A Pan-European evaluation of residential burns camps for children and young people.

INTRODUCTION: Residential camps for children who have experienced a severe burn have existed for over 20 years. The idea stemmed from recognition that children with burns face additional challenges, both physical and psychological, and therefore need long-term psychosocial support away from the acute care setting. Whilst individual programmes have published positive evaluation findings, there have been no cross-regional evaluations undertaken to date. METHODS: Five European burn camp programmes agreed to take part in a cross-regional study to assess the benefits as reported by participants. Shared objectives included: giving children the opportunity to experience success through overcoming challenging activities, enhancing self-esteem and confidence and enabling children to share their experiences of having a burn with peers and staff in a supportive environment. Each site collected qualitative feedback from children, parents and staff using a common framework. Simple Likert scale data were also collected. Each site coded responses into themes which were then collated at one site. RESULTS: 104 children, 57 parent/carers and 50 staff took part in the cross-regional evaluation. Children were aged between 5 and 18 years. 98% of children reported that they had enjoyed camp, in particular the activities and the sense of achievement they brought, along with the ability to gain support and friendship from peers and staff. A large proportion felt that they had benefitted from having the opportunity to share their experiences of having a burn with peers, leaving them feeling less isolated. In addition some comments related to boosting confidence and self-esteem and mastering new skills. Parent/carers again cited the increase in their child's confidence in self and appearance as key benefits of the camps. Staff reports also included the benefits for burn care teams by increasing awareness of patient needs and improving multidisciplinary team working. DISCUSSION: The study highlighted the generic benefits of burn camps by collapsing themes across five different sites. Whilst this minimised the localised differences between camps, further research could be used to analyse these subtle differences in greater detail. Some consideration was made of the language barriers between sites which could have effected the interpretation of some of the individual themes. A multi-methodological approach could be used to reduce this effect in future.

A proof-of-principle gel-free proteomics strategy for the identification of predictive biomarkers for the onset of pre-eclampsia.

OBJECTIVE: Progress in the prevention and treatment of women at risk of pre-eclampsia (PE) still remains hindered by the lack of clinical screening tools that can accurately predict which mothers are at risk. The identification and validation of predictive biomarkers is therefore seen as a critical milestone towards improved healthcare provision and the clinical testing of new therapeutic strategies. Gel-free proteomic technologies offer the capability of analysing hundreds of plasma proteins simultaneously, but as yet these methods have not been applied to pregnancy complications. To assess the feasibility of such an approach to plasma biomarker research in pregnancy we have applied the technique to samples from women with PE to gestation-matched controls. SAMPLE: Pooled plasma samples taken at time of disease from women with PE (n = 23) and gestation-matched controls (n = 23). METHODS: Proteomics strategy for relative quantification of proteins using mass spectrometry. RESULTS: We identified several differences, including elevated levels of endoglin, PAPP-A and PSG1 in PE plasma. Increased levels of endoglin were validated using immunoassay analysis of individual plasma samples. CONCLUSIONS: Although at a relatively early stage, this mass spectrometry-based approach shows promise as a tool to identify global protein changes in plasma. The application of these methods to pre-disease samples is the next step in the identification of clinically useful biomarkers.

Metabolic and proteomic study of NS0 myeloma cell line following the adaptation to protein-free medium.

Proteomics and metabolomics technologies are potentially useful tool for the study of the very complex process of cell adaptation to protein-free medium. In this work, we used the iTRAQ technology to analyze different protein levels in adapted and non-adapted NS0 myeloma cell line. Several proteins with differential expression profile were characterized and quantified. Carbohydrate metabolism, protein synthesis and membrane transport were the principal pathways that change after the adaptation. Changes in lactate production rate with respect to glucose consumption rate were observed according to the changes observed by proteomic.

Identification of early proteomic markers for hepatic steatosis.

The identification of biomarkers for disease state, drug efficacy, and toxicity is becoming increasingly important for drug discovery and development. We have used two-dimensional differential in-gel electrophoresis and mass spectrometry to identify proteomic markers associated with hepatocellular steatosis in rats after dosing with a compound (CDA) in preclinical development. Rats were dosed daily for up to 5 days with CDA for measurement of blood biochemical parameters, histological, and proteomic analysis. Alterations in plasma glucose and liver transaminases were detected from dosing day 3 onward, and livers showed trace levels of hepatocellular vacuolation from 6 h which increased in extent and severity over the 5 day time course. The number of significantly altered protein spots increased over the 5 day time course, and Ingenuity Pathway Analysis showed that the predominant functions altered by CDA treatment were cell death and cellular assembly and organization. This included alterations in secreted proteins, endoplasmic reticulum and mitochondrial chaperones, antioxidant proteins, and enzymes involved in fatty acid biosynthesis. Comparative in vitro dosing studies showed similar alterations to the proteome, neutral lipid accumulation, and mitochondrial dehydrogenase activity in response to CDA treatment of cultured rat hepatocytes. The finding that several proteins showed significant changes in abundance before the onset of overt toxicity in vivo suggested that these could serve as predictive biomarkers of compounds with a propensity to induce liver steatosis. These markers underwent further direct analysis in the in vitro hepatocyte toxicity model to determine their utility in the development of high throughput assays for drug-induced steatosis.

Proapoptotic Bid binds to monolysocardiolipin, a new molecular connection between mitochondrial membranes and cell death.

Recent evidence indicates that the mitochondrial lipid cardiolipin may be instrumental in the proapoptotic action of Bcl-2 family proteins on mitochondrial membranes, leading to the release of apoptogenic factors. However, contrasting evidence indicates that progressive loss of cardiolipin occurs during apoptosis. Here we show that Bid, a crucial proapoptotic protein that integrates the action of other Bcl-2 family members, exhibits discrete specificity for metabolites of cardiolipin, especially monolysocardiolipin (MCL). MCL, normally present in the remodelling of mitochondrial lipids, progressively increases in mitochondria during Fas-mediated apoptosis as a by-product of cardiolipin degradation, and also enhances Bid binding to membranes. MCL may thus play a crucial role in connecting lipid metabolism, relocation of Bid to mitochondria and integrated action of Bcl-2 proteins on mitochondrial membranes. We propose that Bid interaction with MCL 'primes' the mitochondrial outer membrane via segregation of lipid domains, facilitating membrane discontinuity and leakage of apoptogenic factors.

A novel tandem quadrupole mass spectrometer allowing gaseous collisional activation and surface induced dissociation.

A novel tandem quadrupole mass spectrometer is described that enables gaseous collision-induced dissociation (CID) and surface-induced dissociation (SID) experiments. The instrument consists of a commercially available triple quadrupole mass spectrometer connected to an SID region and an additional, orthogonal quadrupole mass analyser. The performance of the instrument was evaluated using leucine-enkephalin, allowing a comparison between CID and SID, and with previous reports of other SID instruments. The reproducibility of SID data was assessed by replicate determinations of the collision energy required for 50% dissociation of leucine-enkephalin; excellent precision was observed (standard deviation of 0.6 eV) though, unexpectedly, the reproducibility of the equivalent figure for CID was superior. Several peptides were analysed using SID in conjunction with liquid secondary-ion mass spectrometry or electrospray; a comparison of the fragmentation of singly protonated peptide ions and the further dissociation of y-type fragments was consistent with the equivalence of the latter fragments to protonated peptides. Few product ions attributable to high-energy cleavages of amino acid side-chains were observed. The SID properties were investigated of a series of peptides differing only in the derivatization of a cysteine residue; similar decomposition efficiencies were observed for all except the cysteic acid analogue, which demonstrated significantly more facile fragmentation.

Chromatographic separations as a prelude to two-dimensional electrophoresis in proteomics analysis.

Current methods of proteome analysis rely almost solely on two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) followed by the excision of individual spots and protein identification using mass spectrometry (MS) and database searching. 2-D PAGE is denaturing in both dimensions and, thus, cannot indicate functional associations between individual proteins. Moreover, less abundant proteins are difficult to identify. To simplify the proteome, and explore functional associations, nondenaturing anion exchange column chromatography was used to separate a soluble protein extract from Escherichia coli. Successive fractions were then analysed using 2-D PAGE and selected spots from both the gels for the start material and the fractionated material were quantified and identified by peptide mass fingerprinting using a MALDI-TOF mass spectrometer. Enrichments of up to 13-fold were attained for individual protein spots and peptide mass fingerprints were of significantly higher quality after chromatographic separation. The marked anomalies between predicted p/and column elution position contrasted with the almost perfect correlation with migration distance on isoelectric focusing (IEF) and were explored further for basic proteins.

Automatic function switching and its usefulness in peptide and protein analysis using direct infusion microspray quadrupole time-of-flight mass spectrometry.

Automatic function switching has been investigated for high-throughput protein identification and sequencing of peptides using direct infusion of tryptic digests on a quadrupole time-of-flight instrument. The increase in speed and the high quality of data make it a favourable technique for tandem mass spectrometry when compared to manual selection of each precursor ion; these advantages are not restricted to combined LC/MS/MS analyses for which the automatic function-switching mode was originally developed. This mode was compared to analyses performed using a slow scan of the quadrupole analyzer with repeated recording of product ion spectra. For the specific purpose of generating product ion data for sequence determination (as opposed to surveying all precursors of a selected product ion), the automatic function-switching mode was, as expected, markedly superior with respect to speed of analysis and quality of data. Furthermore, the automatic function-switching mode provides greater versatility with respect to selection of optimal collision energies.

Physicochemical consequences of the perdeuteriation of glutathione S-transferase from S. japonicum.

Glutathione S:-transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium-tolerant host Escherichia coli strain in the preparation of the protein. 10 heteroatom-bound deuteriums (in addition to the carbon-bound deuteriums) were resistant to exchange when dGST was incubated in protiated buffer. The physicochemical and biological properties of the two proteins were compared. dGST was relatively less stable to heat denaturation and to proteolytic cleavage than was pGST. The midpoint transition temperature for pGST was 54.9 degrees C, whereas that for dGST was 51.0 degrees C. Static light-scattering measurements revealed that the association behavior of dGST is also different from that of pGST. The perdeuteriated enzyme shows a tendency to associate into dimers of the fundamental dimer. This is in contrast with results that have been obtained for other perdeuteriated proteins in which perdeuteriation has been shown to promote dissociation of aggregates. dGST showed a similar K(m) to pGST; similar results had been obtained previously with bacterial alkaline phosphatase. However, whereas the alkaline phosphatase showed a reduced rate of catalysis on deuteriation, dGST exhibited a slightly higher rate of catalysis than pGST. It is clear that the bulk substitution of deuterium for protium has significant effects on the properties of proteins. Until many more examples have been studied, it will be difficult to predict these effects for any given protein. Nevertheless, deuteriation represents an intriguing method of preparing functional analogs of recombinant proteins.

An experimental investigation of thought suppression and anxiety in children.

OBJECTIVES: This study aimed to explore the effects of thought suppression in children--in particular, whether trying to suppress a thought leads to an immediate and/or delayed increase in its occurrence. The influence of anxiety on children's performance in the experimental paradigm was also examined. DESIGN: Participants were allocated to conditions in a between-groups experimental design devised on the basis of thought suppression methodologies reported in the adult literature. METHODS: One-hundred and twelve participants were recruited from local primary schools and randomly allocated to four experimental conditions. Children in all conditions monitored the occurrence of a target thought (either neutral or anxiety-provoking) by tapping their hand on a table each time the thought came to mind during two consecutive experimental periods. During the first period, half the children were asked not to think about the target thought, while the remaining half were asked to think about anything. During the second period, all children were instructed to think about anything. RESULTS: No evidence was found for either an immediate or a delayed increase in frequency of target thoughts as a consequence of suppression attempts. State and trait anxiety were, however, found to influence children's performance during these experimental tasks and were associated with increased intrusions under certain conditions. CONCLUSIONS: The results of this study suggest that the experimental thought suppression paradigm is workable with 7-11-year-old children. The methodological, theoretical and clinical implications of these findings are discussed.

Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation.

Hypoxia-inducible factor (HIF) is a transcriptional complex that plays a central role in the regulation of gene expression by oxygen. In oxygenated and iron replete cells, HIF-alpha subunits are rapidly destroyed by a mechanism that involves ubiquitylation by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. Here we show that the interaction between human pVHL and a specific domain of the HIF-1alpha subunit is regulated through hydroxylation of a proline residue (HIF-1alpha P564) by an enzyme we have termed HIF-alpha prolyl-hydroxylase (HIF-PH). An absolute requirement for dioxygen as a cosubstrate and iron as cofactor suggests that HIF-PH functions directly as a cellular oxygen sensor.

Gas-phase cleavage of PTC-derivatized electrosprayed tryptic peptides in an FT-ICR trapped-ion cell: mass-based protein identification without liquid chromatographic separation.

Condensed phase protein sequencing typically relies on N-terminal labeling with phenylisothiocyanate ("Edman" reagent), followed by cleavage of the N-terminal amino acid. Similar Edman degradation has been observed in the gas phase by collision-activated dissociation of the N-terminal phenyl thiocarbamoyl protonated peptide [1] to yield complementary b1 and y(n-1) fragments, identifying the N-terminal amino acid. By use of infrared multiphoton (rather than collisional) activation, and Fourier transform ion cyclotron resonance (rather than quadrupole) mass analysis, we extend the method to direct analysis of a mixture of tryptic peptides. We validate the approach with bradykinin as a test peptide, and go on to analyze a mixture of 25 peptides produced by tryptic digestion of apomyoglobin. A b1+ ion is observed for three of the Edman-derivatized peptides, thereby identifying their N-terminal amino-acids. Search of the SWISS-PROT database gave a single hit (myoglobin, from the correct biological species), based on accurate-mass FT-ICR MS for as few as one Edman-derivatized tryptic peptide. The method is robust-it succeeds even with partial tryptic digestion, partial Edman derivatization, and partial MS/MS IRMPD cleavage. Improved efficiency and automation should be straightforward.

A combination of chemical derivatisation and improved bioinformatic tools optimises protein identification for proteomics.

The identification of individual protein species within an organism's proteome has been optimised by increasing the information produced from mass spectral analysis through the chemical derivatisation of tryptic peptides and the development of new software tools. Peptide fragments are subjected to two forms of derivatisation. First, lysine residues are converted to homoarginine moieties by guanidination. This procedure has two advantages, first, it usually identifies the C-terminal amino acid of the tryptic peptide and also greatly increases the total information content of the mass spectrum by improving the signal response of C-terminal lysine fragments. Second, an Edman-type phenylthiocarbamoyl (PTC) modification is carried out on the N-terminal amino acid. The renders the first peptide bond highly susceptible to cleavage during mass spectrometry (MS) analysis and consequently allows the ready identification of the N-terminal residue. The utility of the procedure has been demonstrated by developing novel bioinformatic tools to exploit the additional mass spectral data in the identification of proteome proteins from the yeast Saccharomyces cerevisiae. With this combination of novel chemistry and bioinformatics, it should be possible to identify unambiguously any yeast protein spot or band from either two-dimensional or one-dimensional electropheretograms.

Effect of polymorphisms on ligand binding by mouse major urinary proteins.

Mouse urine contains an abundance of major urinary proteins, lipocalins, whose roles include slow release of semiochemicals. These proteins are highly polymorphic, with small sequence differences between individual members. In this study, we purified to homogeneity four of these proteins from two strains of inbred mice and characterized them by mass spectrometry. This analysis has led to the discovery of another variant in this group of proteins. Three of the polymorphic variants that map to the surface have no effect on the binding of a fluorescent probe in the binding cavity, but the fourth, characterized by a Phe to Val substitution in the cavity, shows a substantially lower affinity and fluorescence yield for the probe. These results are interpreted in light of the known crystal structure of the protein and molecular modeling calculations, which rationalize the experimental findings. This work raises the possibility that the calyx-binding site can show specificity for different ligands, the implications of which on pheromone binding and chemical communication are discussed.

Bioinformatic assessment of mass spectrometric chemical derivatisation techniques for proteome database searching.

Identification of proteins from the mass spectra of peptide fragments generated by proteolytic cleavage using database searching has become one of the most powerful techniques in proteome science, capable of rapid and efficient protein identification. Using computer simulation, we have studied how the application of chemical derivatisation techniques may improve the efficiency of protein identification from mass spectrometric data. These approaches enhance ion yield and lead to the promotion of specific ions and fragments, yielding additional database search information. The impact of three alternative techniques has been assessed by searching representative proteome databases for both single proteins and simple protein mixtures. For example, by reliably promoting fragmentation of singly-charged peptide ions at aspartic acid residues after homoarginine derivatisation, 82% of yeast proteins can be unambiguously identified from a single typical peptide-mass datum, with a measured mass accuracy of 50 ppm, by using the associated secondary ion data. The extra search information also provides a means to confidently identify proteins in protein mixtures where only limited data are available. Furthermore, the inclusion of limited sequence information for the peptides can compensate and exceed the search efficiency available via high accuracy searches of around 5 ppm, suggesting that this is a potentially useful approach for simple protein mixtures routinely obtained from two-dimensional gels.

Improved matrix-assisted laser desorption/ionization mass spectrometric analysis of tryptic hydrolysates of proteins following guanidination of lysine-containing peptides.

Analysis of tryptic digests of proteins using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry commonly results in superior detection of arginine-containing peptides compared with lysine-containing counterparts. The effect is attributable in part to the greater stability of the arginine-containing peptide ions associated with the sequestration of the single ionizing proton on the arginine side-chain. Reaction of peptides with O-methylisourea resulted in conversion of lysine to homoarginine residues with consequent improved detection during MALDI-MS. Analysis of the underivatized tryptic digest of the yeast protein, enolase, revealed peptides representing 20% of the protein; the corresponding figure after derivatization was 46%.

Simple horizontal averaging programme enables shade correction for image analysis in psoriasis.

Computer-assisted area measurement of skin lesions would be useful for the assessment of disease severity. Difficulties can be encountered, however, when lesions cannot simply be separated using the normal threshold tool of an image analyser. Owing to body curvature, peripheral sites are usually shadowed. To obtain a well-fitting curved line to account for body curvature-induced glare and shadow, we devised a simple C-programme for averaging neighbouring pixels on the same horizontal line. An image was captured from a colour slide to the image analysis system. C++ was used for programming. Execution of the averaging programme with the original image resulted in a background brightness image file. When this was subtracted from the original image, the lesions were easily detected using the standard threshold tool and the percentage of area involved was calculated. This method is sufficiently accurate for the assessment of disease severity.

Advances in mass spectrometry for proteome analysis.

The most demanding problems in proteomics continue to challenge modern mass spectrometry. Recent developments in instrument design have led to lower limits of detection, while new ion activation techniques and improved understanding of gas-phase ion chemistry have enhanced the capabilities of tandem mass spectrometry for peptide and protein structure elucidation. Future developments must address the., understanding of protein-protein interactions and the characterisation of the dynamic proteome.

5a-formylbicyclomycin: studies on the bicyclomycin-Rho interaction.

Bicyclomycin (1) is a commercial antibiotic whose primary site of action is the rho transcription termination factor. A new bicyclomycin irreversible inactivator, 5a-formylbicyclomycin (3), was prepared to provide information concerning the bicyclomycin-rho inactivation process and the drug's binding pocket within rho. The apparent I(50) value for 3 was 35 microM, showing that 3 was a more effective inhibitor of rho poly C-dependent ATPase activity than 1 (I(50) = 60 microM). Mechanistic studies demonstrated that 3 inhibited poly C-dependent ATP hydrolysis, in part, by a reversible, noncompetitive pathway with respect to ATP (K(i) = 62 microM). Incubation of 3 with rho led to efficient imine formation. Adding excess 1 to solutions containing 3 and rho prevented imine formation, demonstrating that 1 and 3 bind to the same active site in the protein. The 3-rho imine was stabilized by either ATP or ADP or by both, and was converted to the nonreversible 3-rho amine adduct upon treatment with NaBH(4). Mass spectrometric analysis of the amine provided a stoichiometry of approximately five bound 3 per rho hexamer indicating the number of bicyclomycin binding sites for the rho hexamer is between five and six. Monomer exchange experiments using modified 3-rho amine and wild type rho demonstrated that no more than two modified subunits per rho hexamer are sufficient to halt poly C-dependent rho ATPase activity.