Identification of the functional pathways altered by placental cell exposure to high glucose: lessons from the transcript and metabolite interactome.

The specific consequences of hyperglycaemia on placental metabolism and function are incompletely understood but likely contribute to poor pregnancy outcomes associated with diabetes mellitus (DM). This study aimed to identify the functional biochemical pathways perturbed by placental exposure to high glucose levels through integrative analysis of the trophoblast transcriptome and metabolome. The human trophoblast cell line, BeWo, was cultured in 5 or 25 mM glucose, as a model of the placenta in DM. Transcriptomic analysis using microarrays, demonstrated 5632 differentially expressed gene transcripts (≥± 1.3 fold change (FC)) following exposure to high glucose. These genes were used to generate interactome models of transcript response using BioGRID (non-inferred network: 2500 nodes (genes) and 10541 protein-protein interactions). Ultra performance-liquid chromatography-mass spectrometry (MS) and gas chromatography-MS analysis of intracellular extracts and culture medium were used to assess the response of metabolite profiles to high glucose concentration. The interactions of altered genes and metabolites were assessed using the MetScape interactome database, resulting in an integrated model of systemic transcriptome (2969 genes) and metabolome (41 metabolites) response within placental cells exposed to high glucose. The functional pathways which demonstrated significant change in response to high glucose included fatty acid ß-oxidation, phospholipid metabolism and phosphatidylinositol phosphate signalling.

Introduction to metabolomics and its applications in ophthalmology.

Metabolomics is the study of endogenous and exogenous metabolites in biological systems, which aims to provide comparative semi-quantitative information about all metabolites in the system. Metabolomics is an emerging and potentially powerful tool in ophthalmology research. It is therefore important for health professionals and researchers involved in the speciality to understand the basic principles of metabolomics experiments. This article provides an overview of the experimental workflow and examples of its use in ophthalmology research from the study of disease metabolism and pathogenesis to identification of biomarkers.

Mass spectrometry tools and metabolite-specific databases for molecular identification in metabolomics.

The chemical identification of mass spectrometric signals in metabolomic applications is important to provide conversion of analytical data to biological knowledge about metabolic pathways. The complexity of electrospray mass spectrometric data acquired from a range of samples (serum, urine, yeast intracellular extracts, yeast metabolic footprints, placental tissue metabolic footprints) has been investigated and has defined the frequency of different ion types routinely detected. Although some ion types were expected (protonated and deprotonated peaks, isotope peaks, multiply charged peaks) others were not expected (sodium formate adduct ions). In parallel, the Manchester Metabolomics Database (MMD) has been constructed with data from genome scale metabolic reconstructions, HMDB, KEGG, Lipid Maps, BioCyc and DrugBank to provide knowledge on 42,687 endogenous and exogenous metabolite species. The combination of accurate mass data for a large collection of metabolites, theoretical isotope abundance data and knowledge of the different ion types detected provided a greater number of electrospray mass spectrometric signals which were putatively identified and with greater confidence in the samples studied. To provide definitive identification metabolite-specific mass spectral libraries for UPLC-MS and GC-MS have been constructed for 1,065 commercially available authentic standards. The MMD data are available at http://dbkgroup.org/MMD/.

Rapid access chest pain clinics--can they be justified?

Rapid access chest pain clinics are expanding across the country with marked resource implications despite a paucity of data regarding their efficacy. Early assessment of patients in this manner potentially delays review of patients referred via the traditional route. We conducted a prospective observational study of patients referred with chest pain to the Cardiology Outpatient Department over a four-week period in a District General Hospital to compare demographics and outcomes in patients referred to the rapid access with those referred to the general cardiology clinics. There were no significant differences in baseline demographics, exercise test result or clinic outcome. Both populations were low risk. Discussion is needed between primary and secondary care to achieve a consensus as to the purpose of a rapid access system and how best to utilise the service appropriately. Further studies are required to assess the efficacy and health economics of this system.

Plasma metabolites indicate energy metabolism disruption during the preclinical phase of bovine spongiform encephalopathy infection.

During the preclinical phase of bovine spongiform encephalopathy (BSE), significantly increased concentrations of lactic acid were measured in the blood of infected dairy cows. Other plasma metabolites, including alanine, leucine, serine, and glutamic acid, also showed significantly altered concentrations in the preclinical BSE animals compared to a control group. This appears consistent with the exaggerated stress response observed in clinical BSE, and precedes the development of clinical signs and overt behavioural changes. A number of other plasma metabolites including other amino acids and components of the plasma fatty acid content showed no association with BSE status.

Effectiveness of elevator service signs: measurement of perceived understandability, willingness to comply and behaviour.

This research examines the effectiveness of four elevator service signs. The signs' purpose is to reduce delays for longer distance riders by dissuading people from using the elevator when they are only going up one floor or down two floors. Three of the four signs were described in Chapanis' (1965, Human Factors 7, 1-17) seminal treatise entitled 'Words, words, words ...': an original sign and two others that he suggested as possibly being better. The fourth was an enhanced sign incorporating human factor principles that were derived from research since Chapanis' article. The enhancements involved the use of colour, a signal word panel, icons/pictorial, and direct, explicit wording of the required behaviour. In Experiment 1, participants rated the understandability of each sign and their willingness to obey its instructions. The pattern of the means was the same for both questions. The original sign was rated lowest and the enhanced sign was rated highest, with the two other signs receiving intermediate ratings. In Experiment 2, the signs were placed on each floor of six multi-story buildings adjacent to the elevator call buttons. People's use of the elevators during the posting of each sign and during no-sign (control) periods was measured. The experimenter rode the elevators and counted the total number of passengers using the elevators as well as the number who rode up only one floor or down one or two floors (noncompliers). The new enhanced sign increased compliance compared to the other three signs and the no-sign period. These results suggest that design principles derived from recent research can help to promote comprehension, motivation and compliance behaviour to signs.

Evaluating materials used for constructing soft earmoulds.

The use of penetrometry for the assessment of materials employed for soft earmoulds is investigated. Selected test conditions are used for the characterisation of 10 materials of five types (cold-cured methacrylate, heat-cured methacrylate, heat-cured silicone, polyvinyl chloride co-polymer and polyethylene copolymer) used in clinical practice. The results are compared with the outcome of subjective assessments surveyed for four representative materials. The effect of ageing is assessed for a heat-cured silicone (Molloplast B) and a heat-cured methacrylate (Coe Super-Soft).

Femtogram detection of perfluorocarbon tracers using capillary gas chromatography-electron-capture negative ion chemical ionisation mass spectrometry.

An ultrasensitive and selective method has been developed for the detection of a range of perfluorocarbon tracers suitable for long-range atmospheric studies. Following direct injection onto a capillary column the perfluorocarbons are separated and detected using mass spectrometry with electron-capture negative ion chemical ionisation (ECNICI-MS) with methane as reagent gas and selected-ion monitoring. All nine perfluorocarbons tested underwent non-dissociative resonance electron capture to form the molecular anion which was the base peak in all cases. Using this technique, detection limits (signal-to-noise ratio of 2:1) of 3 and 2 fg were obtained for perfluoromethylcyclohexane and perfluoro-1,3-dimethylcyclohexane respectively. The corresponding detection limits using a conventional electron-capture detector were 8 and 9 fg respectively. In addition to increased sensitivity, the ECNICI-MS technique allows temperature programming for the simultaneous analysis of a range of perfluorocarbons and has superior selectivity being less prone to coeluting interferences. Furthermore the method is potentially one to two orders of magnitude more sensitive than the current gas chromatography-MS methods using 13C2H4 tracers.

Detection of trace levels of triclopyr using capillary gas chromatography-electron-capture negative-ion chemical ionization mass spectrometry.

Triclopyr, after esterification, is shown to be a suitable candidate for detection by gas chromatography-electron-capture negative-ion chemical ionization mass spectrometry forming a characteristic carboxylate anion which offers a high detection sensitivity. A detection limit of 70 fg reaching the ionizer is indicated. Low backgrounds and an absence of chemical interferences are shown for vegetation extracts, using a simple method of extraction and derivatisation. A similar behaviour is demonstrated for 2,4-D and 2,4,5-T.

Detection of trace levels of trichothecenes in human blood using capillary gas chromatography-electron-capture negative ion chemical ionisation mass spectrometry.

A sensitive and selective method has been developed for the simultaneous detection in blood of eleven trichothecenes of widely varying polarity. The procedure involved precipitation of blood proteins with acetone followed by a clean-up using reversed-phase Sep-Pak C18 cartridges. The extracted trichothecenes were derivatised as their pentafluoropropionyl esters, separated using capillary gas chromatography and detected using electron-capture negative ion chemical ionisation with methane reagent gas and selected-ion monitoring. Optimum sensitivity and selectivity were obtained using low source temperatures (60 degrees C indicated) and high source pressures (1 Torr indicated). Detection limits on 1-ml blood samples were in the range 0.1-5 ppb. The method was readily adaptable to the detection of other trichothecenes. A protocol was used which minimised the risk of cross-contamination. The method was validated in collaborative studies by the successful analysis of 42 blood samples spiked and submitted blind by two independent laboratories for analysis.