Massively parallel sequencing and analysis of expressed sequence tags in a successful invasive plant

Background Invasive species pose a significant threat to global economies, agriculture and biodiversity. Despite progress towards understanding the ecological factors associated with plant invasions, limited genomic resources have made it difficult to elucidate the evolutionary and genetic factors responsible for invasiveness. This study presents the first expressed sequence tag (EST) collection for Senecio madagascariensis, a globally invasive plant species. Methods We used pyrosequencing of one normalized and two subtractive libraries, derived from one native and one invasive population, to generate an EST collection. ESTs were assembled into contigs, annotated by BLAST comparison with the NCBI non-redundant protein database and assigned gene ontology (GO) terms from the Plant GO Slim ontologies. Key Results Assembly of the 221 746 sequence reads resulted in 12 442 contigs. Over 50 % (6183) of 12 442 contigs showed significant homology to proteins in the NCBI database, representing approx. 4800 independent transcripts. The molecular transducer GO term was significantly over-represented in the native (South African) subtractive library compared with the invasive (Australian) library. Based on NCBI BLAST hits and literature searches, 40 % of the molecular transducer genes identified in the South African subtractive library are likely to be involved in response to biotic stimuli, such as fungal, bacterial and viral pathogens. Conclusions This EST collection is the first representation of the S. madagascariensis transcriptome and provides an important resource for the discovery of candidate genes associated with plant invasiveness. The over-representation of molecular transducer genes associated with defence responses in the native subtractive library provides preliminary support for aspects of the enemy release and evolution of increased competitive ability hypotheses in this successful invasive. This study highlights the contribution of next-generation sequencing to better understanding the molecular mechanisms underlying ecological hypotheses that are important in successful plant invasions.

Phenomenological modeling of cell-to-cell and beat-to-beat variability in isolated Guinea Pig ventricular myocytes

Experimental action potential (AP) recordings in isolated ventricular myoctes display significant temporal beat-to-beat variability in morphology and duration. Furthermore, significant cell-to-cell differences in AP also exist even for isolated cells originating from the same region of the same heart. However, current mathematical models of ventricular AP fail to replicate the temporal and cell-to-cell variability in AP observed experimentally. In this study, we propose a novel mathematical framework for the development of phenomenological AP models capable of capturing cell-to-cell and temporal variabilty in cardiac APs. A novel stochastic phenomenological model of the AP is developed, based on the deterministic Bueno-Orovio/Fentonmodel. Experimental recordings of AP are fit to the model to produce AP models of individual cells from the apex and the base of the guinea-pig ventricles. Our results show that the phenomenological model is able to capture the considerable differences in AP recorded from isolated cells originating from the location. We demonstrate the closeness of fit to the available experimental data which may be achieved using a phenomenological model, and also demonstrate the ability of the stochastic form of the model to capture the observed beat-to-beat variablity in action potential duration.

Towards condition-invariant sequence-based route recognition

In this paper we present a novel algorithm for localization during navigation that performs matching over local image sequences. Instead of calculating the single location most likely to correspond to a current visual scene, the approach finds candidate matching locations within every section (subroute) of all learned routes. Through this approach, we reduce the demands upon the image processing front-end, requiring it to only be able to correctly pick the best matching image from within a short local image sequence, rather than globally. We applied this algorithm to a challenging downhill mountainbiking visual dataset where there was significant perceptual or environment change between repeated traverses of the environment, and compared performance to applying the feature-based algorithm FAB-MAP. The results demonstrate the potential for localization using visual sequences, even when there are no visual features that can be reliably detected.

Interactive axial shortening of columns and walls in high rise buildings

Concrete is commonly used as a primary construction material for tall building construction. Load bearing components such as columns and walls in concrete buildings are subjected to instantaneous and long term axial shortening caused by the time dependent effects of "shrinkage", "creep" and "elastic" deformations. Reinforcing steel content, variable concrete modulus, volume to surface area ratio of the elements and environmental conditions govern axial shortening. The impact of differential axial shortening among columns and core shear walls escalate with increasing building height. Differential axial shortening of gravity loaded elements in geometrically complex and irregular buildings result in permanent distortion and deflection of the structural frame which have a significant impact on building envelopes, building services, secondary systems and the life time serviceability and performance of a building. Existing numerical methods commonly used in design to quantify axial shortening are mainly based on elastic analytical techniques and therefore unable to capture the complexity of non-linear time dependent effect. Ambient measurements of axial shortening using vibrating wire, external mechanical strain, and electronic strain gauges are methods that are available to verify pre-estimated values from the design stage. Installing these gauges permanently embedded in or on the surface of concrete components for continuous measurements during and after construction with adequate protection is uneconomical, inconvenient and unreliable. Therefore such methods are rarely if ever used in actual practice of building construction. This research project has developed a rigorous numerical procedure that encompasses linear and non-linear time dependent phenomena for prediction of axial shortening of reinforced concrete structural components at design stage. This procedure takes into consideration (i) construction sequence, (ii) time varying values of Young's Modulus of reinforced concrete and (iii) creep and shrinkage models that account for variability resulting from environmental effects. The capabilities of the procedure are illustrated through examples. In order to update previous predictions of axial shortening during the construction and service stages of the building, this research has also developed a vibration based procedure using ambient measurements. This procedure takes into consideration the changes in vibration characteristic of structure during and after construction. The application of this procedure is illustrated through numerical examples which also highlight the features. The vibration based procedure can also be used as a tool to assess structural health/performance of key structural components in the building during construction and service life.

A sequence analytic model of entrepreneurial discovery and exploitation process

This study examined the effect that temporal order within the entrepreneurial discovery exploitation process has on the outcomes of venture creation. Consistent with sequential theories of discovery-exploitation, the general flow of venture creation was found to be directed from discovery toward exploitation in a random sample of nascent ventures. However, venture creation attempts which specifically follow this sequence derive poor outcomes. Moreover, simultaneous discovery-exploitation was the most prevalent temporal order observed, and venture attempts that proceed in this manner more likely become operational. These findings suggest that venture creation is a multi-scale phenomenon that is at once directional in time, and simultaneously driven by symbiotically coupled discovery and exploitation.

Tinamous and moa flock together : mitochondrial genome sequence analysis reveals independent losses of flight among ratites

Ratites are large, flightless birds and include the ostrich, rheas, kiwi, emu, and cassowaries, along with extinct members, such as moa and elephant birds. Previous phylogenetic analyses of complete mitochondrial genome sequences have reinforced the traditional belief that ratites are monophyletic and tinamous are their sister group. However, in these studies ratite monophyly was enforced in the analyses that modeled rate heterogeneity among variable sites. Relaxing this topological constraint results in strong support for the tinamous (which fly) nesting within ratites. Furthermore, upon reducing base compositional bias and partitioning models of sequence evolution among protein codon positions and RNA structures, the tinamou–moa clade grouped with kiwi, emu, and cassowaries to the exclusion of the successively more divergent rheas and ostrich. These relationships are consistent with recent results from a large nuclear data set, whereas our strongly supported finding of a tinamou–moa grouping further resolves palaeognath phylogeny. We infer flight to have been lost among ratites multiple times in temporally close association with the Cretaceous–Tertiary extinction event. This circumvents requirements for transient microcontinents and island chains to explain discordance between ratite phylogeny and patterns of continental breakup. Ostriches may have dispersed to Africa from Eurasia, putting in question the status of ratites as an iconic Gondwanan relict taxon. [Base composition; flightless; Gondwana; mitochondrial genome; Palaeognathae; phylogeny; ratites.]

Viruses of banana in East Africa

Bananas are one of the world's most important food crops, providing sustenance and income for millions of people in developing countries and supporting large export industries. Viruses are considered major constraints to banana production, germplasm multiplication and exchange, and to genetic improvement of banana through traditional breeding. In Africa, the two most important virus diseases are bunchy top, caused by Banana bunchy top virus (BBTV), and banana streak disease, caused by Banana streak virus (BSV). BBTV is a serious production constraint in a number of countries within/bordering East Africa, such as Burundi, Democratic Republic of Congo, Malawi, Mozambique, Rwanda and Zambia, but is not present in Kenya, Tanzania and Uganda. Additionally, epidemics of banana streak disease are occurring in Kenya and Uganda. The rapidly growing tissue culture (TC) industry within East Africa, aiming to provide planting material to banana farmers, has stimulated discussion about the need for virus indexing to certify planting material as virus-free. Diagnostic methods for BBTV and BSV have been reported and, for BBTV, PCR-based assays are reliable and relatively straightforward. However for BSV, high levels of serological and genetic variability and the presence of endogenous virus sequences within the banana genome complicate diagnosis. Uganda has been shown to contain the greatest diversity in BSV isolates found anywhere in the world. A broad-spectrum diagnostic test for BSV detection, which can discriminate between endogenous and episomal BSV sequences, is a priority. This PhD project aimed to establish diagnostic methods for banana viruses, with a particular focus on the development of novel methods for BSV detection, and to use these diagnostic methods for the detection and characterisation of banana viruses in East Africa. A novel rolling-circle amplification (RCA) method was developed for the detection of BSV. Using samples of Banana streak MY virus (BSMYV) and Banana streak OL virus (BSOLV) from Australia, this method was shown to distinguish between endogenous and episomal BSV sequences in banana plants. The RCA assay was used to screen a collection of 56 banana samples from south-west Uganda for BSV. RCA detected at least five distinct BSV isolates in these samples, including BSOLV and Banana streak GF virus (BSGFV) as well as three BSV isolates (Banana streak Uganda-I, -L and -M virus) for which only partial sequences had been previously reported. These latter three BSV had only been detected using immuno-capture (IC)-PCR and thus were possible endogenous sequences. In addition to its ability to detect BSV, the RCA protocol was also demonstrated to detect other viruses within the family Caulimoviridae, including Sugar cane bacilliform virus, and Cauliflower mosaic virus. Using the novel RCA method, three distinct BSV isolates from both Kenya and Uganda were identified and characterised. The complete genome of these isolates was sequenced and annotated. All six isolates were shown to have a characteristic badnavirus genome organisation with three open reading frames (ORFs) and the large polyprotein encoded by ORF 3 was shown to contain conserved amino acid motifs for movement, aspartic protease, reverse transcriptase and ribonuclease H activities. As well, several sequences important for expression and replication of the virus genome were identified including the conserved tRNAmet primer binding site present in the intergenic region of all badnaviruses. Based on the International Committee on Taxonomy of Viruses (ICTV) guidelines for species demarcation in the genus Badnavirus, these six isolates were proposed as distinct species, and named Banana streak UA virus (BSUAV), Banana streak UI virus (BSUIV), Banana streak UL virus (BSULV), Banana streak UM virus (BSUMV), Banana streak CA virus (BSCAV) and Banana streak IM virus (BSIMV). Using PCR with species-specific primers designed to each isolate, a genotypically diverse collection of 12 virus-free banana cultivars were tested for the presence of endogenous sequences. For five of the BSV no amplification was observed in any cultivar tested, while for BSIMV, four positive samples were identified in cultivars with a B-genome component. During field visits to Kenya, Tanzania and Uganda, 143 samples were collected and assayed for BSV. PCR using nine sets of species-specific primers, and RCA, were compared for BSV detection. For five BSV species with no known endogenous counterpart (namely BSCAV, BSUAV, BSUIV, BSULV and BSUMV), PCR was used to detect 30 infections from the 143 samples. Using RCA, 96.4% of these samples were considered positive, with one additional sample detected using RCA which was not positive using PCR. For these five BSV, PCR and RCA were both useful for identifying infected samples, irrespective of the host cultivar genotype (Musa A- or B-genome components). For four additional BSV with known endogenous counterparts in the M. balbisiana genome (BSOLV, BSGFV, BSMYV and BSIMV), PCR was shown to detect 75 infections from the 143 samples. In 30 samples from cultivars with an A-only genome component there was 96.3% agreement between PCR positive samples and detection using RCA, again demonstrating either PCR or RCA are suitable methods for detection. However, in 45 samples from cultivars with some B-genome component, the level of agreement between PCR positive samples and RCA positive samples was 70.5%. This suggests that, in cultivars with some B-genome component, many infections were detected using PCR which were the result of amplification of endogenous sequences. In these latter cases, RCA or another method which discriminates between endogenous and episomal sequences, such as immuno-capture PCR, is needed to diagnose episomal BSV infection. Field visits were made to Malawi and Rwanda to collect local isolates of BBTV for validation of a PCR-based diagnostic assay. The presence of BBTV in samples of bananas with bunchy top disease was confirmed in 28 out of 39 samples from Malawi and all nine samples collected in Rwanda, using PCR and RCA. For three isolates, one from Malawi and two from Rwanda, the complete nucleotide sequences were determined and shown to have a similar genome organisation to previously published BBTV isolates. The two isolates from Rwanda had at least 98.1% nucleotide sequence identity between each of the six DNA components, while the similarity between isolates from Rwanda and Malawi was between 96.2% and 99.4% depending on the DNA component. At the amino acid level, similarities in the putative proteins encoded by DNA-R, -S, -M, - C and -N were found to range between 98.8% to 100%. In a phylogenetic analysis, the three East African isolates clustered together within the South Pacific subgroup of BBTV isolates. Nucleotide sequence comparison to isolates of BBTV from outside Africa identified India as the possible origin of East African isolates of BBTV.

Environmental factors controlling temporal and spatial variability in the soil-atmosphere exchange of CO2, CH4 and N2O from an Australian subtropical rainforest

The temporal variations in CO2, CH4 and N2O fluxes were measured over two consecutive years from February 2007 to March 2009 from a subtropical rainforest in south-eastern Queensland, Australia, using an automated sampling system. A concurrent study using an additional 30 manual chambers examined the spatial variability of emissions distributed across three nearby remnant rainforest sites with similar vegetation and climatic conditions. Interannual variation in fluxes of all gases over the 2 years was minimal, despite large discrepancies in rainfall, whereas a pronounced seasonal variation could only be observed for CO2 fluxes. High infiltration, drainage and subsequent high soil aeration under the rainforest limited N2O loss while promoting substantial CH4 uptake. The average annual N2O loss of 0.5 ± 0.1 kg N2O-N ha−1 over the 2-year measurement period was at the lower end of reported fluxes from rainforest soils. The rainforest soil functioned as a sink for atmospheric CH4 throughout the entire 2-year period, despite periods of substantial rainfall. A clear linear correlation between soil moisture and CH4 uptake was found. Rates of uptake ranged from greater than 15 g CH4-C ha−1 day−1 during extended dry periods to less than 2–5 g CH4-C ha−1 day−1 when soil water content was high. The calculated annual CH4 uptake at the site was 3.65 kg CH4-C ha−1 yr−1. This is amongst the highest reported for rainforest systems, reiterating the ability of aerated subtropical rainforests to act as substantial sinks of CH4. The spatial study showed N2O fluxes almost eight times higher, and CH4 uptake reduced by over one-third, as clay content of the rainforest soil increased from 12% to more than 23%. This demonstrates that for some rainforest ecosystems, soil texture and related water infiltration and drainage capacity constraints may play a more important role in controlling fluxes than either vegetation or seasonal variability

Reply to the discussion by Karen Johannesson on “Rare earth element geochemistry of scleractinian coral skeleton during meteoric diagenesis: a sequence through neomorphism of aragonite to calcite” by Webb et al., Sedimentology, 56, 1433-1463

Webb et al. (2009) described a late Pleistocenecoral sample wherein the diagenetic stabilization of original coral aragonite to meteoric calcite was halted more or less mid-way through the process, allowing direct comparison of pre-diagenetic and post-diagenetic microstructure and trace element distributions. Those authors found that the rare earth elements (REEs) were relatively stable during meteoric diagenesis, unlike divalent cations such as Sr,and it was thus concluded that original, in this case marine, REE distributions potentially could be preserved through the meteoric carbonate stabilization process that must have affected many, if not most, ancient limestones. Although this was not the case in the analysed sample, they noted that where such diagenesis took place in laterally transported groundwater, trace elements derived from that groundwater could be incorporated into diagenetic calcite, thus altering the initial REE distribution (Banner et al., 1988). Hence, the paper was concerned with the diagenetic behaviour of REEs in a groundwater-dominated karst system. The comment offered by Johannesson (2011) does not question those research results, but rather, seeks to clarify an interpretation made by Webb et al. (2009) of an earlier paper, Johannesson et al. (2006).

Sensitivity of the NMR density matrix to pulse sequence parameters : a simplified analytic approach

We present a formalism for the analysis of sensitivity of nuclear magnetic resonance pulse sequences to variations of pulse sequence parameters, such as radiofrequency pulses, gradient pulses or evolution delays. The formalism enables the calculation of compact, analytic expressions for the derivatives of the density matrix and the observed signal with respect to the parameters varied. The analysis is based on two constructs computed in the course of modified density-matrix simulations: the error interrogation operators and error commutators. The approach presented is consequently named the Error Commutator Formalism (ECF). It is used to evaluate the sensitivity of the density matrix to parameter variation based on the simulations carried out for the ideal parameters, obviating the need for finite-difference calculations of signal errors. The ECF analysis therefore carries a computational cost comparable to a single density-matrix or product-operator simulation. Its application is illustrated using a number of examples from basic NMR spectroscopy. We show that the strength of the ECF is its ability to provide analytic insights into the propagation of errors through pulse sequences and the behaviour of signal errors under phase cycling. Furthermore, the approach is algorithmic and easily amenable to implementation in the form of a programming code. It is envisaged that it could be incorporated into standard NMR product-operator simulation packages.

Socio-environmental variability and Barmah Forest virus disease transmission : a review of epidemiological evidence and future prospects

Barmah Forest Virus (BFV) disease is the most rapidly emerging mosquito-borne disease in Australia. BFV transmission depends on factors such as climate, virus, vector and the human population. However, the impact of climatic and social factors on BFV remains to be determined. This paper provided an overview of current research and discusses the future research directions on the BFV transmission. These research findings could be regarded as an impetus towards BFV prevention and control strategies.

Preliminary validation of a novel high-resolution melt-based typing method based on the multilocus sequence typing scheme of Streptococcus pyogenes

The major limitation of current typing methods for Streptococcus pyogenes, such as emm sequence typing and T typing, is that these are based on regions subject to considerable selective pressure. Multilocus sequence typing (MLST) is a better indicator of the genetic backbone of a strain but is not widely used due to high costs. The objective of this study was to develop a robust and cost-effective alternative to S. pyogenes MLST. A 10-member single nucleotide polymorphism (SNP) set that provides a Simpson’s Index of Diversity (D) of 0.99 with respect to the S. pyogenes MLST database was derived. A typing format involving high-resolution melting (HRM) analysis of small fragments nucleated by each of the resolution-optimized SNPs was developed. The fragments were 59–119 bp in size and, based on differences in G+C content, were predicted to generate three to six resolvable HRM curves. The combination of curves across each of the 10 fragments can be used to generate a melt type (MelT) for each sequence type (ST). The 525 STs currently in the S. pyogenes MLST database are predicted to resolve into 298 distinct MelTs and the method is calculated to provide a D of 0.996 against the MLST database. The MelTs are concordant with the S. pyogenes population structure. To validate the method we examined clinical isolates of S. pyogenes of 70 STs. Curves were generated as predicted by G+C content discriminating the 70 STs into 65 distinct MelTs.

Consecutive days of cold water immersion: effects on cycling performance and heart rate variability.

We investigated performance and heart rate (HR) variability (HRV) over consecutive days of cycling with post-exercise cold water immersion (CWI) or passive recovery (PAS). In a crossover design, 11 cyclists completed two separate 3-day training blocks (120 min cycling per day, 66 maximal sprints, 9 min time trialling [TT]), followed by 2 days of recovery-based training. The cyclists recovered from each training session by standing in cold water (10 °C) or at room temperature (27 °C) for 5 min. Mean power for sprints, total TT work and HR were assessed during each session. Resting vagal-HRV (natural logarithm of square-root of mean squared differences of successive R-R intervals; ln rMSSD) was assessed after exercise, after the recovery intervention, during sleep and upon waking. CWI allowed better maintenance of mean sprint power (between-trial difference [90 % confidence limits] +12.4 % [5.9; 18.9]), cadence (+2.0 % [0.6; 3.5]), and mean HR during exercise (+1.6 % [0.0; 3.2]) compared with PAS. ln rMSSD immediately following CWI was higher (+144 % [92; 211]) compared with PAS. There was no difference between the trials in TT performance (-0.2 % [-3.5; 3.0]) or waking ln rMSSD (-1.2 % [-5.9; 3.4]). CWI helps to maintain sprint performance during consecutive days of training, whereas its effects on vagal-HRV vary over time and depend on prior exercise intensity.