Functional and structural characterization of a heparanase.

We report the structural and functional characterization of a novel heparanase (BpHep) from the invasive pathogenic bacterium Burkholderia pseudomallei (Bp), showing ∼24% sequence identity with human heparanase (hHep). Site-directed mutagenesis studies confirmed the active site resi-dues essential for activity, and we found that BpHep has specificity for heparan sulfate. Finally, we describe the first heparanase X-ray crystal structure, which provides new insight into both substrate recognition and inhibitor design.

Rapid screening of cellular stress responses in recombinant Pichia pastoris strains using metabolite profiling.

Heterologous protein production in the yeast Pichia pastoris can be limited by biological responses to high expression levels; the unfolded protein response (UPR) is a key determinant of the success of protein production in this organism. Here, we used untargeted NMR metabolic profiling (metabolomics) of a number of different recombinant strains, carried out in a miniaturized format suitable for screening-level experiments. We identified a number of metabolites (from both cell extracts and supernatants) which correlated well with UPR-relevant gene transcripts, and so could be potential biomarkers for future high-throughput screening of large numbers of P. pastoris clones.

convISA: A simple, convoluted method for isotopomer spectral analysis of fatty acids and cholesterol.

Isotopomer spectral analysis (ISA) is a simple approach for modelling the cellular synthesis of fatty acids and cholesterol in a stable isotope labelling experiment. In the simplest model, fatty acid biosynthesis is described by two key parameters: the fractional enrichment of acetyl-CoA from the labelled substrate, D, and the fractional de novo synthesis of the fatty acid during the exposure to the labelled substrate, g(t). The model can also be readily extended to include synthesis via elongation of unlabelled shorter fatty acids. This modelling strategy is less complex than metabolic flux analysis and only requires the measurement of the mass isotopologues of a single metabolite. However, software tools to perform these calculations are not freely available. We have developed an algorithm (convISA), implemented in MATLAB(™), which employs the convolution (Cauchy product) of mass isotopologue distributions (MIDs) for ISA of fatty acids and cholesterol. In our method, the MIDs of each molecule are constructed as a single entity rather than deriving equations for individual isotopologues. The flexibility of this method allows the model to be applied to raw data as well as to data that has been corrected for natural isotope abundance. To test the algorithm, convISA was applied to 238 MIDs of methyl palmitate available from the literature, for which ISA parameters had been calculated via other methods. A very high correlation was observed between estimates of the D and g(t) parameters from convISA with both published values, and estimates generated by our own metabolic flux analysis using a simplified stoichiometric model (r=0.981 and 0.944, and 0.996 and 0.942). We also demonstrate the application of the convolution ISA approach to cholesterol biosynthesis; the model was applied to measurements made on MCF7 cells cultured in U-(13)C-glucose. In conclusion, we believe that convISA offers a convenient, flexible and transparent framework for metabolic modelling that will help facilitate the application of ISA to future experiments.

Metabolomic characterization of nipple aspirate fluid by (1)H NMR spectroscopy and GC-MS.

Nipple aspirate fluid (NAF) is a noninvasively obtained biofluid from the duct openings of the breast. NAF components are constantly secreted, metabolized, and reabsorbed by the epithelial lining of the lactiferous ducts of the breast. NAF has been studied as a potential breast tissue surrogate for the discovery of novel breast cancer risk, early detection, and treatment response biomarkers. We report the first unsupervised metabolite characterization of nipple aspirate fluid using NMR and GC-MS using convenience samples previously collected from four premenopausal and four postmenopausal women. A total of 38 metabolites were identified using the two analytical techniques, including amino acids, organic acids, fatty acids, and carbohydrates. Analytical reproducibility of metabolites in NAF by GC-MS was high across different extraction and analysis days. Overall, 31 metabolites had a coefficient of variation below 20%. By GC-MS, there were eight metabolites unique to NAF, 19 unique to plasma, and 24 shared metabolites. Correlative analysis of shared metabolites between matched NAF and plasma samples from pre- and postmenopausal women shows almost no correlations, with the exception being lactic acid, which was significantly negatively correlated (R(2) = 0.57; P = 0.03). These results suggest that NAF is metabolically distinct from plasma and that the application of metabolomic strategies may be useful for future studies investigating breast cancer risk and intervention response biomarkers.

Between-person comparison of metabolite fitting for NMR-based quantitative metabolomics.

Nuclear magnetic resonance (NMR) spectroscopy is widely used as an analytical platform for metabolomics. Many studies make use of 1D spectra, which have the advantages of relative simplicity and rapid acquisition times. The spectral data can then be analyzed either with a chemometric workflow or by an initial deconvolution or fitting step to generate a list of identified metabolites and associated sample concentrations. Various software tools exist to simplify the fitting process, but at least for 1D spectra, this still requires a degree of skilled operator input. It is of critical importance that we know how much person-to-person variability affects the results, in order to be able to judge between different studies. Here we tested a commercially available software package (Chenomx' NMR Suite) for fitting metabolites to a set of NMR spectra of yeast extracts and compared the output of five different people for both metabolite identification and quantitation. An initial comparison showed good agreement for a restricted set of common metabolites with characteristic well-resolved resonances but wide divergence in the overall identities and number of compounds fitted; refitting according to an agreed set of metabolites and spectral processing approach increased the total number of metabolites fitted but did not dramatically increase the quality of the metabolites that could be fitted without prior knowledge about peak identity. Hence, robust peak assignments are required in advance of manual deconvolution, when the widest range of metabolites is desired. However, very low concentration metabolites still had high coefficients of variation even with shared information on peak assignment. Overall, the effect of the person was less than the experimental group (in this case, sampling method) for almost all of the metabolites.

A software complement to AMDIS for processing GC-MS metabolomic data.

The software package AMDIS performs gas chromatography-mass spectrometry (GC-MS) peak deconvolution but tends to produce false positives and leaves missing values where peaks are found in only a proportion of a set of chromatograms. We have developed a software complement to AMDIS that (i) allows rapid manual inspection of chromatographic peaks across all samples to confirm data quality and (ii) for a given sample set, integrates peak areas across all samples even where AMDIS deconvolution would leave missing values. The freely available package runs within the commercial Matlab environment and is useful where GC-MS is used to profile complex mixtures.

Poly-3-hydroxyoctanoate P(3HO), a medium chain length polyhydroxyalkanoate homopolymer from Pseudomonas mendocina.

Pseudomonas mendocina was found to produce a unique homopolymer of poly(3-hydroxyoctanoate), P(3HO), rather than a copolymer, when grown on sodium octanoate as the sole carbon source. Although this polymer has been produced by other organisms, interestingly this is the first time an absolute homopolymer has been produced by a wild type organism. In addition, a detailed study on the effects of different extraction methods on the yield, molecular weight, thermal properties, and lipopolysaccharide content of P(3HO) has been carried out. The organism was able to accumulate P(3HO) up to 31.38% of its dry cell weight within 48 h in mineral salt medium. Characterization of the monomer was carried out using FTIR, GC-MS, (13)C, (1)H, and HSQC NMR spectroscopy. The polymer had a crystallinity of 37.5%, Young's modulus value of 11.6 MPa and contact angle of 77.3°. Microstructural studies of solvent cast polymer films revealed a smooth surface topography with a root-mean-square roughness value of 0.238 µm.

In vivo tumour imaging employing regional delivery of novel gallium radiolabelled polymer composites.

BACKGROUND: Understanding the regional vascular delivery of particles to tumour sites is a prerequisite for developing new diagnostic and therapeutic composites for treatment of oncology patients. We describe a novel imageable 67Ga-radiolabelled polymer composite that is biocompatible in an animal tumour model and can be used for preclinical imaging investigations of the transit of different sized particles through arterial networks of normal and tumour-bearing organs. RESULTS: Radiolabelling of polymer microspheres with 67Ga was achieved using a simple mix and wash method, with tannic acid as an immobilising agent. Final in vitro binding yields after autoclaving averaged 94.7%. In vivo stability of the composite was demonstrated in New Zealand white rabbits by intravenous administration, and intrahepatic artery instillations were made in normal and VX2 tumour implanted rabbit livers. Stability of radiolabel was sufficient for rabbit lung and liver imaging over at least 3 hours and 1 hour respectively, with lung retention of radiolabel over 91%, and retention in both normal and VX2 implanted livers of over 95%. SPECT-CT imaging of anaesthetised animals and planar imaging of excised livers showed visible accumulation of radiolabel in tumours. Importantly, microsphere administration and complete liver dispersal was more easily achieved with 8 µm diameter MS than with 30 µm MS, and the smaller microspheres provided more distinct and localised tumour imaging. CONCLUSION: This method of producing 67Ga-radiolabelled polymer microspheres is suitable for SPECT-CT imaging of the regional vascular delivery of microspheres to tumour sites in animal models. Sharper distinction of model tumours from normal liver was obtained with smaller MS, and tumour resolution may be further improved by the use of 68Ga instead of 67Ga, to enable PET imaging.

99mTc-radiolabeled composites enabling in vivo imaging of arterial dispersal and retention of microspheres in the vascular network of rabbit lungs, liver, and liver tumors.

PURPOSE: Selective internal radiation therapy (SIRT) is an effective treatment option for liver tumors, using Y-90-loaded polymer microspheres that are delivered via catheterization of the hepatic artery. Since Y-90 is a beta emitter and not conveniently imaged by standard clinical instrumentation, dosimetry is currently evaluated in each patient using a surrogate particle, 99mTechnetium-labeled macroaggregated albumin (99mTc-MAA). We report a new composite consisting of 99mTc-labeled nanoparticles attached to the same polymer microspheres as used for SIRT, which can be imaged with standard SPECT. METHODS: Carbon nanoparticles with an encapsulated core of 99mTc were coated with the polycation protamine sulfate to provide electrostatic attachment to anionic polystyrene sulfonate microspheres of different sizes (30, 12, and 8 µm). The in vivo stability of these composites was determined via intravenous injection and entrapment in the capillary network of normal rabbit lungs for up to 3 hours. Furthermore, we evaluated their biodistribution in normal rabbit livers, and livers implanted with VX2 tumors, following intrahepatic artery instillation. RESULTS: We report distribution tests for three different sizes of radiolabeled microspheres and compare the results with those obtained using 99mTc-MAA. Lung retention of the radiolabeled microspheres ranged from 72.8% to 92.9%, with the smaller diameter microspheres showing the lowest retention. Liver retention of the microspheres was higher, with retention in normal livers ranging from 99.2% to 99.8%, and in livers with VX2 tumors from 98.2% to 99.2%. The radiolabeled microspheres clearly demonstrated preferential uptake at tumor sites due to the increased arterial perfusion produced by angiogenesis. CONCLUSION: We describe a novel use of radiolabeled carbon nanoparticles to generate an imageable microsphere that is stable in vivo under the shear stress conditions of arterial networks. Following intra-arterial instillation in the normal rabbit liver, they distribute in a distinct segmented pattern, with the smaller microspheres extending throughout the organ in finer detail, while still being well retained within the liver. Furthermore, in livers hosting an implanted VX2 tumor, they reveal the increased arterial perfusion of tumor tissue resulting from angiogenesis. These novel composites may have potential as a more representative mimic of the vascular distribution of therapeutic microspheres in patients undergoing SIRT.

Persistence of Epigenomic Effects After Recovery From Repeated Treatment With Two Nephrocarcinogens.

The discovery of the epigenetic regulation of transcription has provided a new source of mechanistic understanding to long lasting effects of chemicals. However, this information is still seldom exploited in a toxicological context and studies of chemical effect after washout remain rare. Here we studied the effects of two nephrocarcinogens on the human proximal tubule cell line RPTEC/TERT1 using high-content mRNA microarrays coupled with miRNA, histone acetylation (HA) and DNA methylation (DM) arrays and metabolomics during a 5-day repeat-dose exposure and 3 days after washout. The mycotoxin ochratoxin A (OTA) was chosen as a model compound for its known impact on HA and DM. The foremost effect observed was the modulation of thousands of mRNAs and histones by OTA during and after exposure. In comparison, the oxidant potassium bromate (KBrO3) had a milder impact on gene expression and epigenetics. However, there was no strong correlation between epigenetic modifications and mRNA changes with OTA while with KBrO3 the gene expression data correlated better with HA for both up- and down-regulated genes. Even when focusing on the genes with persistent epigenetic modifications after washout, only half were coupled to matching changes in gene expression induced by OTA, suggesting that while OTA causes a major effect on the two epigenetic mechanisms studied, these alone cannot explain its impact on gene expression. Mechanistic analysis confirmed the known activation of Nrf2 and p53 by KBrO3, while OTA inhibited most of the same genes, and genes involved in the unfolded protein response. A few miRNAs could be linked to these effects of OTA, albeit without clear contribution of epigenetics to the modulation of the pathways at large. Metabolomics revealed disturbances in amino acid balance, energy catabolism, nucleotide metabolism and polyamine metabolism with both chemicals. In conclusion, the large impact of OTA on transcription was confirmed at the mRNA level but also with two high-content epigenomic methodologies. Transcriptomic data confirmed the previously reported activation (by KBrO3) and inhibition (by OTA) of protective pathways. However, the integration of omic datasets suggested that HA and DM were not driving forces in the gene expression changes induced by either chemical.

Metabolomic characterisation of the effects of oncogenic PIK3CA transformation in a breast epithelial cell line.

Somatic mutations in PIK3CA are frequently found in a number of human cancers, including breast cancer, altering cellular physiology and tumour sensitivity to chemotherapy. This renders PIK3CA an attractive molecular target for early detection and personalised therapy. Using 1H Nuclear Magnetic Resonance spectroscopy (NMR) and Gas Chromatography - Mass Spectrometery (GC-MS) together with 13C stable isotope-labelled glucose and glutamine as metabolic tracers, we probed the phenotypic changes in metabolism following a single copy knock-in of mutant PIK3CA (H1047R) in the MCF10A cell line, an important cell model for studying oncogenic transformation in breast tissues. We observed effects in several metabolic pathways, including a decrease in glycerophosphocholine level together with increases in glutaminolysis, de novo fatty acid synthesis and pyruvate entry into the tricarboxylic acid cycle. Our findings highlight altered glyceroplipid metabolism and lipogenesis, as key metabolic phenotypes of mutant PIK3CA transformation that are recapitulated in the MCF10A cellular model.

Modelling the acid/base 1H NMR chemical shift limits of metabolites in human urine.

INTRODUCTION: Despite the use of buffering agents the 1H NMR spectra of biofluid samples in metabolic profiling investigations typically suffer from extensive peak frequency shifting between spectra. These chemical shift changes are mainly due to differences in pH and divalent metal ion concentrations between the samples. This frequency shifting results in a correspondence problem: it can be hard to register the same peak as belonging to the same molecule across multiple samples. The problem is especially acute for urine, which can have a wide range of ionic concentrations between different samples. OBJECTIVES: To investigate the acid, base and metal ion dependent 1H NMR chemical shift variations and limits of the main metabolites in a complex biological mixture. METHODS: Urine samples from five different individuals were collected and pooled, and pre-treated with Chelex-100 ion exchange resin. Urine samples were either treated with either HCl or NaOH, or were supplemented with various concentrations of CaCl2, MgCl2, NaCl or KCl, and their 1H NMR spectra were acquired. RESULTS: Nonlinear fitting was used to derive acid dissociation constants and acid and base chemical shift limits for peaks from 33 identified metabolites. Peak pH titration curves for a further 65 unidentified peaks were also obtained for future reference. Furthermore, the peak variations induced by the main metal ions present in urine, Na+, K+, Ca2+ and Mg2+, were also measured. CONCLUSION: These data will be a valuable resource for 1H NMR metabolite profiling experiments and for the development of automated metabolite alignment and identification algorithms for 1H NMR spectra.

Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of Pichia pastoris.

RESULTS: We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation. CONCLUSION: Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.

The development of metabolomic sampling procedures for Pichia pastoris, and baseline metabolome data.

Metabolic profiling is increasingly being used to investigate a diverse range of biological questions. Due to the rapid turnover of intracellular metabolites it is important to have reliable, reproducible techniques for sampling and sample treatment. Through the use of non-targeted analytical techniques such as NMR and GC-MS we have performed a comprehensive quantitative investigation of sampling techniques for Pichia pastoris. It was clear that quenching metabolism using solutions based on the standard cold methanol protocol caused some metabolite losses from P. pastoris cells. However, these were at a low level, with the NMR results indicating metabolite increases in the quenching solution below 5% of their intracellular level for 75% of metabolites identified; while the GC-MS results suggest a slightly higher level with increases below 15% of their intracellular values. There were subtle differences between the four quenching solutions investigated but broadly, they all gave similar results. Total culture extraction of cells + broth using high cell density cultures typical of P. pastoris fermentations, was an efficient sampling technique for NMR analysis and provided a gold standard of intracellular metabolite levels; however, salts in the media affected the GC-MS analysis. Furthermore, there was no benefit in including an additional washing step in the quenching process, as the results were essentially identical to those obtained just by a single centrifugation step. We have identified the major high-concentration metabolites found in both the extra- and intracellular locations of P. pastoris cultures by NMR spectroscopy and GC-MS. This has provided us with a baseline metabolome for P. pastoris for future studies. The P. pastoris metabolome is significantly different from that of Saccharomyces cerevisiae, with the most notable difference being the production of high concentrations of arabitol by P. pastoris.