Dynamic analysis of drug-induced cytotoxicity using chip-based dielectrophoretic cell immobilization technology

Quantification of programmed and accidental cell death provides useful end-points for the anticancer drug efficacy assessment. Cell death is, however, a stochastic process. Therefore, the opportunity to dynamically quantify individual cellular states is advantageous over the commonly employed static, end-point assays. In this work, we describe the development and application of a microfabricated, dielectrophoretic (DEP) cell immobilization platform for the realtime analysis of cancer drug-induced cytotoxicity. Microelectrode arrays were designed to generate weak electro-thermal vortices that support efficient drug mixing and rapid cell immobilization at the delta-shape regions of strong electric field formed between the opposite microelectrodes. We applied this technology to the dynamic analysis of hematopoietic tumor cells that represent a particular challenge for real-time imaging due to their dislodgement during image acquisition. The present study was designed to provide a comprehensive mechanistic rationale for accelerated cell-based assays on DEP chips using real-time labeling with cell permeability markers. In this context, we provide data on the complex behavior of viable vs dying cells in the DEP fields and probe the effects of DEP fields upon cell responses to anticancer drugs and overall bioassay performance. Results indicate that simple DEP cell immobilization technology can be readily applied for the dynamic analysis of investigational drugs in hematopoietic cancer cells. This ability is of particular importance in studying the outcome of patient derived cancer cells, when exposed to therapeutic drugs, as these cells are often rare and difficult to collect, purify and immobilize.

Interfacing cell-based assays in environmental scanning electron microscopy using dielectrophoresis

Development of the dielectrophoretic (DEP) live cell trapping technology and its interfacing with the environmental scanning electron microscopy (ESEM) is described. DEP microelectrode arrays were fabricated on glass substrate using photolithography and lift-off. Chip-based arrays were applied for ESEM analysis of DEP-trapped human leukemic cells. This work provides proof-of-concept interfacing of the DEP cell retention and trapping technology with ESEM to provide a high-resolution analysis of individual nonadherent cells.

Investigation of GMP signalling systems in lower vertebrates

This research examined aspects of the cyclic GMP signalling system in fish and amphibians. It contributed new knowledge to our understanding of the role of the system in the development of zebrafish, a model fish species, and in control of the vasculature by the regulatory molecule, nitric oxide.

Context reinstatement effects in children’s cued recall of strongly and weakly associated word pairs

Typically, asking people to reinstate the context of events increases their recall of those events; however, research findings have been mixed with children. We tested whether the principle underlying context reinstatement applies to children as it does to adults. This underlying principle, encoding specificity, suggests that the greater the overlap between study context cues and retrieval context cues, the more information that people should recall. In the current experiment, four age groups (7-year-olds, 9-year-olds, 11-year-olds and adults) took part in an encoding specificity procedure. At study, participants saw cue– target word pairs in which the cue word was either a strong or a weak associate of the target word (e.g., ice–COLD; blow–COLD). During an immediate cued recall test, participants were presented with the same strong or weak cue words and new, extra-list cue words. Overall, children and adults recalled more targets when they were presented with the same cue words at study and test, regardless of whether the cues were strong or weak. This finding suggests that encoding specificity applies to children as well as adults. We discuss the implications of these results.

Trapping and imaging of micron-sized embryos using dielectrophoresis

Development of dielectrophoretic (DEP) arrays for real-time imaging of embryonic organisms is described. Microelectrode arrays were used for trapping both embryonated eggs and larval stages of Antarctic nematode Panagrolaimus davidi. Ellipsoid single-shell model was also applied to study the interactions between DEP fields and developing multicellular organisms. This work provides proof-of-concept application of chip-based technologies for the analysis of individual embryos trapped under DEP force.

Visy interviews data

This data collection consists of:
• Raw interview data from semi-structured interviews
• Transcriptions of interviews
• Photographs
• Sound files
• Diary data from the learning object development process

Data is drawn from respondents in the Visy Industrial Packaging factory site. The data is themed according to the Deakin graduate attributes.

Dielectrophoresis of micro/nano particles using curved microelectrodes

Dielectrophoresis, the induced motion of polarisable particles in non-homogenous electric field, has been proven as a versatile mechanism to transport, immobilise, sort and characterise micro/nano scale particle in microfluidic platforms. The performance of dielectrophoretic (DEP) systems depend on two parameters: the configuration of microelectrodes designed to produce the DEP force and the operating strategies devised to employ this force in such processes. This work summarises the unique features of curved microelectrodes for the DEP manipulation of target particles in microfluidic systems. The curved microelectrodes demonstrate exceptional capabilities including (i) creating strong electric fields over a large portion of their structure, (ii) minimising electro-thermal vortices and undesired disturbances at their tips, (iii) covering the entire width of the microchannel influencing all passing particles, and (iv) providing a large trapping area at their entrance region, as evidenced by extensive numerical and experimental analyses. These microelectrodes have been successfully applied for a variety of engineering and biomedical applications including (i) sorting and trapping model polystyrene particles based on their dimensions, (ii) patterning carbon nanotubes to trap low-conductive particles, (iii) sorting live and dead cells based on their dielectric properties, (iv) real-time analysis of drug-induced cell death, and (v) interfacing tumour cells with environmental scanning electron microscopy to study their morphological properties. The DEP systems based on curved microelectrodes have a great potential to be integrated with the future lab-on-a-chip systems.

Histological and global gene expression analysis of the ‘lactating’ pigeon crop

Background: Both male and female pigeons have the ability to produce a nutrient solution in their crop for the nourishment of their young. The production of the nutrient solution has been likened to lactation in mammals, and hence the product has been called pigeon ‘milk’. It has been shown that pigeon ‘milk’ is essential for growth and development of the pigeon squab, and without it they fail to thrive. Studies have investigated the nutritional value of pigeon ‘milk’ but very little else is known about what it is or how it is produced. This study aimed to gain insight into the process by studying gene expression in the ‘lactating’ crop.
Results: Macroscopic comparison of ‘lactating’ and non-’lactating’ crop reveals that the ‘lactating’ crop is enlarged and thickened with two very obvious lateral lobes that contain discrete rice-shaped pellets of pigeon ‘milk’. This was characterised histologically by an increase in the number and depth of rete pegs extending from the basal layer of the epithelium to the lamina propria, and extensive proliferation and folding of the germinal layer into the superficial epithelium. A global gene expression profile comparison between ‘lactating’ crop and non-’lactating’ crop showed that 542 genes are up-regulated in the ‘lactating’ crop, and 639 genes are down-regulated. Pathway analysis revealed that genes up-regulated in ‘lactating’ crop were involved in the proliferation of melanocytes, extracellular matrix-receptor interaction, the adherens junction and the wingless (wnt) signalling pathway. Gene ontology analysis showed that antioxidant response and microtubule transport were enriched in ‘lactating’ crop.
Conclusions: There is a hyperplastic response in the pigeon crop epithelium during ‘lactation’ that leads to localised cellular stress and expression of antioxidant protein-encoding genes. The differentiated, cornified cells that form the pigeon ‘milk’ are of keratinocyte lineage and contain triglycerides that are likely endocytosed as very low density lipoprotein (VLDL) and repackaged as triglyceride in vesicles that are transported intracellularly by microtubules. This mechanism is an interesting example of the evolution of a system with analogies to mammalian lactation, as pigeon ‘milk’ fulfils a similar function to mammalian milk, but is produced by a different mechanism.

Does free recall moderate the effect of mental context reinstatement instructions on children's cued recall?

Cognitive Interview instructions increase children's recall of events; one important instruction is the mental reinstatement of context. We examined one factor that may affect mental context reinstatement: whether children had the opportunity to freely recall the event before answering cued recall questions. One hundred and fifty-two children aged 6, 9, or 11 years were interviewed twice about a staged event. The event consisted of an argument between two adults about whose turn it was to show the children a film. One week after the event, some of the children received mental context reinstatement instructions before having their cued recall tested. Some children also received a free recall test immediately before the cued recall test. In the second interview, 2 weeks after the first interview, all children freely recalled the event. The results showed no effects of mental context reinstatement instructions and no moderating effect of free recall on children's cued recall. The implications of these findings and directions for future research are discussed.

Water deprivation induces appetite and alters metabolic strategy in Notomys alexis : unique mechanisms for water production in the desert

Like many desert animals, the spinifex hopping mouse, Notomys alexis, can maintain water balance without drinking water. The role of the kidney in producing a small volume of highly concentrated urine has been well-documented, but little is known about the physiological mechanisms underpinning the metabolic production of water to offset obligatory water loss. In Notomys, we found that water deprivation (WD) induced a sustained high food intake that exceeded the pre-deprivation level, which was driven by parallel changes in plasma leptin and ghrelin and the expression of orexigenic and anorectic neuropeptide genes in the hypothalamus; these changed in a direction that would stimulate appetite. As the period of WD was prolonged, body fat disappeared but body mass increased gradually, which was attributed to hepatic glycogen storage. Switching metabolic strategy from lipids to carbohydrates would enhance metabolic water production per oxygen molecule, thus providing a mechanism to minimize respiratory water loss. The changes observed in appetite control and metabolic strategy in Notomys were absent or less prominent in laboratory mice. This study reveals novel mechanisms for appetite regulation and energy metabolism that could be essential for desert rodents to survive in xeric environments.