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The detection, identification and quantification of drug metabolites plays a key role in drug discovery and development. Liquid chromatography (LC) coupled to mass spectrometry (MS) has become the primary technology for these studies due to its sensitivity and specificity. However, the presence of transition metals in the chromatography system and columns can result in non-specific and unwanted interactions with the drug and/or its metabolites, via electron-pair donation, leading to poor chromatography and analyte loss. The use of a hybrid organic/inorganic surface applied to the metal surfaces of the chromatography system and column has been demonstrated to reduce or eliminate these effects. When employed for the analysis of mouse urine, derived from the oral dosing of mice with the EGFR inhibitor gefitinib, we observed more symmetrical LC peaks. This resulted in a 33 % improvement in peak capacity for a 10 min reversed – phase gradient separation, a two-fold increase in MS response, cleaner MS spectra and improved peak response reproducibility. This hybrid surface barrier appears to offer significant advantages in the analysis of low-concentration metabolites, potentially facilitating the accurate determination of the elimination phase of the pharmacokinetic (PK) curve and detection of drug metabolites in microdosing or microsampling studies.

Background Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease characterized by fibrosis and progressive loss of lung function. The pathophysiological pathways involved in IPF are not well understood. Abnormal lipid metabolism has been described in various other chronic lung diseases including asthma and chronic obstructive pulmonary disease (COPD). However, its potential role in IPF pathogenesis remains unclear. Methods In this study, we used ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) to characterize lipid changes in plasma derived from IPF patients with stable and progressive disease. We further applied a data-independent acquisition (DIA) technique called SONAR, to improve the specificity of lipid identification. Results Statistical modelling showed variable discrimination between the stable and progressive subjects, revealing differences in the detection of triglycerides (TG) and phosphatidylcholines (PC) between progressors and stable IPF groups, which was further confirmed by mass spectrometry imaging (MSI) in IPF tissue. Conclusion This is the first study to characterise lipid metabolism between stable and progressive IPF, with results suggesting disparities in the circulating lipidome with disease progression.

Chronic obstructive pulmonary disease (COPD) is characterised by airway inflammation and progressive airflow limitation, whereas idiopathic pulmonary fibrosis (IPF) is characterised by a restrictive pattern due to fibrosis and impaired gas exchange. We undertook metabolomic analysis of blood samples in IPF, COPD and healthy controls (HC) to determine differences in circulating molecules and identify novel pathogenic pathways. An untargeted metabolomics using an ultra‐high‐performance liquid chromatography‐quadrupole time‐of‐flight mass spectrometer (UHPLC‐QTOF‐MS) was performed to profile plasma of patients with COPD (n = 21), and IPF (n = 24) in comparison to plasma from healthy controls (HC; n = 20). The most significant features were identified using multiple database matching. One‐way ANOVA and variable importance in projection (VIP) scores were also used to highlight metabolites that influence the specific disease groups. Non‐polar metabolites such as fatty acids (FA) and membrane lipids were well resolved and a total of 4805 features were identified. The most prominent metabolite composition differences in lipid mediators identified at ∼2–3 fold higher in both diseases compared to HC were palmitoleic acid, oleic acid and linoleic acid; and dihydrotestosterone was lower in both diseases. We demonstrated that COPD and IPF were characterised by systemic changes in lipid constituents such as essential FA sampled from circulating plasma.

Introduction/Aim: Differentiation of preschool wheeze into asthma and non‐asthma would allow targeted treatment for those more likely to benefit. We aimed to use metabolic biochemical profiling to discover novel urinary biomarkers of asthma in school‐age children (6‐10y) and investigate the potential to predict future development of asthma in preschool children (2‐4y). Methods: 211 children were recruited. Healthy preschool(n=25); preschool wheeze (n=87); school‐aged healthy(n=43) and school‐aged asthma(n=56). Urinary metabolic profiles at baseline and during exacerbations were characterized using liquid chromatography mass spectrometry. The utility of each individual metabolite as a biomarker of asthma at school‐age was tested using one‐way ANOVA. Canonical Variate Analysis (CVA), followed by multivariate regression, was performed to identify a specific urinary profile for effectively discriminating school‐age asthma from healthy controls. This model was then mapped to the urinary profiles of the preschool‐wheeze children, collected under identical analytical and data processing protocols. Results: 162 putatively identified metabolites were measured within approved levels of analytical repeatability. There was a significant (p<0.05) disease effect for 34 metabolites. CVA uncovered a strong exacerbation profile correlated to the preschool‐wheeze exacerbation sample. Baseline and exacerbation regression models showed strong discrimination (AUROC=0.91&0.98 respectively). When preschool urinary profiles were projected through these models the proportion of preschool wheeze participants classified as “asthma”(~40%) was in‐line with clinical expectations(~30%). Conclusion: A putative multifactorial urinary biomarker of asthma has shown promise of predicting the onset of childhood asthma. Longitudinal follow‐up is ongoing to determine predictive accuracy of the model. Targeted chemical assay development is underway. Grant Support: Telethon Perth Children's Hospital Resear

Microalgae are important natural sources of interesting high value compounds that could be used in pharmaceutical and nutraceutical applications. Of particular interest is the production and exploitation of microalgal phytosterols; compounds with known biological benefits such as cholesterol reduction, anti-inflammatory activity and even anti-cancer properties. The global market for phytosterols is expected to reach $USD 935 million by 2022. Phytosterols of commercial importance are β-sitosterol, campesterol, brassicasterol, stigmasterol and ergosterol. The current sources of phytosterols are the vegetable and tall oils harvested from land plants. However, it is anticipated that the terrestrial sources will not be able to meet increased demand through 2030 and beyond. Calculations of estimated lipid and phytosterol productivity suggest that microalgae could yield 678–6035 kg. ha−1. y−1 of phytosterol which is more than the current productivity of phytosterols from rapeseed plants, highlighting the economic potential for sourcing phytosterols from environmentally sustainable saline microalgae production. However, there are many challenges that need to be met. The sterol content of microalgae varies according to species and, at present, there is little information on how to manipulate culturing conditions to enhance sterol production in microalgae. Approaches could include nutrient limitation, and/or changing salinity, light and temperature. Molecular approaches such as genetic modification, knocking down or over expression of specific genes and blocking competitive biosynthetic pathways would be desirable. However, an improved understanding of the pathway of sterol biosynthesis in microalgae is necessary for effective use of these molecular approaches. This paper discusses the potential for sterol production from microalgae as an adjunct to current plant-based sources and highlights those areas where we need more information to produce these high value products in an

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Use of herbal medicines and supplements by consumers to prevent or treat disease, particularly chronic conditions continues to grow, leading to increased awareness of the minimal regulation standards in many countries. Fraudulent, adulterated and contaminated herbal and traditional medicines and dietary supplements are a risk to consumer health, with adverse effects and events including overdose, drug-herb interactions and hospitalisation. The scope of the risk has been difficult to determine, prompting calls for new approaches, such as the combination of DNA metabarcoding and mass spectrometry used in this study. Here we show that nearly 50% of products tested had contamination issues, in terms of DNA, chemical composition or both. Two samples were clear cases of pharmaceutical adulteration, including a combination of paracetamol and chlorpheniramine in one product and trace amounts of buclizine, a drug no longer in use in Australia, in another. Other issues include the undeclared presence of stimulants such as caffeine, synephrine or ephedrine. DNA data highlighted potential allergy concerns (nuts, wheat), presence of potential toxins (Neem oil) and animal ingredients (reindeer, frog, shrew), and possible substitution of bird cartilage in place of shark. Only 21% of the tested products were able to have at least one ingredient corroborated by DNA sequencing. This study demonstrates that, despite current monitoring approaches, contaminated and adulterated products are still reaching the consumer. We suggest that a better solution is stronger pre-market evaluation, using techniques such as that outlined in this study.

Global consumption of complementary and alternative medicines, including herbal medicines, has increased substantially, and recent reports of adulteration demonstrate the need for high throughput and extensive pharmacovigilance to ensure product safety and quality. Three different standard reference materials and five previously analyzed herbal medicines have been used as a proof of concept for the application of adulteration/contamination screening using a Direct Sample Analysis (DSA) ion source with TOF MS on the Perkin Elmer AxION 2 TOF. This technique offers the advantages of minimum sample preparation, rapid analysis, and mass accuracies of 5 ppm. The DSA TOF analysis correlates well with the previous analysis on the initial sample set (which found undeclared herbal ingredients), with the added advantage of detecting previously untargeted compounds, including species-specific flavonoids and alkaloids. The rapid analysis using the DSA-TOF facilitates screening for hundreds of compounds in minutes with minimal sample preparation, generating a comprehensive profile for each sample.

Chronic lung diseases represent a significant global burden. Their increasing incidence and complexity render a comprehensive, multidisciplinary and personalized approach to each patient, critically important. Most recently, unique biochemical pathways and disease markers have been identified through large-scale metabolomic studies. Metabolomics is the study of metabolic pathways and the measurement of unique biomolecules in a living system. Analysing samples from different compartments such as bronchoalveolar lavage fluid (BALF) and plasma has proven useful for the characterization of a number of pathological conditions and offers promise as a clinical tool. For example, several studies using mass spectrometry (MS) have shown alterations in the sphingolipid metabolism of chronic obstructive pulmonary disease (COPD) sufferers. In this article, we present a practical review of the application of metabolomics to the study of chronic lung diseases (CLD): COPD, idiopathic pulmonary fibrosis (IPF) and asthma. The insights, which the analytical strategies employed in metabolomics, have provided to the dissection of the biochemistry of CLD and future clinical biomarkers are explored.

Introduction As large scale metabolic phenotyping is increasingly employed in preclinical studies and in the investigation of human health and disease the current LC–MS/MS profiling methodologies adopted for large sample sets can result in lengthy analysis times, putting strain on available resources. As a result of these pressures rapid methods of untargeted analysis may have value where large numbers of samples require screening. Objectives To develop, characterise and evaluate a rapid UHP-HILIC-MS-based method for the analysis of polar metabolites in rat urine and then extend the capabilities of this approach by the addition of IMS to the system. Methods A rapid untargeted HILIC LC–MS/MS profiling method for the analysis of small polar molecules has been developed. The 3.3 min separation used a Waters BEH amide (1 mm ID) analytical column on a Waters Synapt G2-Si Q-Tof enabled with ion mobility spectrometry (IMS). The methodology, was applied to the metabolic profiling of a series of rodent urine samples from vehicle-treated control rats and animals administered tienilic acid. The same separation was subsequently linked to IMS and MS to evaluate the benefits that IMS might provide for metabolome characterisation. Results The rapid HILIC–MS method was successfully applied to rapid analysis of rat urine and found, based on the data generated from the data acquired for the pooled quality control samples analysed at regular intervals throughout the analysis, to be robust. Peak area and retention times for the compounds detected in these samples showed good reproducibility across the batch. When used to profile the urine samples obtained from vehicle-dosed control and those administered tienilic acid the HILIC-MS method detected 3007 mass/retention time features. Analysis of the same samples using HILIC–IMS–MS enabled the detection of 6711 features. Provisional metabolite identification for a number of compounds was performed using the high collision energy MS/MS info

Biomass burning is a significant process in the Earth system, driving ecosystem dynamics and changes in global vegetation, and affecting the carbon cycle and climate. Projections of future fire activities require an understanding of the connection between fire history and climate in the past. Polar snow/ice contain long-term records of past climates and fire activity and hold great promise to improve our understanding of wildfire patterns. Here, using ultra-high-performance liquid chromatography-tandem mass spectrometry techniques, we quantified three organic compounds (levoglucosan, vanillic, and syringic acids) released by biomass burning in snow samples collected along a 1250-km transect from the coast to the ice sheet summit Dome A in East Antarctica. Results indicate that these tracers are ubiquitous and have reached the ice sheet summit from the continental emissions in the Southern Hemisphere. These compounds showed high levels close to the coastal areas and decreased to a low level on the Antarctic plateau. The snow samples had similar levoglucosan/vanillic acid (∼45) and levoglucosan/syringic acid ratios (∼243) as aerosols from biomass burning. Multivariate analysis indicates that these compounds were likely derived from the burning of grasses and evergreen broadleaf trees that are widespread in Southern Hemisphere than from evergreen conifers that dominate northern hemisphere fire-prone ecosystems. Snow accumulation rate influenced the levels of these compounds, while coexisting ions had little effect on compound contents in the snow. The low concentrations of levoglucosan at inland sites (mean of 2.7 pg mL−1; versus 3.5 and 3.7 pg mL−1 in coastal and transition zones, respectively) could be associated with the oxidation by OH radicals under sunlight. Our analysis demonstrated that the ubiquity of multiple biomarkers from biomass burning in East Antarctic surface snow can provide baseline concentrations for future studies in Antarctica.

Diseases of the kidney are difficult to diagnose and treat. This review summarises the definition, cause, epidemiology and treatment of some of these diseases including chronic kidney disease, diabetic nephropathy, acute kidney injury, kidney cancer, kidney transplantation and polycystic kidney diseases. Numerous studies have adopted a metabolomics approach to uncover new small molecule biomarkers of kidney diseases to improve specificity and sensitivity of diagnosis and to uncover biochemical mechanisms that may elucidate the cause and progression of these diseases. This work includes a description of mass spectrometry-based metabolomics approaches, including some of the currently available tools, and emphasises findings from metabolomics studies of kidney diseases. We have included a varied selection of studies (disease, model, sample number, analytical platform) and focused on metabolites which were commonly reported as discriminating features between kidney disease and a control. These metabolites are likely to be robust indicators of kidney disease processes, and therefore potential biomarkers, warranting further investigation.

Background Both anaemia and red blood cell (RBC) transfusion are common and associated with adverse outcomes in patients admitted to the intensive care unit (ICU). The aim of this study was to determine whether serum hepcidin concentration, measured early after ICU admission in patients with anaemia, could identify a group in whom intravenous (IV) iron therapy decreased the subsequent RBC transfusion requirement. Methods We conducted a prospective observational study nested within a multicenter randomized controlled trial (RCT) of IV iron versus placebo. The study was conducted in the ICUs of four tertiary hospitals in Perth, Western Australia. Critically ill patients with haemoglobin (Hb) of < 100 g/L and within 48 h of admission to the ICU were eligible for participation after enrolment in the IRONMAN RCT. The response to IV iron therapy compared with placebo was assessed according to tertile of hepcidin concentration. Results Hepcidin concentration was measured within 48 h of ICU admission in 133 patients. For patients in the lower two tertiles of hepcidin concentration (< 53.0 μg), IV iron therapy compared with placebo was associated with a significant decrease in RBC transfusion requirement [risk ratio 0.48 (95% CI 0.26–0.85), p = 0.013]. Conclusions In critically ill patients with anaemia admitted to an ICU, baseline hepcidin concentration predicts RBC transfusion requirement and is able to identify a group of patients in whom IV iron compared with placebo is associated with a significant decrease in RBC transfusion requirement.

Rodent and cell‐culture models support a role for iron‐related adipokine dysregulation and insulin resistance in the pathogenesis of nonalcoholic fatty liver disease (NAFLD); however, substantial human data are lacking. We examined the relationship between measures of iron status, adipokines, and insulin resistance in patients with NAFLD in the presence and absence of venesection. This study forms part of the Impact of Iron on Insulin Resistance and Liver Histology in Nonalcoholic Steatohepatitis (IIRON2) study, a prospective randomized controlled trial of venesection for adults with NAFLD. Paired serum samples at baseline and 6 months (end of treatment) in controls (n = 28) and patients who had venesection (n = 23) were assayed for adiponectin, leptin, resistin, retinol binding protein‐4, tumor necrosis factor α, and interleukin‐6, using a Quantibody, customized, multiplexed enzyme‐linked immunosorbent assay array. Hepatic iron concentration (HIC) was determined using MR FerriScan. Unexpectedly, analysis revealed a significant positive correlation between baseline serum adiponectin concentration and HIC, which strengthened after correction for age, sex, and body mass index (rho = 0.36; P = 0.007). In addition, there were significant inverse correlations between HIC and measures of insulin resistance (adipose tissue insulin resistance (Adipo‐IR), serum insulin, serum glucose, homeostasis model assessment of insulin resistance, hemoglobin A1c, and hepatic steatosis), whereas a positive correlation was noted with the insulin sensitivity index. Changes in serum adipokines over 6 months did not differ between the control and venesection groups. Conclusion: HIC positively correlates with serum adiponectin and insulin sensitivity in patients with NAFLD. Further study is required to establish causality and mechanistic explanations for these associations and their relevance in the pathogenesis of insulin resistance and NAFLD.

Introduction The role of herbal medicine in the treatment of common psychiatric disorders such as anxiety, depression and insomnia has become more established over the past decade. Some herbal preparations such as St John’s wort (Hypericum perforatum) have demonstrated clinical evidence but have also been included in recent reports of widespread adulteration and contamination. Herbal medicines sold in Australia are required to be listed on the Therapeutic Goods Administration’s (TGA) Australian Register of Therapeutic Goods (ARTG) and must comply with strict ingredient and manufacturing guidelines to assure quality and safety. Objective The aim of this research was to assess whether pharmaceutical adulterants and contaminants were present in psychotropic herbal medicines available in Australia, as a measure of quality, and the effectiveness of regulation. Methods A two-pronged approach combining next-generation DNA sequencing and small-molecule analysis techniques was undertaken to audit a subset of herbal medicines for the presence of prescription medications, illicit drugs, pesticides, herbicides, heavy metals and contaminant DNA. Small-molecule analysis included liquid chromatography with quadrupole time-of-flight mass spectrometer (LC-QTOF-MS) detection, liquid chromatography with UV/vis diode array (LC-UV) detection, gas chromatography with nitrogen–phosphorus and mass spectrometer detection (GC-NPD/MS) and heavy metal analysis using inductively coupled plasma with mass spectrometer (ICP-MS) detection. Results In total, 49% (29 of 59) of the investigated herbal medicines had one or more materials not listed on their labels or ARTG registration, including Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES)-listed material (one medicine), heavy metals (12%) or components that could trigger food sensitivity, such as wheat (12%). In contrast to previous studies, no prescription pharmaceutical adulterants were detected, although 1

Methamphetamine is an illicit psychostimulant drug that is linked to a number of diseases of the nervous system. The downstream biochemical effects of its primary mechanisms are not well understood, and the objective of this study was to investigate whether untargeted metabolomic analysis of an in vitro model could generate data relevant to what is already known about this drug. Rat B50 neuroblastoma cells were treated with 1 mM methamphetamine for 48 h, and both intracellular and extracellular metabolites were profiled using gas chromatography–mass spectrometry. Principal component analysis of the data identified 35 metabolites that contributed most to the difference in metabolite profiles. Of these metabolites, the most notable changes were in amino acids, with significant increases observed in glutamate, aspartate and methionine, and decreases in phenylalanine and serine. The data demonstrated that glutamate release and, subsequently, excitotoxicity and oxidative stress were important in the response of the neuronal cell to methamphetamine. Following this, the cells appeared to engage amino acid-based mechanisms to reduce glutamate levels. The potential of untargeted metabolomic analysis has been highlighted, as it has generated biochemically relevant data and identified pathways significantly affected by methamphetamine. This combination of technologies has clear uses as a model for the study of neuronal toxicology.

Human milk contains a complex combination of lipids, proteins, carbohydrates, and minerals, which are essential for infant growth and development. While the lipid portion constitutes only 5% of the total human milk composition, it accounts for over 50% of the infant’s daily energy intake. Human milk lipids vary throughout a feed, day, and through different stages of lactation, resulting in difficulties in sampling standardization and, like blood, human milk is bioactive containing endogenous lipases, therefore appropriate storage is critical in order to prevent lipolysis. Suitable sample preparation, often not described in studies, must also be chosen to achieve the aims of the study. Gas chromatography methods have classically been carried out to investigate the fatty acid composition of human milk lipids, but with the advancement of other chromatographic techniques, such as liquid and supercritical fluid chromatography, as well as mass spectrometry, intact lipids can also be characterized. Despite the known importance, concise and comprehensive analysis of the human milk lipidome is limited, with gaps existing in all areas of human milk lipidomics, discussed in this review. With appropriate methodology and instrumentation, further understanding of the human milk lipidome and the influence it has on infant outcomes can be achieved.

Three experiments were conducted to develop a bioassay method for assessing the bioavailability of prosulfocarb, pyroxasulfone and trifluralin in both crop residue and soil. In preliminary experiments, Italian ryegrass (Lolium multiflorum Lam.), cucumber (Cucumis sativus L.) and beetroot (Beta vulgaris L.) were tested as bioassay plant species for the three pre-emergent herbicides. Four growth parameters (shoot length, root length, fresh weight and dry weight) were measured for all plant species. Shoot-length inhibition was identified as the most responsive to the herbicide application rates. Italian ryegrass was the most sensitive species to all tested herbicides, whereas beetroot and cucumber had lower and similar sensitivity to shoot inhibition for the three herbicides. The bioassay species performed similarly in wheat and canola residues collected a few days after harvest. In bioassay calibration experiments, dose–response curves were developed for prosulfocarb, pyroxasulfone and trifluralin in a sandy loam soil typical of the grain belt of Western Australia and with wheat residue. The developed bioassay uses ryegrass shoot inhibition for relatively low suspected concentrations of herbicide, and cucumber shoot inhibition for higher rates. The bioassay was validated by spraying the three herbicides separately onto wheat residue and soil and comparing the concentrations derived from chemical analysis with those from the bioassay. All of the linear correlations between concentrations derived from chemical analyses and the bioassays were highly significant. These results indicate that the bioassay calibration curves are suitable for estimating herbicide concentrations in crop residue collected soon after harvest and a sandy-loam soil, low in organic matter.

A full understanding of the origin, formation and degradation of volatile compounds that contribute to wine aroma is required before wine style can be effectively managed. Fractionation of grapes represents a convenient and robust method to simplify the grape matrix to enhance our understanding of the grape contribution to volatile compound production during yeast fermentation. In this study, acetone extracts of both Riesling and Cabernet Sauvignon grape berries were fractionated and model wines produced by spiking aliquots of these grape fractions into model grape juice must and fermented. Non-targeted SPME-GCMS analyses of the wines showed that several medium chain fatty acid ethyl esters were more abundant in wines made by fermenting model musts spiked with certain fractions. Further fractionation of the non-polar fractions and fermentation of model must after addition of these fractions led to the identification of a mixture of polyunsaturated triacylglycerides that, when added to fermenting model must, increase the concentration of medium chain fatty acid ethyl esters in wines. Dosage-response fermentation studies with commercially-available trilinolein revealed that the concentration of medium chain fatty acid ethyl esters can be increased by the addition of this triacylglyceride to model musts. This work suggests that grape triacylglycerides can enhance the production of fermentation-derived ethyl esters and show that this fractionation method is effective in segregating precursors or factors involved in altering the concentration of fermentation volatiles.

Introduction: The role of herbal medicine in the treatment of common psychiatric disorders such as anxiety, depression and insomnia has become more established over the past decade. Some herbal preparations such as St John’s wort (Hypericum perforatum) have demonstrated clinical evidence but have also been included in recent reports of widespread adulteration and contamination. Herbal medicines sold in Australia are required to be listed on the Therapeutic Goods Administration’s (TGA) Australian Register of Therapeutic Goods (ARTG) and must comply with strict ingredient and manufacturing guidelines to assure quality and safety. Objective: The aim of this research was to assess whether pharmaceutical adulterants and contaminants were present in psychotropic herbal medicines available in Australia, as a measure of quality, and the effectiveness of regulation. Methods: A two-pronged approach combining next-generation DNA sequencing and small-molecule analysis techniques was undertaken to audit a subset of herbal medicines for the presence of prescription medications, illicit drugs, pesticides, herbicides, heavy metals and contaminant DNA. Small-molecule analysis included liquid chromatography with quadrupole time-of-flight mass spectrometer (LC-QTOF-MS) detection, liquid chromatography with UV/vis diode array (LC-UV) detection, gas chromatography with nitrogen–phosphorus and mass spectrometer detection (GC-NPD/MS) and heavy metal analysis using inductively coupled plasma with mass spectrometer (ICP-MS) detection. Results: In total, 49% (29 of 59) of the investigated herbal medicines had one or more materials not listed on their labels or ARTG registration, including Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES)-listed material (one medicine), heavy metals (12%) or components that could trigger food sensitivity, such as wheat (12%). In contrast to previous studies, no prescription pharmaceutical adulterants were detected, althou

Background: Pentoxifylline has been shown to increase haemoglobin levels in patients with chronic kidney disease (CKD) and erythropoietin-stimulating agent (ESA)-hyporesponsive anaemia in the Handling Erythropoietin Resistance with Oxpentifylline multicentre double-blind, randomized controlled trial. The present sub-study evaluated the effects of pentoxifylline on the iron-regulatory hormone hepcidin in patients with ESA-hyporesponsive CKD. Methods: This sub-study included 13 patients in the pentoxifylline arm (400 mg daily) and 13 in the matched placebo arm. Hepcidin-25 was measured by ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry following isolation from patient serum. Serum hepcidin-25, serum iron biomarkers, haemoglobin and ESA dosage were compared within and between the two groups. Results: Hepcidin-25 concentration at 4 months adjusted for baseline did not differ significantly in pentoxifylline versus placebo treated patients (adjusted mean difference (MD) −7.9 nmol, P = 0.114), although the difference between the groups mean translated into a >25% reduction of circulating hepcidin-25 due to pentoxifylline compared with the placebo baseline. In paired analysis, serum hepcidin-25 levels were significantly decreased at 4 months compared with baseline in the pentoxifylline group (−5.47 ± 2.27 nmol/l, P < 0.05) but not in the placebo group (2.82 ± 4.29 nmol/l, P = 0.24). Pentoxifylline did not significantly alter serum ferritin (MD 55.4 mcg/l), transferrin saturation (MD 4.04%), the dosage of ESA (MD −9.93 U/kg per week) or haemoglobin concentration (MD 5.75 g/l). Conclusion: The reduction of circulating hepcidin-25 due to pentoxifylline did not reach statistical significance; however, the magnitude of the difference suggests that pentoxifylline may be a clinically and biologically meaningful modulator of hepcidin-25 in dialysis of patients with ESA-hyporesponsive anaemia.

Background: Pentoxifylline has been shown to increase haemoglobin levels in patients with chronic kidney disease (CKD) and erythropoietin-stimulating agent (ESA)-hyporesponsive anaemia in the Handling Erythropoietin Resistance with Oxpentifylline multicentre double-blind, randomized controlled trial. The present sub-study evaluated the effects of pentoxifylline on the iron-regulatory hormone hepcidin in patients with ESA-hyporesponsive CKD. Methods: This sub-study included 13 patients in the pentoxifylline arm (400 mg daily) and 13 in the matched placebo arm. Hepcidin-25 was measured by ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry following isolation from patient serum. Serum hepcidin-25, serum iron biomarkers, haemoglobin and ESA dosage were compared within and between the two groups. Results: Hepcidin-25 concentration at 4 months adjusted for baseline did not differ significantly in pentoxifylline versus placebo treated patients (adjusted mean difference (MD) −7.9 nmol, P = 0.114), although the difference between the groups mean translated into a >25% reduction of circulating hepcidin-25 due to pentoxifylline compared with the placebo baseline. In paired analysis, serum hepcidin-25 levels were significantly decreased at 4 months compared with baseline in the pentoxifylline group (−5.47 ± 2.27 nmol/l, P < 0.05) but not in the placebo group (2.82 ± 4.29 nmol/l, P = 0.24). Pentoxifylline did not significantly alter serum ferritin (MD 55.4 mcg/l), transferrin saturation (MD 4.04%), the dosage of ESA (MD −9.93 U/kg per week) or haemoglobin concentration (MD 5.75 g/l). Conclusion: The reduction of circulating hepcidin-25 due to pentoxifylline did not reach statistical significance; however, the magnitude of the difference suggests that pentoxifylline may be a clinically and biologically meaningful modulator of hepcidin-25 in dialysis of patients with ESA-hyporesponsive anaemia.

Herbal medicines are growing in popularity, use and commercial value; however, there remain problems with the quality and consequently safety of these products. Adulterated, contaminated and fraudulent products are often found on the market, a risk compounded by the fact that these products are available to consumers with little or no medical advice. Current regulations and quality control methods are lacking in their ability to combat these serious problems. Metabolomics is a biochemical profiling tool that may help address these issues if applied to quality control of both raw ingredients and final products. Using the example of the popular herbal medicine, ginseng, this essay offers an overview of the potential use of metabolomics for quality control in herbal medicines and also highlights where more research is needed.

Purpose Both anaemia and allogenic red blood cell transfusion are common and potentially harmful in patients admitted to the intensive care unit. Whilst intravenous iron may decrease anaemia and RBC transfusion requirement, the safety and efficacy of administering iron intravenously to critically ill patients is uncertain. Methods The multicentre, randomized, placebo-controlled, blinded Intravenous Iron or Placebo for Anaemia in Intensive Care (IRONMAN) study was designed to test the hypothesis that, in anaemic critically ill patients admitted to the intensive care unit, early administration of intravenous iron, compared with placebo, reduces allogeneic red blood cell transfusion during hospital stay and increases the haemoglobin level at the time of hospital discharge. Results Of 140 patients enrolled, 70 were assigned to intravenous iron and 70 to placebo. The iron group received 97 red blood cell units versus 136 red blood cell units in the placebo group, yielding an incidence rate ratio of 0.71 [95 % confidence interval (0.43–1.18), P = 0.19]. Overall, median haemoglobin at hospital discharge was significantly higher in the intravenous iron group than in the placebo group [107 (interquartile ratio IQR 97–115) vs. 100 g/L (IQR 89–111),P = 0.02]. There was no significant difference between the groups in any safety outcome. Conclusions In patients admitted to the intensive care unit who were anaemic, intravenous iron, compared with placebo, did not result in a significant lowering of red blood cell transfusion requirement during hospital stay. Patients who received intravenous iron had a significantly higher haemoglobin concentration at hospital discharge.

© Mary Ann Liebert, Inc.Anemia is a complication of interferon-containing hepatitis C treatments. We characterized effects of interferon-based therapy on hepcidin and erythropoietin (EPO) production, iron metabolism, hemolysis, and hematopoiesis. Standard hemopoiesis [reticulocyte hemoglobin (Hb), reticulocyte production index (RPI), free Hb, and haptoglobin], iron biochemistry, hepcidin, and EPO levels were measured in 10 subjects over 12 weeks. There was a rapid decline in Hb during treatment, from a mean pretreatment (t = 0 weeks) Hb of 158.6 to 125.2 g/L at week 4 (P = 0.003) and 122.8 g/L at week 12 (P = 0.005). Paradoxically, the RPI (a measure of bone marrow responsiveness to EPO) decreased on initiation of hepatitis C virus treatment from 0.78% to 0.53% (P = 0.04). Despite worsening anemia, there was no significant increase in EPO levels. Hepcidin levels increased to >20nM in 3 subjects from 5.8 to 27.5nM (P = 0.009) compared with 9.6 to 12.3nM (P = 0.5) for the remainder of subjects. Hepcidin levels peaked at week 1 before returning to baseline levels at week 4. Subjects who responded with a rise in serum hepcidin levels to >20nM had a significantly greater drop in Hb (27.2 g/L, P = 0.008) and reticulocyte Hb (-1.4 g/L, P = 0.013) compared with the subjects who did not exhibit any change in hepcidin production. In conclusion, 30% of subjects treated with interferon exhibited significant transient increase in serum hepcidin levels, which was associated with more extreme anemia and decreased iron availability as evidenced by decreased reticulocyte Hb. In addition, there was a failure to upregulate EPO production in response to anemia and hemolysis (https://clinicaltrials.gov trial NCT01726400).

Purpose: Both anaemia and allogenic red blood cell transfusion are common and potentially harmful in patients admitted to the intensive care unit. Whilst intravenous iron may decrease anaemia and RBC transfusion requirement, the safety and efficacy of administering iron intravenously to critically ill patients is uncertain. Methods: The multicentre, randomized, placebo-controlled, blinded Intravenous Iron or Placebo for Anaemia in Intensive Care (IRONMAN) study was designed to test the hypothesis that, in anaemic critically ill patients admitted to the intensive care unit, early administration of intravenous iron, compared with placebo, reduces allogeneic red blood cell transfusion during hospital stay and increases the haemoglobin level at the time of hospital discharge. Results: Of 140 patients enrolled, 70 were assigned to intravenous iron and 70 to placebo. The iron group received 97 red blood cell units versus 136 red blood cell units in the placebo group, yielding an incidence rate ratio of 0.71 [95 % confidence interval (0.43–1.18), P = 0.19]. Overall, median haemoglobin at hospital discharge was significantly higher in the intravenous iron group than in the placebo group [107 (interquartile ratio IQR 97–115) vs. 100 g/L (IQR 89–111), P = 0.02]. There was no significant difference between the groups in any safety outcome. Conclusions: In patients admitted to the intensive care unit who were anaemic, intravenous iron, compared with placebo, did not result in a significant lowering of red blood cell transfusion requirement during hospital stay. Patients who received intravenous iron had a significantly higher haemoglobin concentration at hospital discharge. The trial was registered at http://www.anzctr.org.au as # ACTRN12612001249842.

Parastagonospora nodorum is a pathogen of wheat that affects yields globally. Previous transcriptional analysis identified a partially reducing polyketide synthase (PR-PKS) gene, SNOG_00477 (SN477), in P. nodorum that is highly upregulated during infection of wheat leaves. Disruption of the corresponding SN477 gene resulted in the loss of production of two compounds, which we identified as (R)-mellein and (R)-O-methylmellein. Using a Saccharomyces cerevisiae yeast heterologous expression system, we successfully demonstrated that SN477 is the only enzyme required for the production of (R)-mellein. This is the first identification of a fungal PKS that is responsible for the synthesis of (R)-mellein. The P. nodorum ΔSN477 mutant did not show any significant difference from the wild-type strain in its virulence against wheat. However, (R)-mellein at 200 μg/ml inhibited the germination of wheat (Triticum aestivum) and barrel medic (Medicago truncatula) seeds. Comparative sequence analysis identified the presence of mellein synthase (MLNS) homologues in several Dothideomycetes and two sodariomycete genera. Phylogenetic analysis suggests that the MLNSs in fungi and bacteria evolved convergently from fungal and bacterial 6-methylsalicylic acid synthases.

Gas chromatography–mass spectrometry (GC–MS) is a commonly used metabolomic platform for the analysis of urine. A key step in the preparation of samples for GC–MS is derivatization, in particular, methoximation and trimethylsilylation. This paper presents an assessment of automated derivatization protocols for GC–MS-based untargeted metabolomic analysis of rat urine. Automated batch and in-time (a sample ready for injection every 70 min) derivatization protocols were tested using N,O-bis(trimethylsilyl)trifluoroacetamide and N-methyl-N(trimethylsilyl)trifluoroacetamide. Principal component analysis determined differences based upon protocol tested (PC-1, 19 %) and silylation reagent (PC-2, 17 %) used. Of 249 compounds, 40 compounds were significantly different (P < 0.05) based upon reagent and 154 compounds were significantly different (P < 0.05) based upon protocol. This study confirms that derivatization reagent and protocol are key factors affecting the reproducibility and intensity of individual urinary metabolites. Additionally, we have identified the majority of the compounds in urine at least to class level, scoring the identifications using the proposed metabolite identification metrics.

Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

Globally, there has been an increase in the use of herbal remedies including traditional Chinese medicine (TCM). There is a perception that products are natural, safe and effectively regulated, however, regulatory agencies are hampered by a lack of a toolkit to audit ingredient lists, adulterants and constituent active compounds. Here, for the first time, a multidisciplinary approach to assessing the molecular content of 26 TCMs is described. Next generation DNA sequencing is combined with toxicological and heavy metal screening by separation techniques and mass spectrometry (MS) to provide a comprehensive audit. Genetic analysis revealed that 50% of samples contained DNA of undeclared plant or animal taxa, including an endangered species of Panthera (snow leopard). In 50% of the TCMs, an undeclared pharmaceutical agent was detected including warfarin, dexamethasone, diclofenac, cyproheptadine and paracetamol. Mass spectrometry revealed heavy metals including arsenic, lead and cadmium, one with a level of arsenic >10 times the acceptable limit. The study showed 92% of the TCMs examined were found to have some form of contamination and/or substitution. This study demonstrates that a combination of molecular methodologies can provide an effective means by which to audit complementary and alternative medicines.

Phytohormones are central components of complex signalling networks in plants. The interplay between these metabolites, which include abscisic acid (ABA), auxin (IAA), ethylene, jasmonic acid (JA) and salicylic acid (SA), regulate plant growth and development and modulate responses to biotic and abiotic stress. Few methods of phytohormone profiling can adequately quantify a large range of plant hormones simultaneously and without the requirement for laborious or highly specialised extraction protocols. Here we describe the development and validation of a phytohormone profiling protocol, based on methyl-chloroformate derivatisation of the plant metabolites and analysis by gas chromatography/mass spectrometry (GC–MS). We describe the analysis of 11 metabolites, either plant phytohormones or intermediates of phytohormone metabolism; ABA, azelaic acid, IAA, JA and SA, and the phytohormone precursors 1-aminocyclopropane 1-carboxylic acid, benzoic acid, cinnamic acid, 13-epi-12-oxophytodienoic acid (13-epi-OPDA), linoleic acid and linolenic acid, and validate the isolation from foliar tissue of the model legume Medicago truncatula. The preparation is insensitive to the presence of water, facilitating measurement of the volatile metabolites. Quantitation was linear over four orders of magnitude, and the limits of detection between two and 10 ng/mL for all measured metabolites using a single quadrupole GC–MS.

A robust technique was developed to identify Phytophthora cinnamomi using headspace solid-phase microextraction (HS-SPME) combined with gas chromatography (GC) coupled to a flame ionization detector (FID) for analyzing volatile organic compounds (VOCs). Six fiber types were evaluated and results indicated that the three-phase fiber 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/ PDMS) had the highest extraction efficiency for both polar and nonpolar GC columns. The maximum extraction efficiency (equilibrium absorption) was achieved 16 h after fiber exposure in the HS. Absorbed compounds on the fiber were completely desorbed in the GC injector after 5 min at 250°C. Compared with the nonpolar column, the polar column showed optimum separation of VOCs released from P. cinnamomi. Under the optimized HS-SPME and GC/FID conditions, lower detection limits for the four external standards was found to be between 1.57 to 27.36 ng/liter. Relative standard deviations <9.010% showed that the method is precise and reliable. The method also showed good linearity for the concentration range that was analyzed using four standards, with regression coefficients between 0.989 and 0.995, and the sensitivity of the method was 104 times greater than that of the conventional HS method. In this study, the VOC profiles of six Phytophthora spp. and one Pythium sp. were characterized by the optimized HS-SPME-GC method. The combination of the VOCs creates a unique pattern for each pathogen; the chromatograms of different isolates of P. cinnamomi were the same and the specific VOC pattern of P. cinnamomi remained consistently independent of the growth medium used. The chromatograms and morphological studies showed that P. cinnamomi released specific VOCs at different stages of colony development. Using the optimized HS-SPME GC method, identification of P. cinnamomi from 15 in vivo diseased soil samples was as high as 100%. Results from this study demonstrate the feasibility of th

A new Task Group has been formed within the international Metabolomics Society to provide a focal point for discussions related to experimental data quality within metabolomics experiments. The current group, which was formed in May 2014, consists of co-chairs Dan Bearden (NIST, USA) and Richard Beger (FDA, USA), and an international panel of scientists listed above as co-authors. The voluntary service of these members to this Task Group is predicated on a one-year commitment with the possibility of renewal based upon mutual agreement.

Volatile organic compounds (VOCs) from Phytophthora cinnamomi- infected lupin seedlings were collected by headspace solid-phase microextraction (HS-SPME). The sampling was done 28 to 44, 52 to 68, and 76 to 92 h after inoculation (HAI). The HS-SPME samples were analyzed by gas chromatography-flame ionization detector (GCFID) to assess the differences in volatile compounds between the P. cinnamomi-infected lupin seedlings and the control. Three specific peaks were identified after 52 to 68 h with the infected lupin seedlings, at which time there were no visible aboveground symptoms of infection. Subsequently, the VOCs of five different substrates (V8A, PDA, lupin seedlings, soil, and soil + lupin seedlings) infected with P. cinnamomi and the corresponding controls were analyzed by gas chromatography- mass spectrometry (GC/MS). A total of 87 VOCs were identified. Of these, the five most abundant that were unique to all five inoculated substrates included: 4-ethyl-2-methoxyphenol, 4-ethylphenol, butyrolactone, phenylethyl alcohol, and 3-hydroxy-2-butanone. Therefore, these metabolites can be used as markers for the identification of P. cinnamomi in different growing environments. Some VOCs were specific to a particular substrate; for example, 2,4,6-rrimethylheptanes, dl-6-methyl-5-hepten-2-ol, dimethyl trisulfide, 6,10-dimethyl- 5,9-undecadien-2-ol, and 2-methoxy-4-vinylphenol were specific to P. cinnamomi + V8A; heptanes and 5-methyl-3-heptaneone were specific to P. cinnamomi + PDA; 3-methyl-1-butanol, ethyl acetate, 2- methyl-propanoic acid, ethyl ester, and ethyl ester 2-methyl-butanoic acid were specific to P. cinnamomi-inoculated lupin seedlings; and benzyl alcohol and 4-ethyl-1, 2-dimethoxybenzene were only detected in the headspace of inoculated soil + lupin seedlings. Results from this investigation have multiple impacts as the volatile organic profiles produced by the pathogen can be utilized as an early warning system to detect the pathogen from contaminated

Cryptosporidiosis is a gastrointestinal disease in humans and animals caused by infection with the protozoan parasite Cryptosporidium. In healthy individuals, the disease manifests mainly as acute self-limiting diarrhoea, but may be chronic and life threatening for those with compromised immune systems. Control and treatment of the disease is challenged by the lack of sensitive diagnostic tools and broad-spectrum chemotherapy. Metabolomics, or metabolite profiling, is an emerging field of study, which enables characterisation of the end products of regulatory processes in a biological system. Analysis of changes in metabolite patterns reflects changes in biochemical regulation, production and control, and may contribute to understanding the effects of Cryptosporidium infection in the host environment. In the present study, metabolomic analysis of faecal samples from experimentally infected mice was carried out to assess metabolite profiles pertaining to the infection. Gas-chromatography mass spectrometry (GC-MS) carried out on faecal samples from a group of C. parvum infected mice and a group of uninfected control mice detected a mean total of 220 compounds. Multivariate analyses showed distinct differences between the profiles of C. parvum infected mice and uninfected control mice,identifying a total of 40 compounds, or metabolites that contributed most to the variance between the two groups. These metabolites consisted of amino acids (n = 17), carbohydrates (n = 8), lipids (n = 7), organic acids (n = 3) and other various metabolites (n = 5), which showed significant differences in levels of metabolite abundance between the infected and uninfected mice groups (p < 0.05). The metabolites detected in this study as well as the differences in abundance between the C. parvum infected and the uninfected control mice, highlights the effects of the infection on intestinal permeability and the fate of the metabolites as a result of nutrient scavenging by the parasite to sup

AIM: Treatment of chronic kidney disease (CKD) includes parenteral iron therapy, and these infusions can lead to iron overload. Secondary iron overload is typically treated with iron chelators, of which deferasirox is one of the most promising. However, it has not been studied in patients with CKD and iron overload.METHODS: A pilot study was conducted to evaluate the pharmacokinetics and safety of deferasirox in eight haemodialysis-dependent patients, who were receiving intravenous iron for treatment of anaemia of CKD. Deferasirox was administered at two doses (10 mg/kg and 15 mg/kg), either acute (once daily for 2 days) or steady-state (once daily for 2 weeks).RESULTS: A dose of 10 mg/kg in either protocol was not sufficient to achieve a plasma concentration in the therapeutic range (acute peak 14.1 and steady-state 22.8 mmol/L), while 15 mg/kg in either protocol maintained plasma concentration well above this range (acute peak 216 and steady-state 171 mmol/L). Plasma concentration observed at 15 mg/kg was well above that expected for this dose (40–50 mmol/L), although no adverse clinical events were observed.CONCLUSION: This study highlights the need to profile drugs such as deferasirox in specific patient groups, such as those with CKD and iron overload.

Phytophthora cinnamomi (P. c) is a plant pathogenic oomycete that is capable of damaging plants in commercial production systems and natural ecosystems worldwide. The most common methods for the detection and diagnosis of P. c infection are expensive, elaborate and time consuming. This study was carried out to examine whether species specific and life cycle specific volatile organic compounds (VOCs) can be absorbed by solid-phase microextraction fibers and detected by gas chromatography that are produced by P. c and another oomycete Pythium dissotocum. A headspace solid-phase microextraction (HS-SPME) together with gas chromatography (GC) method was developed and optimized for the identification of the VOCs released by P. c. The optimized parameters included type of fiber, exposure time, desorption temperature and desorption time. Optimization was achieved with the analytes of P. c+V8A and V8A alone. To perform the HS-SPME, six types of fiber were assayed and compared: 7μm Polydimethylsiloxane (PDMS), 100μm Polydimethylsiloxane (PDMS), 50/30μm Divinylbenzene/CarboxenTM/Polydimethylsiloxane DVB/CAR/PDMS), 65μm Polydimethylsiloxane/Divinylbenzene (PDMS/DVB), 85μm Polyacrylate (PA) fibre and 85μm CarboxenTM/Polydimethylsiloxane (Carboxen™/PDMS). In a comparison of the efficacy of the fibers, the bipolar fiber DVB/CAR/PDMS had a higher extraction efficiency than the other fibers. An exposure time of 16h with DVB/CAR/PDMS fiber in the sample headspace was enough to reach the maximum extraction efficiency. A desorption time of 3min in the GC injector with the desorption temperature of 250°C was enough for the fiber to desorb the compounds of interest. The chromatograms and morphology study confirmed that the VOCs from P. c+V8A had distinct differences from V8A alone, as did different life cycle stages of P. c and different taxa such as Pythium dissotocum. The study proved that P. c has species and life cycle specific VOCs, which in turn demonstrated the feasibility of thi

Headspace solid phase micro-extraction (HS-SPME) coupled with gas chromatography (GC) is a useful sample preparation, volatile extraction and separation method for analysis of volatile compounds from stored grain insects and their hosts. However, for using this high-quality analytical method, there is a need to optimize a range of factors to ensure good extraction efficiency. These factors include fibre selection, column selection and sample preparation. In this paper, six types of polar and non-polar fibre (100µm PDMS, 85 µm PA, 85 µm CAR/PDMS, 65 µm PDMS/DVB, 50/30 µm PDMS/CAR/DVB and 7 µm PDMS) were used to conduct the HS-SPME of volatile chemicals from wheat, wheat flour and two species of stored grain insects Tribolium castaneum (Herbst) and Rhyzopertha dominica (Fabricius). The results showed that the 50/30 µm PDMS/CAR/DVB fibre not only extracted the maximum number of volatile organic chemicals (VOCs), but also captured the largest mass of VOCs for subsequent detection by GC. Optimum sample sealing time, fibre extraction time, desorption time and temperature were 24 h, 4 h, 5 min and 250°, respectively. The GC results of volatiles from different samples gave different patterns of GC spectrum, which indicated that different volatile compounds were released from the different samples. Therefore, this study provides a detailed sequence of HS-SPME-GC optimization steps that can be applied towards the development of HS-SPME-GC methods to detect stored grain insects.

GC-MS based pesticide residue testing in grains often results in enhanced response, with the degree of enhancement being dependent on the type of grain. Some grain matrices cause matrix interference, leading to false positives and false negatives, as well as significant limitations in limits of detection (LOD) and quantification, particularly when published multiple reaction monitoring (MRM) assays are used without validation. Matrix-matched standards and analyte protectants are used routinely in pesticide analysis to combat enhancement and suppression, and to provide a more accurate indication of the residue levels present. Certain pesticide and matrix combinations are well known for illustrating the problems listed above.1,2 It is important to remember that published MRMs should only be used following validation to demonstrate that they are free from matrix interference in the matrix system under study.

Gene expression changes and metabolite abundances were measured during the interaction of Medicago truncatula with the fungal necrotrophic pathogen Phoma medicaginis in leaf tissue of susceptible and resistant accessions. Over 330 genes were differentially expressed in plants infected with P. medicaginis relative to mock-inoculated plants at 12 h post-inoculation. Of these, 191 were induced in either the resistant or the susceptible accession, with 143 genes repressed. Expression changes were observed in genes involved in the oxidative burst, cell wall strengthening and lipid metabolism, as well as several transcription factors. Genes related to salicylic acid, jasmonate and ethylene responses were up-regulated, as well as genes leading to the production of jasmonic acid. Significant induction of genes in the phenylpropanoid pathway leading to lignin and isoflavonoid biosynthesis occurred. High-pressure liquid chromatography with UV detection (HPLC-UV) identified several phenolic compounds induced by P. medicaginis, as well as constitutively higher levels of phenolic compounds, in the resistant M. truncatula accession. Differentially regulated genes induced in both the resistant and susceptible accessions, but with different kinetics, and constitutively more highly expressed and induced phenolic compounds provide candidates for functional analysis. Taken together, these results highlight the importance of the octadecanoid and phenylpropanoid pathways in defence against this necrotrophic pathogen.

Proteomics and transcriptomics are established functional genomics tools commonly used to study filamentous fungi. Metabolomics has recently emerged as another option to complement existing techniques and provide detailed information on metabolic regulation and secondary metabolism. Here, we describe broad generic protocols that can be used to undertake metabolomics studies in filamentous fungi.

Cyanide is well known for its toxicity towards living organisms. Many plants use cyanide as a defensive agent against herbivores, releasing it through the enzymatic hydrolysis of endogenous cyanogenic compounds. At low concentrations, cyanide has been proposed to have a regulatory role in many plant processes including stimulation of seed germination. However, no ecological role for cyanide in seed germination has been established. In the present study, we show that burning plant material produces the cyanohydrin, glyceronitrile. We also show that, in the presence of water, glyceronitrile is slowly hydrolysed to release cyanide that stimulates seed germination of a diverse range of fire-responsive species from different continents. We propose that glyceronitrile serves as an ecological store for cyanide and is an important cue for stimulating seed germination and landscape regeneration after fires.

The Stagonospora nodorum StuA transcription factor gene SnStuA was identified by homology searching inthe genome of the wheat pathogen Stagonospora nodorum. Gene expression analysis revealed that SnStuAtranscript abundance increased throughout infection and in vitro growth to peak during sporulation. Toinvestigate its role, the gene was deleted by homologous recombination. The growth of the resulting mutantswas retarded on glucose compared to the wild-type growth, and the mutants also failed to sporulate. Glutamateas a sole carbon source restored the growth rate defect observed on glucose, although sporulation remainedimpaired. The SnstuA strains were essentially nonpathogenic, with only minor growth observed around thepoint of inoculation. The role of SnstuA was investigated using metabolomics, which revealed that this gene’sproduct played a key role in regulating central carbon metabolism, with glycolysis, the TCA cycle, and aminoacid synthesis all affected in the mutants. SnStuA was also found to positively regulate the synthesis of themycotoxin alternariol. Gene expression studies on the recently identified effectors in Stagonospora nodorumfound that SnStuA was a positive regulator of SnTox3 but was not required for the expression of ToxA. Thisstudy has uncovered a multitude of novel regulatory targets of SnStuA and has highlighted the critical role ofthis gene product in the pathogenicity of Stagonospora nodorum