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.

A genome-wide linkage scan provides evidence for both new and previously reported loci influencing common migraine

Latent class analysis was performed on migraine symptom data collected in a Dutch population sample (N = 12,210, 59% female) in order to obtain empirical groupings of individuals suffering from symptoms of migraine headache. Based on these heritable groupings (h(2) = 0.49, 95% CI: 0.41-0.57) individuals were classified as affected (migrainous headache) or unaffected. Genome-wide linkage analysis was performed using genotype data from 105 families with at least 2 affected siblings. In addition to this primary phenotype, linkage analyses were performed for the individual migraine symptoms. Significance levels, corrected for the analysis of multiple traits, were determined empirically via a novel simulation approach. Suggestive linkage for migrainous headache was found on chromosomes 1 (LOD = 1.63; pointwise P = 0.0031), 13 (LOD = 1.63; P = 0.0031), and 20 (LOD = 1.85; P = 0.0018). Interestingly, the chromosome 1 peak was located close to the ATP1A2 gene, associated with familial hemiplegic migraine type 2 (FHM2). Individual symptom analysis produced a LOD score of 1.97 (P = 0.0013) on chromosome 5 (photo/phonophobia), a LOD score of 2.13 (P = 0.0009) on chromosome 10 (moderate/severe pain intensity) and a near significant LOD score of 3.31 (P = 0.00005) on chromosome 13 (pulsating headache). These peaks were all located near regions previously reported in migraine linkage studies. Our results provide important replication and support for the presence of migraine susceptibility genes within these regions, and further support the utility of an LCA-based phenotyping approach and analysis of individual symptoms in migraine genetic research. Additionally, our novel "2-step" analysis and simulation approach provides a powerful means to investigate linkage to individual trait components.

A reanalysis of 409 European-Ancestry and African American schizophrenia pedigrees reveals significant linkage to 8p23.3 with evidence of locus heterogeneity

The detection and replication of schizophrenia risk loci can require substantial sample sizes, which has prompted various collaborative efforts for combining multiple samples. However, pooled samples may comprise sub-samples with substantial population genetic differences, including allele frequency differences. We investigated the impact of population differences via linkage reanalysis of Molecular Genetics of Schizophrenia 1 (MGS1) affected sibling-pair data, comprising two samples of distinct ancestral origin: European (EA: 263 pedigrees) and African-American (AA: 146 pedigrees). To exploit the linkage information contained within these distinct continental samples, we performed separate analyses of the individual samples, allowing for within-sample locus heterogeneity, and the pooled sample, allowing for both within-sample and between-sample heterogeneity. Significance levels, corrected for the multiple tests, were determined empirically. For all suggestive peaks, stronger linkage evidence was obtained in either the EA or AA sample than the combined sample, regardless of how heterogeneity was modeled for the latter. Notably, we report genomewide significant linkage of schizophrenia to 8p23.3 and evidence for a second, independent susceptibility locus, reaching suggestive linkage, 29 cM away on 8p21.3. We also detected suggestive linkage on chromosomes 5p13.3 and 7q36.2. Many regions showed pronounced differences in the extent of linkage between the EA and AA samples. This reanalysis highlights the potential impact of population differences upon linkage evidence in pooled data and demonstrates a useful approach for the analysis of samples drawn from distinct continental groups.

Building capacity for cancer nursing research and evidence based practice: The cancer nursing professorial precinct initiative

The demand for cancer care is growing due to the increasing incidence of cancer and the improved effectiveness of cancer treatments. It is important that cancer nurses continue to improve patient outcomes through research and the use of evidence in practice development, education and policy. This paper describes a case report of a collaborative academic healthcare model that creates capacity for cancer nursing research and evidence-based practice. The Cancer Nursing Professorial Precinct is a strategic collaboration between the Royal Brisbane and Women’s Hospital (RBWH) and Queensland University of Technology (QUT), in Brisbane Australia. The outcomes of this initiative has been remarkable. The principles and strategies used in this initiative may be useful for cancer services in other countries.

Significant evidence of one or more susceptibility loci for endometriosis with near-Mendelian inheritance on chromosome 7p13-15

BACKGROUND: Endometriosis is a common disease with a heritable component. The collaborative International Endogene Study consists of two data sets (Oxford and Australia) comprising 1176 families with multiple affected. The aim was to investigate whether the apparent concentration of cases in a proportion of families could be explained by one or more rare variants with (near-)Mendelian autosomal inheritance. METHODS AND RESULTS: Linkage analyses (aimed at finding chromosomal regions harbouring disease-predisposing genes) were conducted in families with three or more affected (Oxford: n = 52; Australia: n = 196). In the Oxford data set, a non-parametric linkage score (Kong & Cox (K&C) Log of ODds (LOD)) of 3.52 was observed on chromosome 7p (genome-wide significance P = 0.011). A parametric MOD score (equal to maximum LOD maximized over 357 possible inheritance models) of 3.89 was found at 65.72 cM (D7S510) for a dominant model with reduced penetrance. After including the Australian data set, the non-parametric K&C LOD of the combined data set was 1.46 at 57.3 cM; the parametric analysis found an MOD score of 3.30 at D7S484 (empirical significance: P = 0.035) for a recessive model with high penetrance. Critical recombinant analysis narrowed the probable region of linkage down to overlapping 6.4 Mb and 11 Mb intervals containing 48 and 96 genes, respectively. CONCLUSIONS: This is the first report to suggest that there may be one or more high-penetrance susceptibility loci for endometriosis with (near-)Mendelian inheritance.

As clear as mud: A critical review of evidence for the ecological roles of Australian dingoes

Top-predators have been reported to have an important role in structuring food webs and maintaining ecological processes for the benefit of biodiversity at lower trophic levels. This is thought to be achieved through their suppressive effects on sympatric mesopredators and prey. Great scientific and public interest surrounds the potential use of top-predators as biodiversity conservation tools, and it can often be difficult to separate what we think we know and what we really know about their ecological utility. Not all the claims made about the ecological roles of top-predators can be substantiated by current evidence. We review the methodology underpinning empirical data on the ecological roles of Australian dingoes (Canis lupus dingo and hybrids) to provide a comprehensive and objective benchmark for knowledge of the ecological roles of Australia's largest terrestrial predator. From a wide variety of methodological flaws, sampling bias, and experimental design constraints inherent to 38 of the 40 field studies we assessed, we demonstrate that there is presently unreliable and inconclusive evidence for dingoes role as a biodiversity regulator. We also discuss the widespread (both taxonomically and geographically) and direct negative effects of dingoes to native fauna, and the few robust studies investigating their positive roles. In light of the highly variable and context-specific impacts of dingoes on faunal biodiversity and the inconclusive state of the literature, we strongly caution against the positive management of dingoes in the absence of a supporting evidence-base for such action.

Migraine with aura and migraine without aura are not distinct entities: further evidence from a large Dutch population study

It is often debated whether migraine with aura (MA) and migraine without aura (MO) are etiologically distinct disorders. A previous study using latent class analysis (LCA) in Australian twins showed no evidence for separate subtypes of MO and MA. The aim of the present study was to replicate these results in a population of Dutch twins and their parents, siblings and partners (N = 10,144). Latent class analysis of International Headache Society (IHS)-based migraine symptoms resulted in the identification of 4 classes: a class of unaffected subjects (class 0), a mild form of nonmigrainous headache (class 1), a moderately severe type of migraine (class 2), typically without neurological symptoms or aura (8% reporting aura symptoms), and a severe type of migraine (class 3), typically with neurological symptoms, and aura symptoms in approximately half of the cases. Given the overlap of neurological symptoms and nonmutual exclusivity of aura symptoms, these results do not support the MO and MA subtypes as being etiologically distinct. The heritability in female twins of migraine based on LCA classification was estimated at .50 (95% confidence intervals [CI] .27 - .59), similar to IHS-based migraine diagnosis (h2 = .49, 95% CI .19-.57). However, using a dichotomous classification (affected-unaffected) decreased heritability for the IHS-based classification (h2 = .33, 95% CI .00-.60), but not the LCA-based classification (h2 = .51, 95% CI .23-.61). Importantly, use of the LCA-based classification increased the number of subjects classified as affected. The heritability of the screening question was similar to more detailed LCA and IHS classifications, suggesting that the screening procedure is an important determining factor in genetic studies of migraine.

Further evidence for linkage of Gilles de la Tourette syndrome (GTS) susceptibility loci on chromosomes 2p11, 8q22 and 11q23-24 in South African Afrikaners

Utilizing DNA samples from 91 Afrikaner nuclear families with one or more affected children, five genomic regions on chromosomes 2p, 8q, 11q, 20q, and 21q that gave evidence for association with GTS in previous case-control association studies were investigated for linkage and association with GTS. Highly polymorphic markers with mean heterozygosity of 0.77 were typed and resulting genotypes evaluated using single marker transmission disequilibrium (TDT), single marker haplotype relative risk (HRR), and multi-marker "extended" TDT and HRR methods. Single marker TDT analysis showed evidence for linkage or association, with p-values near 0.05, for markers D2S139, GATA28F12, and D11S1377 on chromosomes 2p11, 8q22 and 11q23-24, respectively. Extended, two-locus TDT and HRR analysis provided further evidence for linkage or association on chromosome 2 with p-values of 0.007 and 0.025, and chromosome 8 with p-values of 0.059 and 0.013, respectively. These results provide important additional evidence for the location of GTS susceptibility loci.

Evidence for an X-linked genetic component in familial typical migraine

Migraine is a common complex disorder that shows strong familial aggregation. There is a general increased prevalence of migraine in females compared with males, with recent studies indicating that migraine affects 18% of females compared with 6% of males. This preponderance of females among migraine sufferers coupled with evidence of an increased risk of migraine in first degree relatives of male probands but not in relatives of female probands suggests the possibility of an X-linked dominant gene. We report here the localization of a typical migraine susceptibility locus to the X chromosome. Of three large multigenerational migraine pedigrees two families showed significant excess allele sharing to Xq markers (P = 0.031 and P = 0.012). Overall analysis of data from all three pedigrees gave significant evidence in support of linkage and heterogeneity (HLOD = 3.1). These findings provide conclusive evidence that familial typical migraine is a heterogeneous disorder. We suggest that the localization of a migraine susceptibility locus to the X chromosome could in part explain the increased risk of migraine in relatives of male probands and may be involved in the increased female prevalence of this disorder.

Evidence for a new metastable crystalline compound in Ge---Te system

Pressure dependence of the electrical resistivity of bulk, melt quenched GexTe100−x glasses (15 less-than-or-equals, slant x less-than-or-equals, slant 28) has been studied up to 8GPa pressure. All the glasses exhibit a sharp, discontinuous glass to crystal transition under pressure. The high pressure crystalline phases are identified to have a face centered cubic structure. The value of the cell constant is 0.779nm for 15 less-than-or-equals, slant x less-than-or-equals, slant 17, 0.642nm for x=20 and 0.55lnm for 22 ≤ x ≤ 28 samples respectively. The cell constants of the high pressure crystalline phases suggest the possible existance of a new metastable crystalline compound in the Ge---Te system with F.C.C. structure and cell constant equal to 1.109nm as reported by Moore et al.

Evidence of bat origin for Menangle virus, a zoonotic paramyxovirus first isolated from diseased pigs

Menangle virus (MenPV) is a zoonotic paramyxovirus capable of causing disease in pigs and humans. It was first isolated in 1997 from stillborn piglets at a commercial piggery in New South Wales, Australia, where an outbreak of reproductive disease occurred. Neutralizing antibodies to MenPV were detected in various pteropid bat species in Australia and fruit bats were suspected to be the source of the virus responsible for the outbreak in pigs. However, previous attempts to isolate MenPV from various fruit bat species proved fruitless. Here, we report the isolation of MenPV from urine samples of the black flying fox, Pteropus alecto, using a combination of improved procedures and newly established bat cell lines. The nucleotide sequence of the bat isolate is 94% identical to the pig isolate. This finding provides strong evidence supporting the hypothesis that the MenPV outbreak in pigs originated from viruses in bats roosting near the piggery. © 2012 Printed in Great Britain.

Sustainable management for rangelands in a variable climate: evidence and insights from northern Australia

Inter-annual rainfall variability is a major challenge to sustainable and productive grazing management on rangelands. In Australia, rainfall variability is particularly pronounced and failure to manage appropriately leads to major economic loss and environmental degradation. Recommended strategies to manage sustainably include stocking at long-term carrying capacity (LTCC) or varying stock numbers with forage availability. These strategies are conceptually simple but difficult to implement, given the scale and spatial heterogeneity of grazing properties and the uncertainty of the climate. This paper presents learnings and insights from northern Australia gained from research and modelling on managing for rainfall variability. A method to objectively estimate LTCC in large, heterogeneous paddocks is discussed, and guidelines and tools to tactically adjust stocking rates are presented. The possible use of seasonal climate forecasts (SCF) in management is also considered. Results from a 13-year grazing trial in Queensland show that constant stocking at LTCC was far more profitable and largely maintained land condition compared with heavy stocking (HSR). Variable stocking (VAR) with or without the use of SCF was marginally more profitable, but income variability was greater and land condition poorer than constant stocking at LTCC. Two commercial scale trials in the Northern Territory with breeder cows highlighted the practical difficulties of variable stocking and provided evidence that heavier pasture utilisation rates depress reproductive performance. Simulation modelling across a range of regions in northern Australia also showed a decline in resource condition and profitability under heavy stocking rates. Modelling further suggested that the relative value of variable v. constant stocking depends on stocking rate and land condition. Importantly, variable stocking may possibly allow slightly higher stocking rates without pasture degradation. Enterprise-level simulations run for breeder herds nevertheless show that poor economic performance can occur under constant stocking and even under variable stocking in some circumstances. Modelling and research results both suggest that a form of constrained flexible stocking should be applied to manage for climate variability. Active adaptive management and research will be required as future climate changes make managing for rainfall variability increasingly challenging.

Are high-impact species predictable? An analysis of naturalised grasses in northern Australia

Predicting which species are likely to cause serious impacts in the future is crucial for targeting management efforts, but the characteristics of such species remain largely unconfirmed. We use data and expert opinion on tropical and subtropical grasses naturalised in Australia since European settlement to identify naturalised and high-impact species and subsequently to test whether high-impact species are predictable. High-impact species for the three main affected sectors (environment, pastoral and agriculture) were determined by assessing evidence against pre-defined criteria. Twenty-one of the 155 naturalised species (14%) were classified as high-impact, including four that affected more than one sector. High-impact species were more likely to have faster spread rates (regions invaded per decade) and to be semi-aquatic. Spread rate was best explained by whether species had been actively spread (as pasture), and time since naturalisation, but may not be explanatory as it was tightly correlated with range size and incidence rate. Giving more weight to minimising the chance of overlooking high-impact species, a priority for biosecurity, meant a wider range of predictors was required to identify high-impact species, and the predictive power of the models was reduced. By-sector analysis of predictors of high impact species was limited by their relative rarity, but showed sector differences, including to the universal predictors (spread rate and habitat) and life history. Furthermore, species causing high impact to agriculture have changed in the past 10 years with changes in farming practice, highlighting the importance of context in determining impact. A rationale for invasion ecology is to improve the prediction and response to future threats. Although our study identifies some universal predictors, it suggests improved prediction will require a far greater emphasis on impact rather than invasiveness, and will need to account for the individual circumstances of affected sectors and the relative rarity of high-impact species.

Evidence for Non-coordinated synthesis of 5 S RNA in the thermophilic fungus, Thermomyces lanuginosus

Analysis of ribosomes and the post ribosomal supernatant fraction of actively growing cells ofThermomyces lanuginosus showed the presence of free 5 S RNA in the supernatant fraction. This 5 S RNA was identical to the ribosomal 5 S RNA in its electrophoretic mobility on 10% Polyacrylamide gel and in its base composition. 5 S RNA from both the sources gave evidence for the presence of diphosphate at the 5’ end. Most of the 5 S RNA that appeared in the cytoplasm was that transported from the nucleus during the isolation. This could be prevented by the use of a hexylene glycol-HEPES buffer.

Evidence for reproductive philopatry in the bull shark Carcharhinus leucas

Reproductive philopatry in bull sharks Carcharhinus leucas was investigated by comparing mitochondrial (NADH dehydrogenase subunit 4, 797 base pairs and control region genes 837 base pairs) and nuclear (three microsatellite loci) DNA of juveniles sampled from 13 river systems across northern Australia. High mitochondrial and low microsatellite genetic diversity among juveniles sampled from different rivers (mitochondrial fST = 0.0767, P < 0.05; microsatellite FST = -0.0022, P > 0.05) supported female reproductive philopatry. Genetic structure was not further influenced by geographic distance (P > 0.05) or long-shore barriers to movement (P > 0.05). Additionally, results suggest that C. leucas in northern Australia has a long-term effective population size of 11 000-13 000 females and has undergone population bottlenecks and expansions that coincide with the timing of the last ice-ages.