One night’s CPAP withdrawal in otherwise compliant OSA patients: marked driving impairment but good awareness of increased sleepiness

Purpose Obstructive sleep apnoea (OSA) patients effectively treated by and compliant with continuous positive air pressure (CPAP) occasionally miss a night’s treatment. The purpose of this study was to use a real car interactive driving simulator to assess the effects of such an occurrence on the next day’s driving, including the extent to which these drivers are aware of increased sleepiness. Methods Eleven long-term compliant CPAP-treated 50–75-year-old male OSA participants completed a 2-h afternoon, simulated, realistic monotonous drive in an instrumented car, twice, following one night: (1) normal sleep with CPAP and (2) nil CPAP. Drifting out of road lane (‘incidents’), subjective sleepiness every 200 s and continuous electroencephalogram (EEG) activities indicative of sleepiness and compensatory effort were monitored. Results Withdrawal of CPAP markedly increased sleep disturbance and led to significantly more incidents, a shorter ‘safe’ driving duration, increased alpha and theta EEG power and greater subjective sleepiness. However, increased EEG beta activity indicated that more compensatory effort was being applied. Importantly, under both conditions, there was a highly significant correlation between subjective and EEG measures of sleepiness, to the extent that participants were well aware of the effects of nil CPAP. Conclusions Patients should be aware that compliance with treatment every night is crucial for safe driving.

Understanding the impact of legislative changes on child car seating and restraint practices in regional Queensland

Road crashes contribute to a significant amount of child mortality and morbidity in Australia. In fact, passenger injuries contribute to the majority of child crash road trauma. A number of factors contribute to child injury and death in motor vehicles, including inappropriate seating position, inappropriate choice of restraint, and incorrect installation and use of child restraints. Prior to March 2010, child restraint legislation in Queensland only required children twelve months and younger to be seated in a properly adjusted and fastened child restraint. This legislation left older infants and young children potentially suboptimally protected. From March 2010, new legislation specified seating position and type of child restraint required, depending on the age of the child. This research was underpinned by the Health Belief Model (HBM), which explores health related behaviour, behaviour change, environmental factors influencing behaviour change (including legislative changes) and is flexible enough to be used in relation to parents' health practices for their children, rather than parent health directly. This thesis investigates the extent to which the changes to child restraint legislation have led parents in regional areas of Queensland to use appropriate restraint practices for their children and determines the extent to which the constructs of the HBM, parental perceptions, barriers and environmental factors contribute to the appropriateness of child seating and restraint use. Study One included three sets of observations taken in two regional cities of Queensland prior to the legislative amendment, during an educative period of six months, and after the enactment of the legislation. Each child's seating position and restraint type were recorded. Results showed that the proportion of children observed occupying the front seat decreased by 15.6 per cent with the announcement the legislation. There was no decrease in front seat use at the enactment of the legislation. The proportion of children observed using dedicated child restraints increased by 8.8 per cent with the announcement of the legislation when there was one child in the vehicle. Further, there was a 10.1 per cent increase in the proportion of children observed using a seat belt that fit with the announcement when there was one child in the vehicle and with the enactment of the legislation regardless of the number of children in the vehicle (21.8 per cent for one child, 39.7 per cent for two children and 40.2 per cent for three or more children). Study Two comprised initial intercept interviews, later followed up by telephone, with parents with children aged eight years and younger at the announcement and telephone interviews at the enactment of the legislation in one regional city in Queensland. Parents reported their child restraint practices, and opinions, knowledge and understanding of the requirements of the new legislation. Parent responses were analysed in terms of the constructs in the HBM. When asked which seating position their child 'usually' used, parents reported child front seat use was nil (0.0 per cent) and did not change with the enactment of the legislative amendment. However, when parents were asked whether they allowed children to use the front seat at some point within the six months prior to the interview, reported child front seat use was 7 (5.4 per cent) children at T2 and 10 (9.6 per cent) at T3. Reported use of age-appropriate child restraints did not increase with the enactment of the legislation (p = 0.77, ns). Parents reported restraint practices were classed as either appropriate or inappropriate. Parents who reported appropriate restraint practices were those whose children were sitting in optimal restraints and seating positions for their age according to the requirements of the legislation. Parents who reported inappropriate restraint practices were those who had one or more children who were suboptimally restrained or seated for their age according to the requirements of the legislation. Neither parents' perceptions about their susceptibility of being in a crash nor the likelihood of severity of child injury if involved in a crash yielded significant differences in the appropriateness of reported parent restraint practices over time with the enactment of the legislation. A trend in the data suggested parents perceived a benefit to using appropriate restraint practices was to avoid fines and demerit points. Over 75 per cent of parents who agreed that child restraints provide better protection for children than an adult seat belt reported appropriately seating and restraining their children (2 (1) = 8.093, p<.05). The self-efficacy measure regarding parents' confidence in installing a child restraint showed a significant association with appropriate parental restraint practices (2 (1) = 7.036, p<.05). Results suggested that some parents may have misinterpreted the announcement of the legislative amendment as the announcement of the enforcement of the legislation instead. Some parents who correctly reported details of the legislation did not report appropriate child restraint practices. This finding shows that parents' knowledge of the legislative amendment does not necessarily have an impact on their behaviour to appropriately seat and restrain children. The results of these studies have important implications for road safety and the prevention of road-related injury and death to children in Queensland. Firstly, parents reported feeling unsure of how to install restraints, which suggests that there may be children travelling in restraints that have not been installed correctly, putting them at risk. Interventions to alert and encourage parents to seek advice when unsure about the correct installation of child restraints could be considered. Secondly, some parents in this study although they were using the most appropriate restraint for their children, reported using a type that was not the most appropriate restraint for the child's age according to the legislation. This suggests that intervention may be effective in helping parents make a more accurate choice of the most appropriate type of restraint to use with children, especially as the child ages and child restraint requirements change. Further research could be conducted to ascertain the most effective methods of informing and motivating parents to use the most appropriate restraints and seating positions for their children, as these results show a concerning disparity between reported restraint practices and those that were observed.

Preoperative fasting procedures : let's use the evidence

Today's anaesthetic techniques are improved and pulmonary aspiration in elective surgical patients is rare. The purpose of fasting guidelines for healthy, low risk patients undergoing elective surgery is to minimize the volume of gastric contents while avoiding unnecessary thirst and dehydration. Fasting guidelines should be based on the best available evidence and in the absence of evidence, on the knowledge of gastrointestinal physiology.

Associations between individual and workplace risk factors for self-reported neck pain and disability among female office workers

A cross-sectional survey of female office workers (n=333) was undertaken to determine the level of neck pain and disability (Neck Disability Index—NDI) and to explore the relationship between individual and workplace risk factors with the NDI score and the presence of pain. Workers reported nil (32%), mild (53%), moderate (14%) and severe (1%) neck pain. There were more risk factors associated with the NDI score than the presence of neck pain. The presence of neck pain was associated with a history of neck trauma (OR: 4.8), using a graduated lens (OR: 4.6), and negative affectivity (OR: 2.7) in the multiple regression model. Factors associated with higher NDI score were using the computer mouse for more than 6 h per day, higher negative affectivity, older age and an uncomfortable workstation. These results suggest that measuring the level of neck pain and disability rather than just the presence of neck pain provides more specific directives for the prevention and management of this disorder.

Logic-gate devices based on printed polymer semiconducting nanostripes

The applications of organic semiconductors in complex circuitry such as printed CMOS-like logic circuits demand miniaturization of the active structures to the submicrometric and nanoscale level while enhancing or at least preserving the charge transport properties upon processing. Here, we addressed this issue by using a wet lithographic technique, which exploits and enhances the molecular order in polymers by spatial confinement, to fabricate ambipolar organic field effect transistors and inverter circuits based on nanostructured single component ambipolar polymeric semiconductor. In our devices, the current flows through a precisely defined array of nanostripes made of a highly ordered diketopyrrolopyrrole-benzothiadiazole copolymer with high charge carrier mobility (1.45 cm2 V-1 s-1 for electrons and 0.70 cm2 V-1 s-1 for holes). Finally, we demonstrated the functionality of the ambipolar nanostripe transistors by assembling them into an inverter circuit that exhibits a gain (105) comparable to inverters based on single crystal semiconductors.

Understanding the effects of complex seasonality on suburban daily transit ridership

Fluctuations in transit ridership pattern over the year have always concerned transport planners, operators and researchers. Predominantly, metrological elements have been specified to explain variability in ridership volume. However, the outcome of this research points to new direction to explain ridership fluctuation in Brisbane. It explored the relationship between daily bus ridership, seasonality and weather variables for a one-year period, 2012. Rather than segregating the entire year’s ridership into the four calendar seasons (summer, autumn, spring, and winter), this analysis distributed the yearly ridership into nine complex seasonality blocks. These represent calendar season, school/university (academic) period and their corresponding holidays, as well as other observant holidays such as Christmas. The dominance of complex seasonality over typical calendar season was established through analysis and using Multiple Linear Regression (MLR). This research identified a very strong association between complex seasonality and bus ridership. Furthermore, an expectation that Brisbane’s subtropical summer is unfavourable to transit usage was not supported by the findings of this study. A nil association of precipitation and temperature was observed in this region. Finally, this research developed a ridership estimation model, capable of predicting daily ridership within very limited error range. Following the application of this developed model, the estimated annual time series data of each suburb was analysed using Fourier Transformation to appreciate whether any cyclical effects remained, compared with the original data.

Quality of life in children with Crohn disease

Objectives: Quality of life (QOL) is reportedly poor in children with Crohn disease (CD) but improves with increasing disease duration. This article aims to detail QOL in a cohort of Australian children with CD in relation to disease duration, disease activity, and treatment. MATERIALS AND METHODS: QOL, assessed using the IMPACT-III questionnaire, and disease activity measures, assessed using the Pediatric Crohn's Disease Activity Index (PCDAI), were available in 41 children with CD. For this cohort, a total of 186 measurements of both parameters were available. Results: QOL was found to be significantly lower, and disease activity significantly higher (F = 31.1, P = 0.00), in patients within 6 months of their diagnosis compared with those up to 2.5 years, up to 5 years, and beyond 5 years since diagnosis. Higher disease activity was associated with poorer QOL (r =-0.51, P = 0.00). Total QOL was highest in children on nil medications and lowest in children on enteral nutrition. The PCDAI (t =-6.0, P = 0.00) was a significant predictor of QOL, with the clinical history (t =-6.9, P = 0.00) and examination (t =-2.9, P = 0.01) sections of the PCDAI significantly predicting QOL. Disease duration, age, or sex was neither related to nor significant predictors of QOL, but height z score and type of treatment approached significance. Conclusions: Children with CD within 6 months of their diagnosis have impaired QOL compared with those diagnosed beyond 6 months. These patients, along with those with growth impairment, ongoing elevated disease activity with abdominal pain, diarrhoea and/or perirectal and extraintestinal complications, may benefit from regular assessments of QOL as part of their clinical treatment. © 2010 by European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.

SU-8-Based Microchips for Capillary Electrophoresis and Electrospray Ionization Mass Spectrometry

Miniaturization of analytical instrumentation is attracting growing interest in response to the explosive demand for rapid, yet sensitive analytical methods and low-cost, highly automated instruments for pharmaceutical and bioanalyses and environmental monitoring. Microfabrication technology in particular, has enabled fabrication of low-cost microdevices with a high degree of integrated functions, such as sample preparation, chemical reaction, separation, and detection, on a single microchip. These miniaturized total chemical analysis systems (microTAS or lab-on-a-chip) can also be arrayed for parallel analyses in order to accelerate the sample throughput. Other motivations include reduced sample consumption and waste production as well as increased speed of analysis. One of the most promising hyphenated techniques in analytical chemistry is the combination of a microfluidic separation chip and mass spectrometer (MS). In this work, the emerging polymer microfabrication techniques, ultraviolet lithography in particular, were exploited to develop a capillary electrophoresis (CE) separation chip which incorporates a monolithically integrated electrospray ionization (ESI) emitter for efficient coupling with MS. An epoxy photoresist SU-8 was adopted as structural material and characterized with respect to its physicochemical properties relevant to chip-based CE and ESI/MS, namely surface charge, surface interactions, heat transfer, and solvent compatibility. As a result, SU-8 was found to be a favorable material to substitute for the more commonly used glass and silicon in microfluidic applications. In addition, an infrared (IR) thermography was introduced as direct, non-intrusive method to examine the heat transfer and thermal gradients during microchip-CE. The IR data was validated through numerical modeling. The analytical performance of SU-8-based microchips was established for qualitative and quantitative CE-ESI/MS analysis of small drug compounds, peptides, and proteins. The CE separation efficiency was found to be similar to that of commercial glass microchips and conventional CE systems. Typical analysis times were only 30-90 s per sample indicating feasibility for high-throughput analysis. Moreover, a mass detection limit at the low-attomole level, as low as 10E+5 molecules, was achieved utilizing MS detection. The SU-8 microchips developed in this work could also be mass produced at low cost and with nearly identical performance from chip to chip. Until this work, the attempts to combine CE separation with ESI in a chip-based system, amenable to batch fabrication and capable of high, reproducible analytical performance, have not been successful. Thus, the CE-ESI chip developed in this work is a substantial step toward lab-on-a-chip technology.

Sediment and nutrient losses from exotic Pinus plantation management operations under simulated rainfall

Rainfall simulation experiments were carried out to measure runoff and soil water fluxes of suspended solids, total nitrogen, total phosphorus, dissolved organic carbon and total iron from sites in Pinus plantations on the coastal lowlands of south-eastern Queensland subjected to various operations (treatments). The operations investigated were cultivated and nil-cultivated site preparation, fertilised site preparation, clearfall harvesting and prescribed burning; these treatments were compared with an 8-y-old established plantation. Flow-weighted mean concentrations of total nitrogen and total phosphorus in surface runoff from the cultivated and nil-cultivated site-preparation, clearfall harvest, prescribed burning and 8-y-old established plantation treatments were very similar. However, both the soil water and the runoff from the fertilised site preparation treatment contained more nitrogen (N) and phosphorus (P) than the other treatments - with 3.10 mg N L-1 and 4.32 mg P L-1 (4 and 20 times more) in the runoff. Dissolved organic carbon concentrations in runoff from the nil-cultivated site-preparation and prescribed burn treatments were elevated. Iron concentrations were highest in runoff from the nil-cultivated site-preparation and 8-y-old established plantation treatments. Concentrations of suspended solids in runoff were higher from cultivated site preparation and prescribed burn treatments, and reflect the great disturbance of surface soil at these sites. The concentrations of all analytes were highest in initial runoff from plots, and generally decreased with time. Total nitrogen (mean 7.28, range 0.11-13.27 mg L-1) and total phosphorus (mean 11.60, range 0.06-83.99 mg L-1) concentrations in soil water were between 2 and 10 times greater than in surface runoff, which highlights the potential for nutrient fluxes in interflow (i.e. in the soil above the water table) through the general plantation area. Implications in regard to forest management are discussed, along with results of larger catchment-scale studies.

Effects of rootstock and nitrogen fertiliser on postharvest anthracnose development in Hass avocado

These rootstock and nitrogen fertiliser studies confirmed that rootstock race can significantly affect the development of postharvest disease and mineral nutrient accumulation in Hass avocado fruit. When Hass (Guatemalan race) was grafted to seedling Velvick (West Indian race) rootstock, the severity and incidence of anthracnose in fruit were significantly reduced by up to 64 and 37%, respectively, compared with seedling Duke 6 (Mexican race) rootstock. Stem-end rot was also influenced by rootstock in some seasons, and significant reductions (up to 87%) in the severity and incidence of stem-end rot were recorded in Hass fruit from Velvick compared with Duke 6 rootstock trees. These improvements in postharvest diseases were associated with significantly lower concentrations of nitrogen and potassium, higher concentrations of calcium and magnesium, lower ratios of nitrogen:calcium and higher ratios of calcium + magnesium:potassium in Hass leaves and fruit from Velvick compared with Duke 6 rootstock trees. Altering the rate of nitrogen fertiliser had minimal impact on postharvest disease development. However, in one season, reducing the rate of nitrogen fertiliser to nil significantly reduced the concentration of nitrogen in the fruit skin, decreased the nitrogen:calcium ratio and significantly reduced the severity and incidence of anthracnose in Hass fruit from both Velvick and Duke 6 rootstock trees. The form of nitrogen fertiliser (ammonium compared with nitrate) applied to the trees did not significantly affect the postharvest disease susceptibility of Hass avocado fruit on either Velvick or Duke 6 rootstock. The Guatemalan race rootstocks, Anderson 8 and Anderson 10, were also found to be superior to the Mexican race rootstock, Parida 1, for reducing anthracnose severity. This again, was associated with a better balance of mineral nutrients (significantly lower nitrogen:calcium and higher calcium + magnesium:potassium ratios) in the fruit. This rootstock effect, however, was only observed in the first season of a 3-year experiment, possibly because of a better balance between vegetative growth and fruit production in Parida 1 in the latter two seasons. Significant positive correlations between anthracnose severity and fruit skin nitrogen:calcium ratios were evident across all experiments.

The effect of endophyte on the performance of irrigated perennial ryegrasses in subtropical Australia

The effect of fungal endophyte (Neotyphodium lolii) infection on the performance of perennial ryegrass (Lolium perenne) growing under irrigation in a subtropical environment was investigated. Seed of 4 cultivars, infected with standard (common toxic or wild-type) endophyte or the novel endophyte AR1, or free of endophyte (Nil), was sown in pure swards, which were fertilised with 50 kg N/ha.month. Seasonal and total yield, persistence, and rust susceptibility were assessed over 3 years, along with details of the presence of endophyte and alkaloids in plant shoots. Endophyte occurrence in tillers in both the standard and AR1 treatments was above 95% for Bronsyn and Impact throughout and rose to that level in Samson by the end of the second year. Meridian AR1 only reached 93% while, in the standard treatment, the endophyte had mostly died before sowing. Nil Zendophyte treatments carried an average of ?0.6% infection throughout. Infection of the standard endophyte was associated with increased dry matter (DM) yields in all 3 years compared with no endophyte. AR1 also significantly increased yields in the second and third years. Over the full 3 years, standard and AR1 increased yields by 18% and 11%, respectively. Infection with both endophytes was associated with increased yields in all 4 seasons, the effects increasing in intensity over time. There was 27% better persistence in standard infected plants compared with Nil at the end of the first year, increasing to 198% by the end of the experiment, while for AR1 the improvements were 20 and 134%, respectively. The effect of endophyte on crown rust (Puccinia coronata) infection was inconsistent, with endophyte increasing rust damage on one occasion and reducing it on another. Cultivar differences in rust infection were greater than endophyte effects. Plants infected with the AR1 endophyte had no detectable ergovaline or lolitrem B in leaf, pseudostem, or dead tissue. In standard infected plants, ergovaline and lolitrem B were highest in pseudostem and considerably lower in leaf. Dead tissue had very low or no detectable ergovaline but high lolitrem B concentrations. Peramine concentration was high and at similar levels in leaf and pseudostem, but not detectable in dead material. Concentration was similar in both AR1 and standard infected plants. Endophyte presence appeared to have a similar effect in the subtropics as has been demonstrated in temperate areas, in terms of improving yields and persistence and increasing tolerance of plants to stress factors.

Sorghum stay-green QTL individually reduce post-flowering drought-induced leaf senescence

Sorghum is an important source of food, feed, and biofuel, especially in the semi-arid tropics because this cereal is well adapted to harsh, drought-prone environments. Post-flowering drought adaptation in sorghum is associated with the stay-green phenotype. Alleles that contribute to this complex trait have been mapped to four major QTL, Stg1-Stg4, using a population derived from BTx642 and RTx7000. Near-isogenic RTx7000 lines containing BTx642 DNA spanning one or more of the four stay-green QTL were constructed. The size and location of BTx642 DNA regions in each RTx7000 NIL were analysed using 62 DNA markers spanning the four stay-green QTL. RTx7000 NILs were identified that contained BTx642 DNA completely or partially spanning Stg1, Stg2, Stg3, or Stg4. NILs were also identified that contained sub-portions of each QTL and various combinations of the four major stay-green QTL. Physiological analysis of four RTx7000 NILs containing only Stg1, Stg2, Stg3, or Stg4 showed that BTx642 alleles in each of these loci could contribute to the stay-green phenotype. RTx7000 NILs containing BTx642 DNA corresponding to Stg2 retained more green leaf area at maturity under terminal drought conditions than RTx7000 or the other RTx7000 NILs. Under post-anthesis water deficit, a trend for delayed onset of leaf senescence compared with RTx7000 was also exhibited by the Stg2, Stg3, and Stg4 NILs, while significantly lower rates of leaf senescence in relation to RTx7000 were displayed by all of the Stg NILs to varying degrees, but particularly by the Stg2 NIL. Greener leaves at anthesis relative to RTx7000, indicated by higher SPAD values, were exhibited by the Stg1 and Stg4 NILs. The RTx7000 NILs created in this study provide the starting point for in-depth analysis of stay-green physiology, interaction among stay-green QTL and map-based cloning of the genes that underlie this trait.

Response of perennial ryegrass (Lolium perenne) to renovation in Australian dairy pastures

This study reports on the effect of oversowing perennial ryegrass (Lolium perenne L.) into a degraded perennial ryegrass and white clover (Trifolium repens L.) pasture to extend its productive life using various intensities of seedbed preparation. Sites in New South Wales (NSW), Western Australia (WA), South Australia (SA) and Tasmania (Tas.) were chosen by a local group of farmers as being degraded and in need of renovation. Control (nil renovation) and medium (mulch and graze, spray with glyphosphate and sow) renovation treatments were common to all sites whereas minimum (mulch and graze, and sow) and full seedbed (graze and spray with glyphosphate and then full seedbed preparation) renovation were imposed only at some sites. Plots varied in area from 0.14 to 0.50 ha, and were renovated then sown in March or April 2000 and subsequently grazed by dairy cows. Pasture utilisation was estimated from pre- and post-grazing pasture mass assessed by a rising plate pasture meter. Utilised herbage mass of the renovated treatments was significantly higher than control plots in period 1 (planting to August) and 2 (first spring) at the NSW site only. There was no difference among treatments in period 3 (first summer) at any site, and only at the WA and NSW sites in period 4 (March to July 2001) was there a response to renovation. As a result, renovation at the NSW site only significantly increased ryegrass utilisation over the whole experimental period. Ryegrass plant density was higher at the NSW, WA (excluding minimum renovation) and Tas. (excluding full renovation) sites 6 months after renovation but this was only sustained for 12 months for the minimum and medium treatments at the NSW and Tas. sites, respectively, presumably due to reduced competition from naturalised C4 summer grasses [kikuyu (Pennisetum clandestinum) and paspalum (Paspalum dilatatum)] in NSW At the NSW, WA and SA sites, the original ryegrass plant density was low (<35 plants/m2) compared with the Tas. site where density was around 185/m2. The response to renovating a degraded perennial ryegrass pasture varied between sites in Australia. Positive responses were generally small and were most consistent where renovation removed competing C4 summer grasses.

Growth responses of sugarcane to mycorrhizal spore density and phosphorus rate

Arbuscular mycorrhizal (AM) fungi, commonly found in long-term cane-growing fields in northern Queensland, are linked with both negative and positive growth responses by sugarcane (Saccharum spp.), depending on P supply. A glasshouse trial was established to examine whether AM density might also have an important influence on these growth responses. Mycorrhizal spores (Glomus clarum), isolated from a long-term cane block in northern Queensland, were introduced into a pasteurised low-P cane soil at 5 densities (0, 0.06, 0.25, 1, 4 spores/g soil) and with 4 P treatments (0, 8.2, 25, and 47 mg/kg). At 83 days after planting, sugarcane tops responded positively to P fertilizer, although responses attributable to spore density were rarely observed. In one case, addition of 4 spores/g led to a 53% yield response over those without AM at 8 mgP/kg, or a relative benefit of 17 mg P/kg. Root colonisation was reduced for plants with nil or 74 mg P/kg. For those without AM, P concentration in the topmost visible dewlap (TVD) leaf increased significantly with fertiliser P (0.07 v. 0.15%). However, P concentration increased further with the presence of AM spores. Irrespective of AM, the critical P concentration in the TVD leaf was 0.18%. This study confirms earlier reports that sugarcane is poorly responsive to AM. Spore density, up to 4 spores/g soil, appears unable to influence this responsiveness, either positively or negatively. Attempts to gain P benefits by increasing AM density through rotation seem unlikely to lead to yield increases by sugarcane. Conversely, sugarcane grown in fields with high spore densities and high plant-available P, such as long-term cane-growing soils, is unlikely to suffer a yield reduction from mycorrhizal fungi.

Gene expression evidence for off-target effects caused by RNA interference-mediated gene silencing of Ubiquitin-63E in the cattle tick Rhipicephalus microplus.

Knowledge of cattle tick (Rhipicephalus (Boophilus) microplus; Acari: Ixodidae) molecular and cellular pathways has been hampered by the lack of an annotated genome. In addition, most of the tick expressed sequence tags (ESTs) available to date consist of similar to 50% unassigned sequences without predicted functions. The most common approach to address this has been the application of RNA interference (RNAi) methods to investigate genes and their pathways. This approach has been widely adopted in tick research despite minimal knowledge of the tick RNAi pathway and double-stranded RNA (dsRNA) uptake mechanisms. A strong knockdown phenotype of adult female ticks had previously been observed using a 594 bp dsRNA targeting the cattle tick homologue for the Drosophila Ubiquitin-63E gene leading to nil or deformed eggs. A NimbleGen cattle tick custom microarray based on the BmiGI.V2 database of R. microplus ESTs was used to evaluate the expression of mRNAs harvested from ticks treated with the tick Ubiquitin-63E 594 bp dsRNA compared with controls. A total of 144 ESTs including TC6372 (Ubiquitin-63E) were down-regulated with 136 ESTs up-regulated following treatment. The results obtained substantiated the knockdown phenotype with ESTs identified as being associated with ubiquitin proteolysis as well as oogenesis, embryogenesis, fatty acid synthesis and stress responses. A bioinformatics analysis was undertaken to predict off-target effects (OTE) resulting from the in silico dicing of the 594 bp Ubiquitin-63E dsRNA which identified 10 down-regulated ESTs (including TC6372) within the list of differentially expressed probes on the microarrays. Subsequent knockdown experiments utilising 196 and 109 bp dsRNAs, and a cocktail of short hairpin RNAs (shRNA) targeting Ubiquitin-63E, demonstrated similar phenotypes for the dsRNAs but nil effect following shRNA treatment. Quantitative reverse transcriptase PCR analysis confirmed differential expression of TC6372 and selected ESTs. Our study demonstrated the minimisation of predicted OTEs in the shorter dsRNA treatments (similar to 100-200 bp) and the usefulness of microarrays to study knockdown phenotypes.