Non-invasive sampling techniques : applications for mapping the generic variation of powerful owls

The Powerful Owl (Ninox strenua) is endemic to Australia, being resident in the three eastern mainland states and the Australian Capital Territory. It is classified nationally as of conservation significance and vulnerable in the state of Victoria. The elusive nature of this owl, along with its dispersed distribution, low population density and difficulty in identifying individual birds, limit the collection of ecological data. Molecular methods can be used to obtain crucial ecological information, essential for Powerful Owl conservation.

Non-invasive sampling is a relatively new method used for obtaining genetic material from free-ranging animals. This type of sampling however, is generally overlooked as a potential DNA source. Shed hair and feathers, faeces, urine, skins and eggshells are all potential sources of DNA. Non-invasive sampling regimes may be the only alternative for the genetic analysis of endangered and/or elusive species that are difficult to sample otherwise.

Powerful Owls moult annually. Shed feathers therefore, can be collected from under roost trees and used for genetic analysis. Feathers collected provide DNA that is unique to the individual and can provide additional ecological knowledge of the species.

In this study we collected shed Powerful Owl feathers during 2003 and 2004. In order to obtain samples from across the owl's large distribution, public awareness about the project via the way of flyers, mail-outs, media sources (radio, newspapers and magazines), email lists and public seminars was initiated. Overall, the collection strategy was very successful with over 500 Powerful Owl feather samples being collected.

Genetic information obtained from the analysis of DNA from feathers can enable a more rigorous assessment of population viability of the Powerful Owl. Specifically designed molecular markers will facilitate unequivocal identification of individual birds ("DNA fingerprinting"). Through the application of molecular techniques we can collect ecological information about the Powerful Owl such as, genetic divergence, population structure, dispersal patterns, migration and inbreeding. These questions can not be addressed via traditional data collection and will contribute significantly to the successful conservation of the Powerful Owl and potentially other raptor species.

Sexing accuracy and indicators of maturation status in captive Murray cod Maccullochella peelii peelii using non-invasive ultrasonic imagery

Macroscopic- and histological-based assessments of gonad condition were compared with ultrasound images to determine the feasibility of this technology as a non-invasive diagnostic tool for identifying sex and assessing maturation status of Murray cod. Four age-classes (1⁺, 2⁺, 3⁺ and 6⁺ years), were sub-sampled at monthly intervals throughout their annual reproductive cycle and scanned with a 5 MHz linear transducer. An interpretation of sex was made from the resulting images and maximum cross-sectional gonad diameter and area were recorded. Fish were subsequently dissected to confirm gender, and the weights and maturation status of gonads determined and then compared with their respective image profile. Ovaries of females were usually a distinctive feature in ultrasound images, being particularly obvious in older and/or more developed fish. In contrast, the identification of male testis was more problematic. Nonetheless, identifying sex from ultrasound images was consistently achieved by recording the presence/absence of a female ovary (96% total sexing accuracy). Maximum cross-sectional ovary diameter and area were highly correlated with gonad weight (r² = 0.90 and 0.89, respectively) suggesting that indices of maturation status, comparable to the gonadosomatic index (GSI), can be obtained non-destructively from ultrasound scans of females. A less distinct relationship occurred between these dimensions and weight of testes (r² = 0.41). Significant increases (P < 0.05) in mean gonad index (GI, calculated from gonad diameter) occurred for most gonad development stages. However, differences in mean GI between maturation stages were confounded by phenotypic variability, indicating that GI may be limited to population level studies. Nevertheless, ultrasound images of ovaries at each development stage were visually distinctive and enabled qualitative evaluations of maturity, thereby complementing quantitative GI assessments. Repeated serial-monitoring of the same population using ultrasound appears to have great potential for tracking maturation-induced changes in broodfish.

Relationship of nutritional and metabolic factors to non-invasive indices of macrovascular disease in diabetes

Factors which may account for the high frequency of macrovascular disease in diabetics are age, sex, cigarette smoking, hypertension, obesity, lack of exercise, diet, hyperglycaemia, hyperinsulinaeroia, hypercholesterolaemia, hypertriglyceridaemia, low HDL-cholesterol concentration, elevated free fatty acid concentration and enhanced platelet aggregation. Twenty seven (13 men and 14 women) non-insulin-dependent diabetics and thirty eight age, height and weight matched healthy subjects (10 men and 28 women) were studied. None of the subjects were smokers, or hypertensive. No subject had any clinical evidence of peripheral arterial disease, coronary heart disease or cerebrovascular disease. All had apparently normal peripheral pulses and normal ankle/arm blood pressure indices. Methods for determining arterial compliance in the segment between the left subclavian artery and each common femoral artery, and proximal resistance at the common femoral artery and posterior tibial artery, have been reviewed and developed. An appropriate food intake methodology for deriving food indices from food records was developed. Biochemical determinants have been made of glucose tolerance, glycosylated haemoglobin, serum total cholesterol, HDL-cholesterol, LDL-cholesterol, triglyceride, plasma free fatty acid and insulin. A significant decrease in the arterial compliance, and a significant increase in the arterial proximal resistance at the common femoral artery and posterior tibial artery in non-insulin-dependent diabetics, compared with their healthy controls, have been found. Significant negative correlation between arterial compliance and proximal resistance and, a significant positive correlation between the arterial proximal resistance of common femoral artery and posterior tibial artery were found. Differences between control (healthy subjects) and non-insulin-dependent diabetic groups indicate that preclinical peripheral arterial disease can be recognised even in mild diabetics by non-invasive measurement of arterial compliance or proximal resistance. There were significant and negative correlations between arterial compliance and each of blood glucose, blood glycosylated haemoglobin (HbAlC), plasma free fatty acid and plasma insulin concentration. There were significant and positive correlations between arterial proximal resistance of common femoral artery and posterior tibial artery and each of blood glucose, glycosylated haemoglobin and plasma free fatty acid concentration. Multivariate analysis to examine each of the biochemical factors Including blood glucose, blood glycosylated haemoglobin (HbAlC), plasma free fatty acid, plasma Insulin and lipids, showed that the factor which most influenced the arterial compliance and the proximal resistance of posterior tibial artery was the glucose level in the fasting state or the glucose response after a glucose load. In addition, the factors which most influenced proximal resistance of the common femoral artery were free fatty acid -level in the fasting state or glucose response after a glucose load. The factors which most influenced arterial compliance were glucose level in men, and the insulin level in the fasting state or the plasma free fatty acid response after a glucose load in women. These findings indicate that blood glucose, plasma free fatty acid and plasma insulin are risk factors for changes in arterial wall characteristic at a stage when no clinical evidence of macrovascular disease is apparent. Arterial compliance was decreased and the proximal resistance of posterior tibial artery was increased in those with a low intake of protective foods compared with those with a high intake whether healthy subjects or non-insulin-dependent diabetics. Arterial compliance was decreased in non-fish eaters compared with the fish eaters whether healthy subjects or non-insulin-dependent diabetics. Proximal resistance of the posterior tibia! artery in non-fish eaters was increased compared with fish eaters in healthy subjects. Overall, food variety, a protective food score consumption and fish consumption emerge as importance determinants of arterial wall characteristics at a stage when no clinical evidence of macrovascular disease is apparent.

Non-invasive collection and examination of human corneal epithelial cells

Purpose. To report the development of a new apparatus for non-invasive collection of human corneal epithelial cells.

Methods. Previous methods of non-invasive, irrigative corneal cell collection resulted in low cell yields limiting potential analysis. A new ocular surface cell collection apparatus (OSCCA) was designed to collect more epithelial cells from direct irrigation of the corneal surface to allow for clinical comparisons. Forty-five samples were obtained (unilateral or bilateral over seven visits) from five human participants. Cell yield, size, phenotype, and corneal staining (prior and post eye wash) were examined.

Results. On average 364 ± 230 epithelial cells were collected from the cornea per eye. Epithelial cell sizes ranged from 8.21 to 51.69 μm in diameter, and 67.30 to 2098.85 μm2 area. The proportion of corneal specific cells collected per sample was 75 ± 14% as determined by positive K3 expression with AE5. On average, 77 ± 0.2% of epithelial cells harvested were nucleated, the remainder were non-nucleated ghost cells. Corneal staining was reduced in the OSCCA-washed vs. contralateral non-washed eyes (p = 0.02).

Conclusions. The OSCCA allows collection of human corneal epithelial cells with significantly higher yields, and greater specificity than previously reported. Reduced corneal staining observed post eye-wash demonstrated the safety of the technique, and its ability to remove cells directly from the corneal surface. The OSCCA could provide an objective non-invasive method of investigating pathological changes, effects of topical therapeutics, and impact of contact lenses and care-solutions of the cells of the ocular surface.

Cloud-based non-invasive tele-rehabilitation exercise monitoring

Exercise based rehabilitation plays a vital role in the recovery of various conditions, such as stroke, Parkinson’s disease (PD), chronic pain, and so on. Recently, telerehabilitation has become increasingly popular quantitative nature in assessments particularly for systematic monitoring of progress as well as cost saving for the patients as well as for the health care sector at large. However, challenges do exist in implementing a distributed bio-feedback in a cost-effective and efficient way. In this paper, we present the associated conceptual framework of cloud-based tele-rehabilitation system employing affordable non-invasive Microsoft Kinect® allowing patients to perform rehabilitation exercises in non-clinical setting such as home environments without loosing the quality of patients care. More importantly, different from existing tele-rehabilitation systems, our system not only measures whether patients can perform rehabilitation tasks, but also how well they can finish the tasks. Preliminary experiments validate its potential in training healthy subject to perform exercise motions emulating the physical rehabilitation process.

The double sensor-a non-invasive device to continuously monitor core temperature in humans on earth and in space

The objective of our study was to establish whether rectal temperature recordings in humans could be replaced by a non-invasive skin temperature sensor combined with a heat flux sensor (Double Sensor) located at the forehead to monitor core body temperature changes due to circadian rhythms. Rectal and Double Sensor data were collected continuously for 24h in seven men undertaking strict head-down tilt bed-rest. Individual differences between the two techniques varied between -0.72 and +0.55 degrees C. Nonetheless, when temperature data were approximated by cosinor analysis in order to compare circadian rhythm profiles between methods, it was observed that there were no significant differences between mesor, amplitude, and acrophase (P>0.310). It was therefore concluded that the Double Sensor technology is presently not accurate enough for performing single individual core body temperature measurements under resting conditions at normal ambient room temperature. Yet, it seems to be a valid, non-invasive alternative for monitoring circadian rhythm profiles.

Force measurement capability for robotic assisted minimally invasive surgery systems

An automated laparoscopic instrument capable of non-invasive measurement of tip/tissue interaction forces for direct application in robotic assisted minimally invasive surgery systems_ is introduced in this paper. It has the capability to measure normal grasping forces as well as lateral interaction forces without any sensor mounted on the tip jaws. Further to non-invasive actuation of the tip, the proposed instrument is also able to change the grasping direction during surgical operation. Modular design of the instrument allows conversion between surgical modalities (e.g., grasping, cutting, and dissecting). The main focus of this paper is on evaluation of the grasping force capability of the proposed instrument. The mathematical formulation of fenestrated insert is presented and its non-linear behaviour is studied. In order to measure the stiffness of soft tissues, a device was developed that is also described in this paper. Tissue characterisation experiments were conducted and results are presented and analysed here. The experimental results verify the capability of the proposed instrument in accurately measuring grasping forces and in characterising artificial tissue samples of varying stiffness.

Soft tissue characterisation using a force feedback-enabled instrument for robotic assisted minimally invasive surgery systems

An automated laparoscopic instrument capable of non-invasivemeasurement of tip/tissue interaction forces for direct application in robotic assisted minimally invasive surgery systems is introduced in this chapter. It has the capability to measure normal grasping forces as well as lateral interaction forces without any sensor mounted on the tip jaws. Further to non-invasive actuation of the tip, the proposed instrument is also able to change the grasping direction during surgical operation. Modular design of the instrument allows conversion between surgical modalities (e.g., grasping, cutting, and dissecting). The main focus of thispaper is on evaluation of the grasping force capability of the proposed instrument. The mathematical formulation of fenestrated insert is presented and its non-linear behaviour is studied. In order to measure the stiffness of soft tissues, a device was developed that is also described in this chapter. Tissue characterisation experiments were conducted and results are presented and analysed here. The experimental results verify the capability of the proposed instrument in accurately measuring grasping forces and in characterising artificial tissue samples of varying stiffness.

A PAL1 gene promoter-green fluorescent protein reporter system to analyse defence responses in live cells of Arabidopsis thaliana

Arabidopsis thaliana ecotype Columbia-0 was transformed with a green fluorescent protein (GFP) gene under control of a phenylalanine ammonia-lyase (PAL) promoter. PAL is a key enzyme of the phenylpropanoid pathway and is induced to high levels during plant stress. Constitutive expression of PAL1 promoter-controlled GFP occurred in vascular tissues within stems, leaves and roots and in developing flowers. PAL1 promoter–GFP expression was examined in leaves of transgenic plants subjected to an abiotic elicitor, mechanical wounding or to inoculation with the pathogens Pseudomonas syringae pv. tomato or Peronospora parasitica. Wounding of leaves and treatment with an abiotic elicitor and compatible interactions produced low to moderate levels of GFP. However, in incompatible interactions there were high levels of GFP produced. In incompatible interactions, the intensity of GFP fluorescence was similar to that produced in transgenic plants expressing GFP driven by the CaMV promoter. The bright green fluorescence produced in live cells and tissues was readily visualised using conventional fluorescence microscopy and was quantified using spectroflourometry. This is the first report of the use of GFP as a reporter of defence gene activation against pathogens. It has several advantages over other reporter genes including real time analysis of gene expression and visualisation of defence gene activation in a non-invasive manner.

Does testosterone mediate the trade-off between nestling begging and growth in the canary (Serinus canaria)?

Nestling birds solicit food from their parents with vigorous begging displays, involving posturing, jostling and calling. In some species, such as canaries, begging is especially costly because it causes a trade off against nestling growth. Fitness costs of begging like this are predicted by evolutionary theory because they function to resolve conflicts of interest within the family over the provision of parental investment. However, the mechanism that links these costs with nestling behaviour remains unclear. In the present study, we determine if the relationships between nestling androgen levels, nestling begging intensities and nestling growth rates are consistent with the hypothesis that testosterone is responsible for the trade-off between begging and growth. We test this idea with a correlational study, using fecal androgens as a non-invasive method for assaying nestling androgen levels. Our results show that fecal androgen levels are positively correlated with nestling begging intensity, and reveal marked family differences in each trait. Furthermore, changes in fecal androgen levels between 5 and 8 days after hatching are positively associated with changes in nestling begging intensity, and negatively associated with nestling growth during this time. Although these correlational results support our predictions, we suggest that that experimental manipulations are now required to test the direct or indirect role of testosterone in mediating the trade-off between begging and growth.