Advantages of tandem LC-MS for the rapid assessment of tissue-specific metabolic complexity using a pentafluorophenylpropyl stationary phase

In this study, a tandem LC-MS (Waters Xevo TQ) MRM-based MS method was developed for rapid, broad profiling of hydrophilic metabolites from biological samples, in either positive or negative ion modes without the need for an ion pairing reagent, using a reversed-phase pentafluorophenylpropyl (PFPP) column. The developed method was successfully applied to analyze various biological samples from C57BL/6 mice, including urine, duodenum, liver, plasma, kidney, heart, and skeletal muscle. As result, a total 112 of hydrophilic metabolites were detected within 8 min of running time to obtain a metabolite profile of the biological samples. The analysis of this number of hydrophilic metabolites is significantly faster than previous studies. Classification separation for metabolites from different tissues was globally analyzed by PCA, PLS-DA and HCA biostatistical methods. Overall, most of the hydrophilic metabolites were found to have a "fingerprint" characteristic of tissue dependency. In general, a higher level of most metabolites was found in urine, duodenum, and kidney. Altogether, these results suggest that this method has potential application for targeted metabolomic analyzes of hydrophilic metabolites in a wide ranges of biological samples.

Development of a rapid and validated method for investigating the metabolism of scoparone in rat using ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry

Scoparone (6,7-dimethoxycoumarin) is known to have a wide range of pharmacological properties. In this study, a rapid and validated ultra-performance liquid chromatography/electrospray ionization quadruple time-of-flight mass spectrometry (UPLC/ESI-QTof-MS) method was developed to investigate the metabolism of scoparone in rat for the first time. The new method reduced the sample handling and analytical time by three- to six-fold, and the detection limit by five- to 1000-fold, compared to published methods. Far more metabolites were detected and identified compared to published data, which were preliminarily identified as scopoletin, isoscopoletin, isofraxidin, and fraxidin, respectively, when subjected to tandem mass spectrometry analyses. It is found that the metabolic trajectory of scoparone in rat focused on phase I metabolism which is obviously different from published results, and revealed a wide range of pharmacological properties of scoparone partly attributed to the bioactivities of its metabolites.

Identification of molecular genetic factors that influence migraine

Migraine is a common neurological disorder with a strong genetic basis. However, the complex nature of the disorder has meant that few genes or susceptibility loci have been identified and replicated consistently to confirm their involvement in migraine. Approaches to genetic studies of the disorder have included analysis of the rare migraine subtype, familial hemiplegic migraine with several causal genes identified for this severe subtype. However, the exact genetic contributors to the more common migraine subtypes are still to be deciphered. Genome-wide studies such as genome-wide association studies and linkage analysis as well as candidate genes studies have been employed to investigate genes involved in common migraine. Neurological, hormonal and vascular genes are all considered key factors in the pathophysiology of migraine and are a focus of many of these studies. It is clear that the influence of individual genes on the expression of this disorder will vary. Furthermore, the disorder may be dependent on gene–gene and gene–environment interactions that have not yet been considered. In addition, identifying susceptibility genes may require phenotyping methods outside of the International Classification of Headache Disorders II criteria, such as trait component analysis and latent class analysis to better define the ambit of migraine expression.

A molecular genetic approach for forensic animal species identification

This study investigated potential markers within chromosomal, mitochondrial DNA (mtDNA) and ribosomal RNA (rRNA) with the aim of developing a DNA based method to allow differentiation between animal species. Such discrimination tests may have important applications in the forensic science, agriculture, quarantine and customs fields. DNA samples from five different animal individuals within the same species for 10 species of animal (including human) were analysed. DNA extraction and quantitation followed by PCR amplification and GeneScan visualisation formed the basis of the experimental analysis. Five gene markers from three different types of genes were investigated. These included genomic markers for the β-actin and TP53 tumor suppressor gene. Mitochondrial DNA markers, designed by Bataille et al. [Forensic Sci. Int. 99 (1999) 165], examined the Cytochrome b gene and Hypervariable Displacement Loop (D-Loop) region. Finally, a ribosomal RNA marker for the 28S rRNA gene optimised by Naito et al. [J. Forensic Sci. 37 (1992) 396] was used as a possible marker for speciation. Results showed a difference of only several base pairs between all species for the β-actin and 28S markers, with the exception of Sus scrofa (pig) β-actin fragment length, which produced a significantly smaller fragment. Multiplexing of Cytochrome b and D-Loop markers gave limited species information, although positive discrimination of human DNA was evident. The most specific and discriminatory results were shown using the TP53 gene since this marker produced greatest fragment size differences between animal species studied. Sample differentiation for all species was possible following TP53 amplification, suggesting that this gene could be used as a potential animal species identifier.

Identification and simultaneous analysis of harmane, harmol, isovitexin and vitexin in Passiflora incarnata extracts with a novel HLPC method

A high performance liquid chromatographic method for the simultaneous analysis of two flavonoids (iso-vitexin and vitexin), and three indole alkaloids (harmane, harmine, and harmol) was developed. This method was then utilised to quantitate levels of these five constituents in methanolic extracts of Australian Passiflora incarnata. HPLC analysis was performed using a Waters™ Novapak C18 (150 × 4 mm, 4 μm) column, with a gradient solvent system of methanol-water-acetic acid. Detection was achieved by PDA UV (254 nm) and fluorescence (excitation 254 nm, emission 414 nm), utilising the external standard method to obtain quantification.

Impact of a school-based food garden on attitudes and identification skills regarding vegetables and fruit : a 12-month intervention trial

Objective To determine changes in ability to identify specific vegetables and fruits, and attitudes towards vegetables and fruit, associated with the introduction of a school-based food garden. Design A 12-month intervention trial using a historical control (control n 132, intervention n 120), class-based, self-administered questionnaires requiring one-word answers and 3-point Likert scale responses. Setting A state primary school (grades 4 to 7) in a low socio-economic area of Brisbane, Australia. Intervention The introduction of a school-based food garden, including the funding of a teacher coordinator for 11 h/week to facilitate integration of garden activities into the curriculum. Main outcome measures Ability to identify a series of vegetables and fruits, attitudes towards vegetables and fruit. Analysis Frequency distributions for each item were generated and χ2 analyses were used to determine statistical significance. Exploratory factor analysis was employed to detect major trends in data. Results The intervention led to enhanced ability to identify individual vegetables and fruits, greater attention to origins of produce (garden-grown and fresh), changes to perceived consumption of vegetables and fruits, and enhanced confidence in preparing fruit and vegetable snacks, but decreased interest in trying new fruits. Conclusions The introduction of this school-based food garden was associated with skill and attitudinal changes conducive to enhancing vegetable and fruit consumption. The ways in which such changes might impact on dietary behaviours and intake require further analysis.

Human identification at a distance using iris and face

This research has successfully applied super-resolution and multiple modality fusion techniques to address the major challenges of human identification at a distance using face and iris. The outcome of the research is useful for security applications.

Transcription and expression of Plasmodium falciparum histidine-rich proteins in different stages and strains : implications for rapid diagnostic tests

Background: Although rapid diagnostic tests (RDTs) for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. Methods: Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. Results: Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. Conclusions: The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs.

A microscopic simulation model to estimate bus rapid transit station bus capacity

The Bus Rapid Transit (BRT) station is the interface between passengers and services. The station is crucial to line operation as it is typically the only location where buses can pass each other. Congestion may occur here when buses maneuvering into and out of the platform lane interfere with bus flow, or when a queue of buses forms upstream of the platform lane blocking the passing lane. Further, some systems include operation where express buses do not observe the station, resulting in a proportion of non-stopping buses. It is important to understand the operation of the station under this type of operation and its effect on BRT line capacity. This study uses microscopic traffic simulation modeling to treat the BRT station operation and to analyze the relationship between station bus capacity and BRT line bus capacity. First, the simulation model is developed for the limit state scenario and then a statistical model is defined and calibrated for a specified range of controlled scenarios of dwell time characteristics. A field survey was conducted to verify the parameters such as dwell time, clearance time and coefficient of variation of dwell time to obtain relevant station bus capacity. The proposed model for BRT bus capacity provides a better understanding of BRT line capacity and is useful to transit authorities in BRT planning, design and operation.

Person re-identification using group information

After first observing a person, the task of person re-identification involves recognising an individual at different locations across a network of cameras at a later time. Traditionally, this task has been performed by first extracting appearance features of an individual and then matching these features to the previous observation. However, identifying an individual based solely on appearance can be ambiguous, particularly when people wear similar clothing (i.e. people dressed in uniforms in sporting and school settings). This task is made more difficult when the resolution of the input image is small as is typically the case in multi-camera networks. To circumvent these issues, we need to use other contextual cues. In this paper, we use "group" information as our contextual feature to aid in the re-identification of a person, which is heavily motivated by the fact that people generally move together as a collective group. To encode group context, we learn a linear mapping function to assign each person to a "role" or position within the group structure. We then combine the appearance and group context cues using a weighted summation. We demonstrate how this improves performance of person re-identification in a sports environment over appearance based-features.

A methodology to develop an urban transport disadvantage framework : the case of Brisbane, Australia

Most individuals travel in order to participate in a network of activities which are important for attaining a good standard of living. Because such activities are commonly widely dispersed and not located locally, regular access to a vehicle is important to avoid exclusion. However, planning transport system provisions that can engage members of society in an acceptable degree of activity participation remains a great challenge. The main challenges in most cities of the world are due to significant population growth and rapid urbanisation which produces increased demand for transport. Keeping pace with these challenges in most urban areas is difficult due to the widening gap between supply and demand for transport systems which places the urban population at a transport disadvantage. The key element in mitigating the issue of urban transport disadvantage is to accurately identify the urban transport disadvantaged. Although wide-ranging variables and multi-dimensional methods have been used to identify this group, variables are commonly selected using ad-hoc techniques and unsound methods. This poses questions of whether the current variables used are accurately linked with urban transport disadvantage, and the effectiveness of the current policies. To fill these gaps, the research conducted for this thesis develops an operational urban transport disadvantage framework (UTDAF) based on key statistical urban transport disadvantage variables to accurately identify the urban transport disadvantaged. The thesis develops a methodology based on qualitative and quantitative statistical approaches to develop an urban transport disadvantage framework designed to accurately identify urban transport disadvantage. The reliability and the applicability of the methodology developed is the prime concern rather than the accuracy of the estimations. Relevant concepts that impact on urban transport disadvantage identification and measurement and a wide range of urban transport disadvantage variables were identified through a review of the existing literature. Based on the reviews, a conceptual urban transport disadvantage framework was developed based on the causal theory. Variables identified during the literature review were selected and consolidated based on the recommendations of international and local experts during the Delphi study. Following the literature review, the conceptual urban transport disadvantage framework was statistically assessed to identify key variables. Using the statistical outputs, the key variables were weighted and aggregated to form the UTDAF. Before the variable's weights were finalised, they were adjusted based on results of correlation analysis between elements forming the framework to improve the framework's accuracy. The UTDAF was then applied to three contextual conditions to determine the framework's effectiveness in identifying urban transport disadvantage. The development of the framework is likely to be a robust application measure for policy makers to justify infrastructure investments and to generate awareness about the issue of urban transport disadvantage.

Identification of TFG (TRK-fused gene) as a putative metastatic melanoma tumor suppressor gene

High density SNP arrays can be used to identify DNA copy number changes in tumors such as homozygous deletions of tumor suppressor genes and focal amplifications of oncogenes. Illumina Human CNV370 Bead chip arrays were used to assess the genome for unbalanced chromosomal events occurring in 39 cell lines derived from stage III metastatic melanomas. A number of genes previously recognized to have an important role in the development and progression of melanoma were identified including homozygous deletions of CDKN2A (13 of 39 samples), CDKN2B (10 of 39), PTEN (3 of 39), PTPRD (3 of 39), TP53 (1 of 39), and amplifications of CCND1 (2 of 39), MITF (2 of 39), MDM2 (1 of 39), and NRAS (1 of 39). In addition, a number of focal homozygous deletions potentially targeting novel melanoma tumor suppressor genes were identified. Because of their likely functional significance for melanoma progression, FAS, CH25H, BMPR1A, ACTA2, and TFG were investigated in a larger cohort of melanomas through sequencing. Nonsynonymous mutations were identified in BMPR1A (1 of 43), ACTA2 (3 of 43), and TFG (5 of 103). A number of potentially important mutation events occurred in TFG including the identification of a mini mutation ‘‘hotspot’’ at amino acid residue 380 (P380S and P380L) and the presence of multiple mutations in two melanomas. Mutations in TFG may have important clinical relevance for current therapeutic strategies to treat metastatic melanoma.

Efficacy of proxy definitions for identification of fatigue/sleep-related crashes : an Australian evaluation

Fatigue/sleepiness is recognised as an important contributory factor in fatal and serious injury road traffic incidents (RTIs), however, identifying fatigue/sleepiness as a causal factor remains an uncertain science. Within Australia attending police officers at a RTI report the causal factors; one option is fatigue/sleepiness. In some Australian jurisdictions police incident databases are subject to post hoc analysis using a proxy definition for fatigue/sleepiness. This secondary analysis identifies further RTIs caused by fatigue/sleepiness not initially identified by attending officers. The current study investigates the efficacy of such proxy definitions for attributing fatigue/sleepiness as a RTI causal factor. Over 1600 Australian drivers were surveyed regarding their experience and involvement in fatigue/sleep-related RTIs and near-misses during the past five years. Driving while fatigued/sleepy had been experienced by the majority of participants (66.0% of participants). Fatigue/sleep-related near misses were reported by 19.1% of participants, with 2.4% being involved in a fatigue/sleep-related RTI. Examination of the characteristics for the most recent event (either a near miss or crash) found that the largest proportion of incidents (28.0%) occurred when commuting to or from work, followed by social activities (25.1%), holiday travel (19.8%), or for work purposes (10.1%). The fatigue/sleep related RTI and near-miss experience of a representative sample of Australian drivers does not reflect the proxy definitions used for fatigue/sleepiness identification. In particular those RTIs that occur in urban areas and at slow speeds may not be identified. While important to have a strategy for identifying fatigue/sleepiness related RTIs proxy measures appear best suited to identifying specific subsets of such RTIs.

Identification of a serine protease inhibitor which causes inclusion vacuole reduction and is lethal to Chlamydia trachomatis

The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical biology strategy that has uncovered the first essential protease for this organism. Identification and application of a unique CtHtrA inhibitor (JO146) to cultures of Chlamydia resulted in a complete loss of viable elementary body formation. JO146 treatment during the replicative phase of development resulted in a loss of Chlamydia cell morphology, diminishing inclusion size, and ultimate loss of inclusions from the host cells. This completely prevented the formation of viable Chlamydia elementary bodies. In addition to its effect on the human C. trachomatis strain, JO146 inhibited the viability of the mouse strain, Chlamydia muridarum, both in vitro and in vivo. Thus, we report a chemical biology approach to establish an essential role for Chlamydia CtHtrA. The function of CtHtrA for Chlamydia appears to be essential for maintenance of cell morphology during replicative the phase and these findings provide proof of concept that proteases can be targetted for anti-microbial therapy for intracellular pathogens.