of determining the sex of skeletal remains using the distal humerus.

This study introduces a new method of determining sex based on four morphological features of the posterior, distal humerus. The technique was developed on a 20th century anatomy series, the University of Toronto Grant Skeletal Collection, and was tested on 35 known individuals from the University of New Mexico Documented Collection and 93 individuals from the William M. Bass Donated Skeletal Collection. Four statistically significant characteristics relating to the carrying angle of the arm are identified (p < 0.05). Together, they are capable of determining sex with 92% accuracy.