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Chenopodium quinoa (quinoa) is considered a superfood with its favourable nutrient composition and being gluten free. Quinoa has high tolerance to abiotic stresses, such as salinity, water deficit (drought) and cold. The tolerance mechanisms are yet to be elucidated. Quinoa has Epidermal Bladder Cells (EBCs) that densely cover the shoot surface, particularly the younger parts of the plant. Here, we report on the EBC's primary and secondary metabolomes, as well as the lipidome in control conditions and in response to abiotic stresses. EBCs were isolated from plants after cold, heat, high-light, water deficit and salt treatments. We used untargeted Gas Chromatography-Mass Spectrometry (GC-MS) to analyse metabolites and untargeted and targeted Liquid Chromatography-MS (LC-MS) for lipids and secondary metabolite analyses. We identified 64 primary metabolites, including sugars, organic acids and amino acids, 19 secondary metabolites, including phenolic compounds, betanin and saponins and 240 lipids categorized in five groups including glycerolipids and phospholipids. We found only few changes in the metabolic composition of EBCs in response to abiotic stresses, these were metabolites related with heat, cold and high-light treatments, but not salt stress. Na+ concentrations were low in EBCs with all treatments, and approximately two orders of magnitude lower than K+ concentrations. This article is protected by copyright. All rights reserved.

Metabolic profiling of Pratylenchus thornei resistant and susceptible wheat genotypes indicates that fatty acid, glycerolipid and flavonoid classes of metabolites constitutively expressed in resistant wheat roots could reduce nematode reproduction. The root-lesion nematode Pratylenchus thornei reduces wheat production in many parts of the world. In this study the metabolic profiles of two wheat genotypes ‘QT16258’ (moderately resistant) and ‘Janz’ (susceptible) were compared at 8 weeks post inoculation with or without P. thornei. We performed untargeted liquid chromatography mass spectrometry analysis (LC–MS) of the wheat root samples. A total of 11,704 MS features were identified, out of which 765 MS features were annotated using in-house chemical standards. Principal components analysis (PCA) and partial least square discriminant analysis (PLS-DA) indicated dissimilarity of the metabolome between P. thornei resistant and susceptible genotypes. Two-way analysis of variance indicated that metabolic differences were mainly constitutive rather than induced by inoculation with P. thornei. Eighty-four annotated metabolites were significantly (p ≤ 0.01) higher in relative concentration in ‘QT16258’ than ‘Janz’ and belonged to the following classes of metabolites: flavonoids, fatty acids, glycerolipids, alkaloids, tannins, nucleotides, steroid glycosides and terpenoids. Eighty-five annotated metabolites were significantly (p ≤ 0.01) higher in relative concentration in ‘Janz’ than ‘QT16258’ and belonged to the following classes of metabolites: amino acids, sugars, flavonoids and alkaloids. Several metabolites at higher concentration in ‘QT16258’, including quercetin-3,4'-O-di-beta-glucoside (flavonoid), linoleic acid (fatty acid), lysophosphatidylethanolamine (glycerolipid), hirsutine (alkaloid), 1-methylsulfinylbutenyl-isothiocyanate (glucosinolate), could potentially strengthen the root cell walls to inhibit nematode penetration and/or reduce nematode motility. Some metabolites at higher concentrations in susceptible ‘Janz’, including phenolics, coniferyl alcohol and indole acetic acid conjugates, could be nematode attractants as well as part of a hypersensitive browning reaction to nematode invasion.

Summary Mass spectrometry is the predominant analytical tool used in the field of plant lipidomics. However, there are many challenges associated with the mass spectrometric detection and identification of lipids because of the highly complex nature of plant lipids. Studies into lipid biosynthetic pathways, gene functions in lipid metabolism, lipid changes during plant growth and development, and the holistic examination of the role of plant lipids in environmental stress responses are often hindered. Here, we leveraged a robust pipeline that we previously established to extract and analyze lipid profiles of different tissues and developmental stages from the model plant Arabidopsis thaliana. We analyzed seven tissues at several different developmental stages and identified more than 200 lipids from each tissue analyzed. The data were used to create a web‐accessible in silico lipid map that has been integrated into an electronic Fluorescent Pictograph (eFP) browser. This in silico library of Arabidopsis lipids allows the visualization and exploration of the distribution and changes of lipid levels across selected developmental stages. Furthermore, it provides information on the characteristic fragments of lipids and adducts observed in the mass spectrometer and their retention times, which can be used for lipid identification. The Arabidopsis tissue lipid map can be accessed at http://bar.utoronto.ca/efp_arabidopsis_lipid/cgi‐bin/efpWeb.cgi., Significance Statement With the complexity of plant lipids, a web database collecting information on plant lipids is of great value. Here the most comprehensive and detailed tissue‐specific lipid profile of Arabidopsis thaliana across developmental stages is provided in the form of an open‐source, electronic Fluorescent Pictograph (eFP) browser, along with insights into lipid dynamics across plant growth and development. This resource will significantly advance future research into the Arabidopsis lipidome and facilitate research into other plants.

Lipids are a group of compounds with diverse structures that perform several important functions in plants. To unravel and better understand their in vivo functions, plant biologists have been using various lipidomic technologies including liquid-chromatography (LC)-mass spectrometry (MS). However, there are still significant challenges in LC-MS based plant lipidomics, which need to be addressed. In this review, we provide an overview of the key developments in LC-MS based lipidomic approaches to detect and identify plant lipids with emphasis on areas that can be further improved. Given that the cellular lipidome is estimated to contain hundreds of thousands of lipids,1,2 many of the lipid structures remain to be discovered. Furthermore, the plant lipidome is considered to be significantly more complex compared to that of mammals. Recent technical developments in mass spectrometry have made the detection of novel lipids possible; hence, approaches that can be used for plant lipid discovery are also discussed.

Significance Intense insecticide usage is suggested to be a significant contributor to the observed decline of insect populations around the world. Beneficial insects play essential roles in food production and ecosystem health. It is therefore vital to understand the mechanisms by which low doses of insecticide impact insect biology in order to understand and assess the threat posed. We investigated the impacts of the neonicotinoid insecticide imidacloprid on Drosophila. The binding of the insecticide to receptors in the brain triggers oxidative stress, reduces energy levels, and induces neurodegeneration as well as vision loss. As the receptors targeted by imidacloprid are conserved among insects, and other insecticides have also been shown to cause oxidative stress, these findings have wider significance., Declining insect population sizes are provoking grave concern around the world as insects play essential roles in food production and ecosystems. Environmental contamination by intense insecticide usage is consistently proposed as a significant contributor, among other threats. Many studies have demonstrated impacts of low doses of insecticides on insect behavior, but have not elucidated links to insecticidal activity at the molecular and cellular levels. Here, the histological, physiological, and behavioral impacts of imidacloprid are investigated in Drosophila melanogaster, an experimental organism exposed to insecticides in the field. We show that oxidative stress is a key factor in the mode of action of this insecticide at low doses. Imidacloprid produces an enduring flux of Ca2+ into neurons and a rapid increase in levels of reactive oxygen species (ROS) in the larval brain. It affects mitochondrial function, energy levels, the lipid environment, and transcriptomic profiles. Use of RNAi to induce ROS production in the brain recapitulates insecticide-induced phenotypes in the metabolic tissues, indicating that a signal from neurons is responsible. Chronic low level exposures in adults lead to mitochondrial dysfunction, severe damage to glial cells, and impaired vision. The potent antioxidant, N-acetylcysteine amide (NACA), reduces the severity of a number of the imidacloprid-induced phenotypes, indicating a causal role for oxidative stress. Given that other insecticides are known to generate oxidative stress, this research has wider implications. The systemic impairment of several key biological functions, including vision, reported here would reduce the resilience of insects facing other environmental challenges.

Endemism and rarity have long intrigued scientists. We focused on a rare endemic and critically-endangered species in a global biodiversity hotspot, Grevillea thelemanniana (Proteaceae). We carried out plant and soil analyses of four Proteaceae, including G. thelemanniana, and combined these with glasshouse studies. The analyses related to hydrology and plant water relations as well as soil nutrient concentrations and plant nutrition, with an emphasis on sodium (Na) and calcium (Ca). The local hydrology and matching plant traits related to water relations partially accounted for the distribution of the four Proteaceae. What determined the rarity of G. thelemanniana, however, was its accumulation of Ca. Despite much higher total Ca concentrations in the leaves of the rare G. thelemanniana than in the common Proteaceae, very few Ca crystals were detected in epidermal or mesophyll cells. Instead of crystals, G. thelemanniana epidermal cell vacuoles contained exceptionally high concentrations of noncrystalline Ca. Calcium ameliorated the negative effects of Na on the very salt-sensitive G. thelemanniana. Most importantly, G. thelemanniana required high concentrations of Ca to balance a massively accumulated feeding-deterrent carboxylate, trans-aconitate. This is the first example of a calcicole species accumulating and using Ca to balance accumulation of an antimetabolite.

Chenopodium quinoa (quinoa) is considered a superfood, as it has favourable nutrient composition and is gluten free. Quinoa has high tolerance to several abiotic stresses, i.e. salinity, water deficit (drought) and cold. The tolerance mechanisms are yet to be elucidated. Quinoa has Epidermal Bladder Cells (EBCs) that densely cover the shoot surface, particularly the younger parts of the plant. Here, we report on the EBC’s primary and secondary metabolomes, as well as the lipidome in response to abiotic stresses. EBCs were isolated from plants after cold, heat, high-light, water deficit and salt treatments. We used untargeted Gas Chromatography-Mass Spectrometry (GC-MS) to analyse metabolites and untargeted and targeted Liquid Chromatography-MS (LC-MS) for lipids and secondary metabolite analyses. We identified 64 primary metabolites, including sugars, organic acids and amino acids, 19 secondary metabolites, including phenolic compounds, betanin and saponins and 240 lipids categorized in five groups including glycerolipids and phospholipids. Although we found only few changes in the metabolic composition of bladders in response to abiotic stresses, metabolites related with heat, cold and high-light treatments, but not salt stress, were changed significantly. Na concentrations were low in EBCs with all treatments, and approximately two orders of magnitude lower than K concentrations.

The plight of insect populations around the world and the threats it poses to agriculture and ecosystems has thrown insecticide use into the spotlight. Spinosad is an organic insecticide, considered less harmful to beneficial insects than synthetic insecticides, but its mode of action remains unclear. Using Drosophila, we show that low doses of spinosad reduce cholinergic response in neurons by antagonizing Dα6 nAChRs. Dα6 nAChRs are transported to lysosomes that become enlarged and accumulate upon spinosad treatment. Oxidative stress is initiated in the central nervous system, and spreads to midgut and disturbs lipid storage in metabolic tissues in a Dα6-dependent manner. Spinosad toxicity was ameliorated with the antioxidant N-Acetylcysteine amide (NACA). Chronic exposures lead to mitochondrial defects, severe neurodegeneration and blindness in adult animals. The many deleterious effects of low doses of this insecticide reported here point to an urgent need for rigorous investigation of its impacts on beneficial insects.

BackgroundThe plant lipidome is highly complex, and the composition of lipids in different tissues as well as their specific functions in plant development, growth and stress responses have yet to be fully elucidated. To do this, efficient lipid extraction protocols which deliver target compounds in solution at concentrations adequate for subsequent detection, quantitation and analysis through spectroscopic methods are required. To date, numerous methods are used to extract lipids from plant tissues. However, a comprehensive analysis of the efficiency and reproducibility of these methods to extract multiple lipid classes from diverse tissues of a plant has not been undertaken.ResultsIn this study, we report the comparison of four different lipid extraction procedures in order to determine the most effective lipid extraction protocol to extract lipids from different tissues of the model plantArabidopsis thaliana.ConclusionWhile particular methods were best suited to extract different lipid classes from diverse Arabidopsis tissues, overall a single-step extraction method with a 24 h extraction period, which uses a mixture of chloroform, isopropanol, methanol and water, was the most efficient, reproducible and the least labor-intensive to extract a broad range of lipids for untargeted lipidomic analysis of Arabidopsis tissues. This method extracted a broad range of lipids from leaves, stems, siliques, roots, seeds, seedlings and flowers of Arabidopsis. In addition, appropriate methods for targeted lipid analysis of specific lipids from particular Arabidopsis tissues were also identified.

Increases in soil salinity impacts growth and yield of agricultural plants by inhibiting plant functions. Soil salinization is increasing because of the pressure of a growing population on food supply. Genetically modified crops and plant breeding techniques are being used to produce plants tolerant to salt stress. However, interactions of fungal endophytes with crop plants can also improve tolerance and is a less expensive approach. Here, the role of Trichoderma harzianum T-22 in alleviating NaCl-induced stress in two barley genotypes (cv. Vlamingh and cv. Gairdner) has been investigated. Metabolomics using GC-MS for polar metabolites and LC-MS for lipids was employed to provide insights into the biochemical changes in barley roots inoculated with fungus during the early stages of interaction. T. harzianum increased the root length of both genotypes under controlled and saline conditions. The fungus reduced the relative concentration of sugars in both genotypes and caused no change in organic acids under saline conditions. Amino acids decreased only in cv. Gairdner in fungus-inoculated roots under saline conditions. Lipid analyses suggest that salt stress causes large changes in the lipid profile of roots but that inoculation with fungus greatly reduces the extent of these changes. By studying a tolerant and a sensitive genotype and their responses to salt and inoculation we have been able to develop hypotheses about what lipid species and metabolites may be involved in the tolerant genotype for its tolerance to salt and how fungal inoculation changes the response of the sensitive genotype to improve its tolerance.

Background:Age-related macular degeneration (AMD) is a leading cause of blindness in the ageing population. Without effective treatment strategies that can prevent disease progression, there is an urgent need for novel therapeutic interventions to reduce the burden of vision loss and improve patients’ quality of life. Dysfunctional innate immune responses to oxidative stress observed in AMD can be caused by the formation of oxidised lipids, whilst polyunsaturated fatty acids have shown to increase the risk of AMD and disease progression in affected individuals. Previously, our laboratory has shown that the vegetable-derived isothiocyanate, L-sulforaphane (LSF), can protect human adult pigment epithelial cells from oxidative damage by upregulating gene expression of the oxidative stress enzyme Glutathione-S-Transferase µ1. This study aims to validate the protective effects of LSF on human retinal cells under oxidative stress conditions and to reveal the key players in fatty acid and lipid metabolism that may facilitate this protection.Methods:Thein vitrooxidative stress model of AMD was based on the exposure of an adult retinal pigment epithelium-19 cell line to 200µM hydrogen peroxide. Percentage cell proliferation following LSF treatment was measured using tetrazolium salt-based assays. Untargeted fatty acid profiling was performed by gas chromatography-mass spectrometry. Untargeted lipid profiling was performed by liquid chromatography-mass spectrometry.Results:Under hydrogen peroxide-induced oxidative stress conditions, LSF treatment induced dose-dependent cell proliferation. The key fatty acids that were increased by LSF treatment of the retinal cells include oleic acid and eicosatrienoic acid. LSF treatment also increased levels of the lipid classes phosphatidylcholine, cholesteryl ester and oxo-phytodienoic acid but decreased levels of phosphatidylethanolamine lipids.Conclusions:We propose that retinal cells at risk of oxidative damage and apoptosis can be pre-conditioned with LSF to regulate levels of selected fatty acids and lipids known to be implicated in the pathogenesis and progression of AMD.

Chilling and frost conditions impose major yield restraints to wheat crops in Australia and other temperate climate regions. Unpredictability and variability of field frost events are major impediments for cold tolerance breeding. Metabolome and lipidome profiling were used to compare the cold response in spikes of cold-tolerant Young and sensitive variety Wyalkatchem at the young microspore (YM) stage of pollen development. We aimed to identify metabolite markers that can reliably distinguish cold-tolerant and sensitive wheat varieties for future cold-tolerance phenotyping applications. We scored changes in spike metabolites and lipids for both varieties during cold acclimation after initial and prolonged exposure to combined chilling and freezing cycles (1 and 4 days, respectively) using controlled environment conditions. The two contrasting wheat varieties showed qualitative and quantitative differences in primary metabolites involved in osmoprotection, but differences in lipid accumulation most distinctively separated the cold response of the two wheat lines. These results resemble what we previously observed in flag leaves of the same two wheat varieties. The fact that this response occurs in tissue types with very different functions indicates that chilling and freezing tolerance in these wheat lines is associated with re-modelling of membrane lipid composition to maintain membrane fluidity.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting motor neurons which shows sexual dimorphism in its incidence, age of onset, and progression rate. All steroid hormones, including androgens, estrogens, and progestogens, have been implicated in modulating ALS. Increasing evidence suggests that steroid hormones provide neuroprotective and neurotrophic support to motor neurons, either directly or via surrounding glial cell interactions, by activating their respective nuclear hormone receptors and initiating transcriptional regulatory responses. The SOD1G93A transgenic mouse also shows sex-specific differences in age of onset and progression, and remains the most widely used model in ALS research. To provide a more comprehensive understanding of the influences of steroid hormone signaling in ALS, we systemically characterized sex hormone receptor expression at transcript and protein levels, cellular localization, and the impact of disease course in lumbar spinal cords of male and female SOD1G93A mice. We found that spinal motor neurons highly express nuclear androgen receptor (AR), estrogen receptor (ER)α, ERβ, and progesterone receptor with variations in glial cell expression. AR showed the most robust sex-specific difference in expression and was downregulated in male SOD1G93A mouse spinal cord, in association with depletion in 5α-reductase type 2 isoform, which primarily metabolizes testosterone to 5α-dihydrotestosterone. ERα was highly enriched in reactive astrocytes of SOD1G93A mice and ERβ was strongly upregulated. The 5α-reductase type 1 isoform was upregulated with disease progression and may influence local spinal cord hormone levels. In conclusion, steroid hormone receptor expression is dynamic and cell-type specific in SOD1G93A mice which may provide targets to modulate progression in ALS.

The mechanisms underlying rootzone-localized responses to salinity during early stages of barley development remain elusive. In this study, we performed the analyses of multi-root-omes (transcriptomes, metabolomes, and lipidomes) of a domesticated barley cultivar (Clipper) and a landrace (Sahara) that maintain and restrict seedling root growth under salt stress, respectively. Novel generalized linear models were designed to determine differentially expressed genes (DEGs) and abundant metabolites (DAMs) specific to salt treatments, genotypes, or rootzones (meristematic Z1, elongation Z2, and maturation Z3). Based on pathway over-representation of the DEGs and DAMs, phenylpropanoid biosynthesis is the most statistically enriched biological pathway among all salinity responses observed. Together with histological evidence, an intense salt-induced lignin impregnation was found only at stelic cell wall of Clipper Z2, compared with a unique elevation of suberin deposition across Sahara Z2. This suggests two differential salt-induced modulations of apoplastic flow between the genotypes. Based on the global correlation network of the DEGs and DAMs, callose deposition that potentially adjusted symplastic flow in roots was almost independent of salinity in rootzones of Clipper, and was markedly decreased in Sahara. Taken together, we propose two distinctive salt tolerance mechanisms in Clipper (growth-sustaining) and Sahara (salt-shielding), providing important clues for improving crop plasticity to cope with deteriorating global soil salinization., Integrative analyses of the multi-root-omes (transcriptomes, metabolomes, and lipidomes) of barley genotypes demonstrate two differential salt-induced modulations of apoplastic and symplastic flows between their seedling roots. As demonstrated by the localization of cell wall components (lignin, suberin, and callose), this study illustrates two distinctive rootzone-localized growth and tolerance mechanisms in response to salinity during the early stage of barley development.

Here, we developed a robust lipidomics workflow merging both targeted and untargeted approaches on a single liquid chromatography coupled to quadrupole-time of flight (LC-QqTOF) mass spectrometry platform with parallel reaction monitoring (PRM). PRM assays integrate both untargeted profiling from MS1 scans and targeted profiling obtained from MS/MS data. This workflow enabled the discovery of more than 2300 unidentified features and identification of more than 600 lipid species from 23 lipid classes at the level of fatty acid/long chain base/sterol composition in a barley root extracts. We detected the presence of 142 glycosyl inositol phosphorylceramides (GIPC) with HN(Ac)-HA as the core structure of the polar head, 12 cardiolipins and 17 glucuronosyl diacylglycerols (GlcADG) which have been rarely reported previously for cereal crops. Using a scheduled algorithm with up to 100 precursors multiplexed per duty cycle, the PRM assay was able to achieve a rapid profiling of 291 species based on MS/MS data by a single injection. We used this novel approach to demonstrate the applicability and efficiency of the workflow to study salt stress induced changes in the barley root lipidome. Results show that 221 targeted lipids and 888 unknown features were found to have changed significantly in response to salt stress. This combined targeted and untargeted single workflow approach provides novel applications of lipidomics addressing biological questions.

© 2018, The Author(s), under exclusive licence to Springer Nature Limited. Glycosylation requires activated glycosyl donors in the form of nucleotide sugars to drive processes such as post-translational protein modifications and glycolipid and polysaccharide biosynthesis. Most of these reactions occur in the Golgi, requiring cytosolic-derived nucleotide sugars, which need to be actively transferred into the Golgi lumen by nucleotide sugar transporters. We identified a Golgi-localized nucleotide sugar transporter from Arabidopsis thaliana with affinity for UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) and assigned it UDP-GlcNAc transporter 1 (UGNT1). Profiles of N-glycopeptides revealed that plants carrying the ugnt1 loss-of-function allele are virtually devoid of complex and hybrid N-glycans. Instead, the N-glycopeptide population from these alleles exhibited high-mannose structures, representing structures prior to the addition of the first GlcNAc in the Golgi. Concomitantly, sphingolipid profiling revealed that the biosynthesis of GlcNAc-containing glycosyl inositol phosphorylceramides (GIPCs) is also reliant on this transporter. By contrast, plants carrying the loss-of-function alleles affecting ROCK1, which has been reported to transport UDP-GlcNAc and UDP-N-acetylgalactosamine, exhibit no changes in N-glycan or GIPC profiles. Our findings reveal that plants contain a single UDP-GlcNAc transporter that delivers an essential substrate for the maturation of N-glycans and the GIPC class of sphingolipids.

Salt stress causes dramatic changes in the organization and dynamic properties of membranes, however, little is known about the underlying mechanisms involved. Modified trichomes, known as epidermal bladder cells (EBC), on the leaves and stems of the halophyte Mesembryanthemum crystallinum can be successfully exploited as a single-cell-type system to investigate salt-induced changes to cellular lipid composition. In this study, alterations in key molecular species from different lipid classes highlighted an increase in phospholipid species, particularly those from phosphatidylcholine and phosphatidic acid (PA), where the latter is central to the synthesis of membrane lipids. Triacylglycerol (TG) species decreased during salinity, while there was little change in plastidic galactolipids. EBC transcriptomic and proteomic data mining revealed changes in genes and proteins involved in lipid metabolism and the upregulation of transcripts for PIPKIB, PI5PII, PIPKIII, and phospholipase D delta suggested the induction of signalling processes mediated by phosphoinositides and PA. TEM and flow cytometry showed the dynamic nature of lipid droplets in these cells under salt stress. Altogether, this work indicates that the metabolism of TG might play an important role in EBC response to salinity as either an energy reserve for sodium accumulation and/or driving membrane biosynthesis for EBC expansion.

The mechanisms underlying rootzone-localised responses to salinity stress during early stage of barley development remains fragmentary and elusive. Here, we performed a comprehensive detection of the multi-root-omes (transcriptomes, metabolomes, lipidomes) of a domesticated barley cultivar (Clipper) and a landrace (Sahara) with seedling root growth maintained and restricted in response to salt stress, respectively. Novel generalized linear models were designed to determine differentially expressed genes (DEG) or abundant metabolites (DAM) specific to salt treatments, genotypes, or rootzones (meristematic Z1, elongation Z2, maturation Z3). Based on pathway over-representation of the DEG and DAM, phenylpropanoid biosynthesis is the most statistically over-represented biological pathways among all salinity responses observed. Together with the histological evidence, an intense salt-induced lignin impregnation was found only at the stelic cell wall of Clipper Z2, comparing to a unique elevation of suberin deposition across Sahara Z2. This suggests two differential salt-induced modulations of apoplastic flow between the genotypes. Based on global correlation network construction of the DEG and DAM, callose deposition that potentially adjusted the symplastic flow in roots was almost independent of salinity in rootzones of Clipper, but was markedly decreased in that of Sahara. Through closer examinations of molecular and hormonal clues, we further demonstrate that the salinity response in rootzones of Clipper were mostly at recovery phase, comparing to Sahara with rootzones retained at quiescence. Taken together, we propose that two distinctive salt tolerance mechanisms could exist in Clipper (growth-sustaining) and Sahara (salt-shielding), providing important clues for improving crop plasticity to cope with the deteriorating global salinization of soil.

IntroductionFrost events lead to A$360 million of yield losses annually to the Australian wheat industry, making improvement of chilling and frost tolerance an important trait for breeding.ObjectivesThis study aimed to use metabolomics and lipidomics to explore genetic variation in acclimation potential to chilling and to identify metabolite markers for chilling tolerance in wheat.MethodsWe established a controlled environment screening assay that is able to reproduce field rankings of wheat germplasm for chilling and frost tolerance. This assay, together with targeted metabolomics and lipidomics approaches, were used to compare metabolite and lipid levels in flag leaves of two wheat varieties with contrasting chilling tolerance.ResultsThe sensitive variety Wyalkatchem showed a strong reduction in amino acids after the first cold night, followed by accumulation of osmolytes such as fructose, glucose, putrescine and shikimate over a 4-day period. Accumulation of osmolytes is indicative of acclimation to water stress in Wyalkatchem. This response was not observed for tolerant variety Young. The two varieties also displayed significant differences in lipid accumulation. Variation in two lipid clusters, resulted in a higher unsaturated to saturated lipid ratio in Young after 4 days cold treatment and the lipids PC(34:0), PC(34:1), PC(35:1), PC(38:3), and PI(36:4) were the main contributors to the unsaturated to saturated ratio change. This indicates that Young may have superior ability to maintain membrane fluidity following cold exposure, thereby avoiding membrane damage and water stress observed for Wyalkatchem.ConclusionOur study suggests that metabolomics and lipidomics markers could be used as an alternative phenotyping method to discriminate wheat varieties with differences in cold acclimation.

BACKGROUND AND PURPOSE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease typically more common in males, implicating androgens in progression of both patients and mouse models. Androgen effects are mediated by androgen receptor which is highly expressed in spinal motor neurons and skeletal muscles. To clarify the role of androgen receptors in ALS, we therefore examined the effect of androgen receptor antagonism in the SOD1(G93A) mouse model. EXPERIMENTAL APPROACH: The androgen receptor antagonist, flutamide, was administered to presymptomatic SOD1(G93A) mice as a slow‐release subcutaneous implant (5 mg·day(−1)). Testosterone, flutamide, and metabolite levels were measured in blood and spinal cord tissue by LC–MS–MS. Effects on disease onset and progression were assessed using motor function tests, survival, muscle, and neuropathological analyses. KEY RESULTS: Flutamide was metabolised to 2‐hydroxyflutamide achieving steady‐state plasma levels across the study duration and reached the spinal cord at pharmacologically active concentrations. Flutamide treatment accelerated disease onset and locomotor dysfunction in male SOD1(G93A) mice, but not female mice, without affecting survival. Analysis of hindlimb muscles revealed exacerbation of myofibre atrophy in male SOD1(G93A) mice treated with flutamide, although motor neuron pathology was not affected. CONCLUSION AND IMPLICATIONS: The androgen receptor antagonist accelerated disease onset in male SOD1(G93A) mice, leading to exacerbated muscle pathology, consistent with a role of androgens in modulating disease severity, sexual dimorphism, and peripheral pathology in ALS. These results also demonstrate a key contribution of skeletal muscle pathology to disease onset, but not outcome, in this mouse model of ALS.

Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2, encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen.

Free fatty acids hold important immune-modulatory roles during infection. However, the host’s long-chain polyunsaturated fatty acids, not commonly found in the membranes of bacterial pathogens, also have significant broad-spectrum antibacterial potential. Of these, the omega-6 fatty acid arachidonic acid (AA) and the omega-3 fatty acid decosahexaenoic acid (DHA) are highly abundant; hence, we investigated their effects on the multidrug-resistant human pathogen Acinetobacter baumannii. Our analyses reveal that AA and DHA incorporate into the A. baumannii bacterial membrane and impact bacterial fitness and membrane integrity, with DHA having a more pronounced effect. Through transcriptional profiling and mutant analyses, we show that the A. baumannii β-oxidation pathway plays a protective role against AA and DHA, by limiting their incorporation into the phospholipids of the bacterial membrane. Furthermore, our study identified a second bacterial membrane protection system mediated by the AdeIJK efflux system, which modulates the lipid content of the membrane via direct efflux of lipids other than AA and DHA, thereby providing a novel function for this major efflux system in A. baumannii. This is the first study to examine the antimicrobial effects of host fatty acids on A. baumannii and highlights the potential of AA and DHA to protect against A. baumannii infections. IMPORTANCE A shift in the Western diet since the industrial revolution has resulted in a dramatic increase in the consumption of omega-6 fatty acids, with a concurrent decrease in the consumption of omega-3 fatty acids. This decrease in omega-3 fatty acid consumption has been associated with significant disease burden, including increased susceptibility to infectious diseases. Here we provide evidence that DHA, an omega-3 fatty acid, has superior antimicrobial effects upon the highly drug-resistant pathogen Acinetobacter baumannii, thereby providing insights into one of the potential health benefits of omega-3 fatty acids. The identification and characterization of two novel bacterial membrane protective mechanisms against host fatty acids provide important insights into A. baumannii adaptation during disease. Furthermore, we describe a novel role for the major multidrug efflux system AdeIJK in A. baumannii membrane maintenance and lipid transport. This core function, beyond drug efflux, increases the appeal of AdeIJK as a therapeutic target.

BACKGROUND AND PURPOSE: Endothelium‐derived vasoconstriction is a hallmark of vascular dysfunction in hypertension. In some cases, an overproduction of endothelium‐derived prostacyclin (PGI(2)) can cause contraction rather than relaxation. Relaxin is well known for its vasoprotective actions, but the possibility that this peptide could also reverse endothelium‐derived vasoconstriction has never been investigated. We tested the hypothesis that short‐term relaxin treatment mitigates endothelium‐derived vasoconstriction in spontaneously hypertensive rats (SHR). EXPERIMENTAL APPROACH: Male Wistar Kyoto rats (WKY) and SHR were subcutaneously infused with either vehicle (20 mmol·L(−1) sodium acetate) or relaxin (13.3 μg·kg(−1)·hr(−1)) using osmotic minipumps for 3 days. Vascular reactivity to the endothelium‐dependent agonist ACh was assessed in vitro by wire myography. Quantitative PCR and LC‐MS were used to identify changes in gene expression of prostanoid pathways and PG production, respectively. KEY RESULTS: Relaxin treatment ameliorated hypertension‐induced endothelial dysfunction by increasing NO‐dependent relaxation and reducing endothelium‐dependent contraction. Notably, short‐term relaxin treatment up‐regulated mesenteric PGI(2) receptor (IP) expression, permitting PGI(2)–IP‐mediated vasorelaxation. In the aorta, reversal of contraction was accompanied by suppression of the hypertension‐induced increase in prostanoid‐producing enzymes and reduction in PGI(2)‐evoked contractions. CONCLUSIONS AND IMPLICATIONS: Relaxin has region‐dependent vasoprotective actions in hypertension. Specifically, relaxin has distinct effects on endothelium‐derived contracting factors and their associated vasoconstrictor pathways in mesenteric arteries and the aorta. Taken together, these observations reveal the potential of relaxin as a new therapeutic agent for vascular disorders that are associated with endothelium‐derived vasoconstriction including hypertension.

International audience; Autophagy is an essential component of innate immunity, enabling the detection and elimination of intracellular pathogens. Legionella pneumophila, an intracellular pathogen that can cause a severe pneumonia in humans, is able to modulate autophagy through the action of effector proteins that are translocated into the host cell by the pathogen's Dot/Icm type IV secretion system. Many of these effectors share structural and sequence similarity with eukaryotic proteins. Indeed, phylogenetic analyses have indicated their acquisition by horizontal gene transfer from a eukaryotic host. Here we report that L. pneumophila translocates the effector protein sphingosine-1 phosphate lyase (LpSpl) to target the host sphingosine biosynthesis and to curtail autophagy. Our structural characterization of LpSpl and its comparison with human SPL reveals high structural conservation, thus supporting prior phylogenetic analysis. We show that LpSpl possesses S1P lyase activity that was abrogated by mutation of the catalytic site residues. L. pneumophila triggers the reduction of several sphingolipids critical for macrophage function in an LpSpl-dependent and -independent manner. LpSpl activity alone was sufficient to prevent an increase in sphingosine levels in infected host cells and to inhibit autophagy during macrophage infection. LpSpl was required for efficient infection of A/J mice, highlighting an important virulence role for this effector. Thus, we have uncovered a previously unidentified mechanism used by intracellular pathogens to inhibit autophagy, namely the disruption of host sphingolipid biosynthesis.

Apicomplexa are parasitic protozoa that cause important human diseases including malaria, cryptosporidiosis and toxoplasmosis. The replication of these parasites within their target host cell is dependent on both salvage as well as de novo synthesis of fatty acids. In Toxoplasma gondii, fatty acid synthesis via the apicoplast-localized FASII is essential for pathogenesis, while the role of two other fatty acid biosynthetic complexes remains unclear. Here, we demonstrate that the ER-localized fatty acid elongation (ELO) complexes are essential for parasite growth. Conditional knockdown of the nonredundant hydroxyacyl-CoA dehydratase and enoyl-CoA reductase enzymes in the ELO pathway severely repressed intracellular parasite growth. (13) C-glucose and (13) C-acetate labeling and comprehensive lipidomic analyses of these mutants showed a selective defect in synthesis of unsaturated long and very long-chain fatty acids (LCFAs and VLCFAs) and depletion of phosphatidylinositol and phosphatidylethanolamine species containing unsaturated LCFAs and VLCFAs. This requirement for ELO pathway was bypassed by supplementing the media with specific fatty acids, indicating active but inefficient import of host fatty acids. Our experiments highlight a gap between the fatty acid needs of the parasite and availability of specific fatty acids in the host cell that the parasite has to close using a dedicated synthesis and modification pathway.

Lipids are defined as hydrophobic or amphipathic small molecules which consist of a number of structurally and functionally distinct molecules that span from nonpolar to neutral to polar compounds. Lipidomics is the comprehensive analysis of all lipids in a biological system. Changes in lipid metabolism and composition, as well as of distinct lipid species have been linked with altered plant growth, development, and responses to environmental stresses including salinity. Recently, improved liquid chromatography mass spectrometry (LC-MS)-based techniques have provided the rapid expansion of lipidomics research. Sample preparation and lipid extraction are important steps in lipidomics, and this chapter describes important considerations in lipid monophasic and biphasic extractions from plant tissues prior to untargeted plant lipidomics approaches with LC-MS.

Introduction Mass spectrometry imaging (MSI) is a technology that enables the visualization of the spatial distribution of hundreds to thousands of metabolites in the same tissue section simultaneously. Roots are below-ground plant organs that anchor plants to the soil, take up water and nutrients, and sense and respond to external stresses. Physiological responses to salinity are multifaceted and have predominantly been studied using whole plant tissues that cannot resolve plant salinity responses spatially. Objectives This study aimed to use a comprehensive approach to study the spatial distribution and profiles of metabolites, and to quantify the changes in the elemental content in young developing barley seminal roots before and after salinity stress. Methods Here, we used a combination of liquid chromatography–mass spectrometry (LC–MS), inductively coupled plasma mass spectrometry (ICP–MS), and matrix-assisted laser desorption/ionization (MALDI–MSI) platforms to profile and analyze the spatial distribution of ions, metabolites and lipids across three anatomically different barley root zones before and after a short-term salinity stress (150 mM NaCl). Results We localized, visualized and discriminated compounds in fine detail along longitudinal root sections and compared ion, metabolite, and lipid composition before and after salt stress. Large changes in the phosphatidylcholine (PC) profiles were observed as a response to salt stress with PC 34:n showing an overall reduction in salt treated roots. ICP–MS analysis quantified changes in the elemental content of roots with increases of Na+ and decreases of K+ content. Conclusion Our results established the suitability of combining three mass spectrometry platforms to analyze and map ionic and metabolic responses to salinity stress in plant roots and to elucidate tolerance mechanisms in response to abiotic stress, such as salinity stress. Electronic supplementary material The online version of this article (10.1007/s11306-018-1359-3) contains supplementary material, which is available to authorized users.

Salinity in soils is one of the major factors that adversely affects agriculture by inhibiting plant growth, resulting in low crop productivity. Among cereal crops (eg. rice, wheat), barley (Hordeum vulgare L.) is rated as salt-tolerant, and exhibits a considerable variation in salt tolerance amongst its cultivars. Barley is a food and brewing crop, and as a glycophyte it suffers substantial yield loss when grown under saline conditions. Relatively little is currently understood of salt stress perception and responses in plant roots, which involve complex changes at the physiological, metabolic, molecular, transcriptional, and genetic levels. We aim to develop new tools to unravel how plants respond to the perception of salt stress. Evidence is accumulating that lipid signalling is an integral part of the complex regulatory networks that plants utilize to respond to salinity through modifications of membrane lipids. These occur through changes in activity of such enzymes as phospholipase D and diacylglycerol kinase that produce different classes of lipid and lipid-derived messengers. In addition, abiotic stress provokes enhanced production of reactive oxygen species, resulting in lipid modifications by the oxidation of lipid species. Lipidomics analysis using liquid chromatography mass spectrometry (LC-MS) revealed that roots from tolerant and sensitive cultivars respond differently to salt stress. To investigate the modifications of lipids leading to root responses to salinity, we are using a combination of multiple approaches, such as targeted and untargeted lipidomics of barley roots. Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI-MSI) was also employed to examine the spatial distribution of lipids in barley roots grown under control and saline conditions. The combination of LC-MS and MALDI-MSI identified a large number of metabolites and lipids with a unique spatial distribution. MSI was capable of discriminating salt vs control treated roots, identifying major lipid changes under salt treatment in a spatial manner. Non-uniform spatial distribution of metabolites was observed among different barley cultivars. Major PC lipid species and carbohydrates were identified to change the most in salt treated roots compared to control. Given the lack of fundamental knowledge of the lipids involved in signalling and metabolism under saline stress, our results provide insight into novel mechanisms of how barley roots respond to salt stress.

More than 100 lipid researchers from across Australia participated in the 3rd Australian Lipid Meeting (ALM3), held on the 21st and 22nd of November 2016 in Melbourne, Australia.[...]

© 2017 Vodstrcil et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Background Azithromycin is recommended for the treatment of uncomplicated urogenital chlamydia infection although the standard 1gram dose sometimes fails to eradicate the infection (treatment failure). One hypothesis proposed for treatment failure has been insufficient levels of the antibiotic at the site of infection. We developed an assay using liquid chromatography and tandem mass spectrometry (LC-MS/MS) to measure azithromycin concentration in high-vaginal swabs and monitor how concentration changes over time following routine azithromycin treatment. Methods Azithromycin concentrations were measured in two groups of women either within the first 24h of taking a 1g dose (N = 11) or over 9 days (N = 10). Azithromycin concentrations were normalised to an internal standard (leucine enkephalin), and the bulk lipid species phosphatidylcholine [PC(34:1)], using an Agilent 6490 triple quadrupole instrument in positive ionisation mode. The abundances of azithromycin, PC(34:1), and leu-enkephalin were determined by multiple reaction monitoring and absolute levels of azithromycin estimated using standard curves prepared on vaginal specimens. Results Vaginal azithromycin concentrations of women were rapidly obtained after 5h post-treatment (mean concentration = 1031mcg/mg of lipid, range = 173-2693mcg/mg). In women followed for 9 days, peak concentrations were highest after day 2 (mean concentration = 2206mcg/mg, range = 721-5791mcg/mg), and remained high for at least 9 days with a mean concentration of 384mcg/mg (range = 139-1024mcg/mg) on day 9. Conclusion Our study confirmed that a single 1g dose of azithromycin is rapidly absorbed and remains in the vagina at relatively high levels for at least a week, suggesting that poor antibiotic absorption is unlikely to be an explanation for treatment failure.

Colonic bacteria may mediate the transformation of curcuminoids, but studies of this metabolism are limited. Here, the metabolism of curcuminoids by Escherichia fergusonii (ATCC 35469) and two Escherichia coli strains (ATCC 8739 and DH10B) was examined in modified medium for colon bacteria (mMCB) with or without pig cecal fluid. LC-MS analysis showed that 16-37% of curcumin, 6-16% of demethoxycurcumin (DMC) and 7-15% of bis-demethoxycurcumin (Bis-DMC), and 7-15% of bis-demethoxycurcumin (Bis-DMC) were converted following 36 h of fermentation, with the amount of curcuminoids degraded varying depending on the bacterial strain and medium used. Three metabolites (dihydrocurcumin (DHC), tetrahydrocurcumin (THC), and ferulic acid (FA)) were found in fermentation cultures with all strains used. In addition, a compound with m/z [M - H](-) 470 was found and identified to be a curcumin adduct (curcumin-l-cysteine), using accurate mass FT-ICR-MS. This study provides insights into the bacterial metabolism of curcuminoids.

This study reports a detailed examination of the sustainability of using organic solvents to extract lipid from wet microalgal concentrate ( Tetraselmis suecica ) to produce biodiesel. In the preliminary section of the study, a simple surrogate method that facilitated the rapid determination of lipid yield during organic solvent extraction was developed. Based on monitoring the change in the area of the spectrophotometric absorbance of the microalgal lipid solution, the method appeared to accurately predict the lipid yield measured by a traditional gravimetric technique ( r 2 = 0.97). In the main section of the study, the kinetics of lipid extraction from wet microalgal concentrate was examined by developing a model that described microalgal lipid extraction as a first-order equilibrium-driven process. A monophasic methanol-based mixture or hexane/isopropanol-based mixture was selected as an extraction system. The model was successfully fitted to the evolution of lipid concentration in the extraction solvent obtained from the experiments (0.68 ⩽ r 2 ⩽ 1.00). The lipid extraction efficiency was shown to increase with an increase in the speed of agitation, the extraction temperature, and the degree of cellular disruption. When water content of the extraction mixture was less than 20 vol%, increase in the water content of the extraction mixture did not significantly reduce the lipid extraction efficiency as long as the mixture remained monophasic. This finding was critical for the economics of organic solvent extraction as it removed the prerequisite for dried biomass normally achieved via an energy-intensive drying process. The Hildebrand solubility parameter of the microalgal lipid extracted in the study was estimated to be between 24 and 28 MPa 1/2 . Any monophasic combination of organic solvents with Hildebrand value lying within this range was able to effectively extract lipid from the microalgal biomass with marginal variation in extraction efficiency.

Despite recent studies on health benefits of chia seed owing to its high content of ω-3 fatty acids, little work has been conducted on extractability of its nutrients. We examined the effect of soaking chia seed in water on the extractability of its omega fatty acids and lipids. State-of-the-art mass spectrometry techniques including GC-MS, LC-MS, and MALDI-MSI were utilized to identify and determine the spatial distribution of omega fatty acids and lipids in chia seed. Results showed that 24 h soaking in water improves the extractability of omega fatty acids and the ω-6:ω-3 ratio. Increase in the release levels of triacylglycerols and diacylglycerols and reduction in the release levels of phosphatidylcholines are envisaged to be the result of cell wall weakening and consequently availability of lipids for extraction. Results of MALDI-MSI show that highly abundant lipid species are mainly localised in the chia seed endosperm rather than its mucilage.

Cell disruption is an integral part of the downstream operation required to produce biodiesel from microalgae. This study investigated the use of ultrasonication and high-pressure homogenization (HPH) as cell disruption methods for two microalgal species, Tetraselmis suecica (TS) and Chlorococcum sp. (C sp.). The kinetics of cell disruption followed a first-order model (0.65

Chlamydia is the most common bacterial sexually transmitted infection among men who have sex with men. Repeat infection following treatment with 1g azithromycin is common and treatment failure of up to 22% has been reported. This study measured the pharmacokinetics of azithromycin in rectal tissue in men following a single 1g dose to assess whether azithromycin reaches the rectal site in adequate concentrations to kill chlamydia. Ten healthy men took a single oral dose of 1g azithromycin and provided nine self-collected swabs and one blood sample over 14 days. Participant demographics, medications, sexual behaviour, treatment side effects, lubricant use and douching practices were recorded with each swab. Drug concentration over time was determined using liquid chromatography-mass spectrometry and total exposure (AUC0-∞) was estimated from the concentration-time profiles. Following 1g of azithromycin, rectal concentrations peaked after a median of 24 hours (median 133mcg/g) and remained above the minimum inhibitory concentration for chlamydia (0.125mcg/mL) for at least 14 days in all men. AUC0-∞ was the highest ever reported in human tissue (13103((mcg/g).hr)). Tissue concentrations were not associated with weight (mg/kg), but data suggest that increased gastric pH could increase azithromycin levels and diarrhoea or use of water-based lubricants could decrease concentrations. High and sustained concentrations of azithromycin were found in rectal tissue following a single 1g dose suggesting that inadequate concentrations are unlikely to cause treatment failure. Factors effecting absorption (pH and diarrhoea) or drug depletion (douching and water-based lubricants) may be more important determinants of concentrations in situ.

Epidermal bladder cells (EBCs) have been postulated to assist halophytes in coping with saline environments. However, little direct supporting evidence is available. Here,Chenopodium quinoaplants were grown under saline conditions for 5weeks. One day prior to salinity treatment, EBCs from all leaves and petioles were gently removed by using a soft cosmetic brush and physiological, ionic and metabolic changes in brushed and non‐brushed leaves were compared. Gentle removal of EBC neither initiated wound metabolism nor affected the physiology and biochemistry of control‐grown plants but did have a pronounced effect on salt‐grown plants, resulting in a salt‐sensitive phenotype. Of 91 detected metabolites, more than half were significantly affected by salinity. Removal of EBC dramatically modified these metabolic changes, with the biggest differences reported for gamma‐aminobutyric acid (GABA), proline, sucrose and inositol, affecting ion transport across cellular membranes (as shown in electrophysiological experiments). This work provides the first direct evidence for a role of EBC in salt tolerance in halophytes and attributes this to (1) a key role of EBC as a salt dump for external sequestration of sodium; (2) improved K+retention in leaf mesophyll and (3) EBC as a storage space for several metabolites known to modulate plant ionic relations.

Experimental samples are valuable and can represent a significant investment in time and resources. It is highly desirable at times to obtain as much information as possible from a single sample. This is especially relevant for systems biology approaches in which several ‘omics platforms are studied simultaneously. Unfortunately, each platform has a particular extraction methodology which increases sample number and sample volume requirements when multiple ‘omics are analyzed. We evaluated the integration of a yeast extraction method; specifically we explored whether fractions from a single metabolite extraction could be apportioned to multiple downstream ‘omics analytical platforms. In addition, we examined how variations to a chloroform/methanol yeast metabolite extraction regime influence metabolite recoveries. We show that protein suitable for proteomic analysis can be recovered from a metabolite extraction and that recovery of lipids, while reproducible, are not wholly quantitative. Higher quenching solution temperatures (−30 °C) can be used without significant leakage of intracellular metabolites when lower fermentation temperatures (20 °C) are employed. However, extended residence time in quenching solution, in combination with vigorous washing of quenched cell pellets, leads to extensive leakage of intracellular metabolites. Finally, there is minimal difference in metabolite amounts obtained when metabolite extractions are performed at 4 °C compared to extractions at −20 °C. The evaluated extraction method delivers material suitable for metabolomic and proteomic analyses from the same sample preparation.

SummaryLymphatic metastasis is facilitated by lymphangiogenic growth factors VEGF-C and VEGF-D that are secreted by some primary tumors. We identified regulation of PGDH, the key enzyme in prostaglandin catabolism, in endothelial cells of collecting lymphatics, as a key molecular change during VEGF-D-driven tumor spread. The VEGF-D-dependent regulation of the prostaglandin pathway was supported by the finding that collecting lymphatic vessel dilation and subsequent metastasis were affected by nonsteroidal anti-inflammatory drugs (NSAIDs), known inhibitors of prostaglandin synthesis. Our data suggest a control point for cancer metastasis within the collecting lymphatic endothelium, which links VEGF-D/VEGFR-2/VEGFR-3 and the prostaglandin pathways. Collecting lymphatics therefore play an active and important role in metastasis and may provide a therapeutic target to restrict tumor spread.

The cell wall is essential for viability of fungi and is an effective drug target in pathogens such as Candida albicans. The contribution of post-transcriptional gene regulators to cell wall integrity in C. albicans is unknown. We show that the C. albicans Ccr4-Pop2 mRNA deadenylase, a regulator of mRNA stability and translation, is required for cell wall integrity. The ccr4/pop2 mutants display reduced wall β-glucans and sensitivity to the echinocandin caspofungin. Moreover, the deadenylase mutants are compromised for filamentation and virulence. We demonstrate that defective cell walls in the ccr4/pop2 mutants are linked to dysfunctional mitochondria and phospholipid imbalance. To further understand mitochondrial function in cell wall integrity, we screened a Saccharomyces cerevisiae collection of mitochondrial mutants. We identify several mitochondrial proteins required for caspofungin tolerance and find a connection between mitochondrial phospholipid homeostasis and caspofungin sensitivity. We focus on the mitochondrial outer membrane SAM complex subunit Sam37, demonstrating that it is required for both trafficking of phospholipids between the ER and mitochondria and cell wall integrity. Moreover, in C. albicans also Sam37 is essential for caspofungin tolerance. Our study provides the basis for an integrative view of mitochondrial function in fungal cell wall biogenesis and resistance to echinocandin antifungal drugs.

This paper describes a simple and inexpensive pervaporation flow injection (PFI) system for the on-line determination of arsenic in industrial samples based on amperometric detection. The PFI system uses on-line hydride generation for the separation of As(III) from the sample matrix thus substantially reducing matrix interferences associated with "dirty" industrial samples. A method for the speciation of As(III) and As(V) is also described. The PFI system is validated using hydride generation atomic absorption spectrometry. There was no statistically significant difference at the 95% confidence level between the results for the two methods. The proposed PFI method is characterised by a wide analytical concentration range (0.05-60mgL(-1)), a detection limit for As(III) of 1.0microgL(-1) and a sample throughput of 12h(-1).

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