Membrane Lipids Augment Cell Envelope Stress Signaling via the MadRS System to Defend Against Antimicrobial Peptides and Antibiotics in Enterococcus faecalis.

Enterococci have evolved resistance mechanisms to protect their cell envelopes against bacteriocins and host cationic antimicrobial peptides (CAMPs) produced in the gastrointestinal environment. Activation of the membrane stress response has also been tied to resistance to the lipopeptide antibiotic daptomycin. However, the actual effectors mediating resistance have not been elucidated. Here, we show that the MadRS (formerly YxdJK) membrane antimicrobial peptide defense system controls a network of genes, including a previously uncharacterized three gene operon (madEFG) that protects the E. faecalis cell envelope from antimicrobial peptides. Constitutive activation of the system confers protection against CAMPs and daptomycin in the absence of a functional LiaFSR system and leads to persistence of cardiac microlesions in vivo. Moreover, changes in the lipid cell membrane environment alter CAMP susceptibility and expression of the MadRS system. Thus, we provide a framework supporting a multilayered envelope defense mechanism for resistance and survival coupled to virulence.

Elucidating the impact of bacterial lipases, human serum albumin, and FASII inhibition on the utilization of exogenous fatty acids by Staphylococcus aureus.

IMPORTANCE: Incorporation of host-derived exogenous fatty acids (eFAs), particularly unsaturated fatty acids (UFAs), by Staphylococcus aureus could affect the bacterial membrane fluidity and susceptibility to antimicrobials. In this work, we found that glycerol ester hydrolase (Geh) is the primary lipase hydrolyzing cholesteryl esters and, to a lesser extent, triglycerides and that human serum albumin (HSA) could serve as a buffer of eFAs, where low levels of HSA facilitate the utilization of eFAs but high levels of HSA inhibit it. The fact that the type II fatty acid synthesis (FASII) inhibitor, AFN-1252, leads to an increase in UFA content even in the absence of eFA suggests that membrane property modulation is part of its mechanism of action. Thus, Geh and/or the FASII system look to be promising targets to enhance S. aureus killing in a host environment by restricting eFA utilization or modulating membrane properties, respectively.

Membrane Lipids Augment Cell Envelope Stress Signaling and Resistance to Antibiotics and Antimicrobial Peptides in Enterococcus faecalis.

Enterococci have evolved resistance mechanisms to protect their cell envelopes against bacteriocins and host cationic antimicrobial peptides (CAMPs) produced in the gastrointestinal environment. Activation of the membrane stress response has also been tied to resistance to the lipopeptide antibiotic daptomycin. However, the actual effectors mediating resistance have not been elucidated. Here, we show that the MadRS (formerly YxdJK) membrane antimicrobial peptide defense system controls a network of genes, including a previously uncharacterized three gene operon (madEFG) that protects the E. faecalis cell envelope from antimicrobial peptides. Constitutive activation of the system confers protection against CAMPs and daptomycin in the absence of a functional LiaFSR system and leads to persistence of cardiac microlesions in vivo. Moreover, changes in the lipid cell membrane environment alter CAMP susceptibility and expression of the MadRS system. Thus, we provide a framework supporting a multilayered envelope defense mechanism for resistance and survival coupled to virulence.

Molecular Basis of Cell Membrane Adaptation in Daptomycin-Resistant Enterococcus faecalis.

Daptomycin is a last-resort lipopeptide antibiotic that disrupts cell membrane (CM) and peptidoglycan homeostasis. Enterococcus faecalis has developed a sophisticated mechanism to avoid daptomycin killing by re-distributing CM anionic phospholipids away from the septum. The CM changes are orchestrated by a three-component regulatory system, designated LiaFSR, with a possible contribution of cardiolipin synthase (Cls). However, the mechanism by which LiaFSR controls the CM response and the role of Cls are unknown. Here, we show that cardiolipin synthase activity is essential for anionic phospholipid redistribution and daptomycin resistance since deletion of the two genes ( cls1 and cls2 ) encoding Cls abolished CM remodeling. We identified LiaY, a transmembrane protein regulated by LiaFSR, as an important mediator of CM remodeling required for re-distribution of anionic phospholipid microdomains via interactions with Cls1. Together, our insights provide a mechanistic framework on the enterococcal response to cell envelope antibiotics that could be exploited therapeutically.

High-throughput analysis of lipidomic phenotypes of methicillin-resistant Staphylococcus aureus by coupling in situ 96-well cultivation and HILIC-ion mobility-mass spectrometry.

Antimicrobial resistance is a major threat to human health as resistant pathogens spread globally, and the development of new antimicrobials is slow. Since many antimicrobials function by targeting cell wall and membrane components, high-throughput lipidomics for bacterial phenotyping is of high interest for researchers to unveil lipid-mediated pathways when dealing with a large number of lab-selected or clinical strains. However, current practice for lipidomic analysis requires the cultivation of bacteria on a large scale, which does not replicate the growth conditions for high-throughput bioassays that are normally carried out in 96-well plates, such as susceptibility tests, growth curve measurements, and biofilm quantitation. Analysis of bacteria grown under the same condition as other bioassays would better inform the differences in susceptibility and other biological metrics. In this work, a high-throughput method for cultivation and lipidomic analysis of antimicrobial-resistant bacteria was developed for standard 96-well plates exemplified by methicillin-resistant Staphylococcus aureus (MRSA). By combining a 30-mm liquid chromatography (LC) column with ion mobility (IM) separation, elution time could be dramatically shortened to 3.6 min for a single LC run without losing major lipid features. Peak capacity was largely rescued by the addition of the IM dimension. Through multi-linear calibration, the deviation of retention time can be limited to within 5%, making database-based automatic lipid identification feasible. This high-throughput method was further validated by characterizing the lipidomic phenotypes of antimicrobial-resistant mutants derived from the MRSA strain, W308, grown in a 96-well plate.

Elucidating the Impact of Bacterial Lipases, Human Serum Albumin, and FASII Inhibition on the Utilization of Exogenous Fatty Acids by Staphylococcus aureus.

Staphylococcus aureus only synthesizes straight-chain or branched-chain saturated fatty acids (SCFAs or BCFAs) via the type II fatty acid synthesis (FASII) pathway, but as a highly adaptive pathogen, S. aureus can also utilize host-derived exogenous fatty acids (eFAs), including SCFAs and unsaturated fatty acids (UFAs). S. aureus secretes three lipases, Geh, sal1, and SAUSA300_0641, which could perform the function of releasing fatty acids from host lipids. Once released, the FAs are phosphorylated by the fatty acid kinase, FakA, and incorporated into the bacterial lipids. In this study, we determined the substrate specificity of S. aureus secreted lipases, the effect of human serum albumin (HSA) on eFA incorporation, and the effect of FASII inhibitor, AFN-1252, on eFA incorporation using comprehensive lipidomics. When grown with major donors of fatty acids, cholesteryl esters (CEs) and triglycerides (TGs), Geh was found to be the primary lipase responsible for hydrolyzing CEs, but other lipases could compensate for the function of Geh in hydrolyzing TGs. Lipidomics showed that eFAs were incorporated into all major S. aureus lipid classes and that fatty acid-containing HSA can serve as a source of eFAs. Furthermore, S. aureus grown with UFAs displayed decreased membrane fluidity and increased production of reactive oxygen species (ROS). Exposure to AFN-1252 enhanced UFAs in the bacterial membrane, even without a source of eFAs, indicating a FASII pathway modification. Thus, the incorporation of eFAs alters the S. aureus lipidome, membrane fluidity, and ROS formation, which could affect host-pathogen interactions and susceptibility to membrane-targeting antimicrobials.

Tracking the Metabolic Fate of Exogenous Arachidonic Acid in Ferroptosis Using Dual-Isotope Labeling Lipidomics.

Lipid metabolism is implicated in a variety of diseases, including cancer, cell death, and inflammation, but lipidomics has proven to be challenging due to the vast structural diversity over a narrow range of mass and polarity of lipids. Isotope labeling is often used in metabolomics studies to follow the metabolism of exogenously added labeled compounds because they can be differentiated from endogenous compounds by the mass shift associated with the label. The application of isotope labeling to lipidomics has also been explored as a method to track the metabolism of lipids in various disease states. However, it can be difficult to differentiate a single isotopically labeled lipid from the rest of the lipidome due to the variety of endogenous lipids present over the same mass range. Here we report the development of a dual-isotope deuterium labeling method to track the metabolic fate of exogenous polyunsaturated fatty acids, e.g., arachidonic acid, in the context of ferroptosis using hydrophilic interaction-ion mobility-mass spectrometry (HILIC-IM-MS). Ferroptosis is a type of cell death that is dependent on lipid peroxidation. The use of two isotope labels rather than one enables the identification of labeled species by a signature doublet peak in the resulting mass spectra. A Python-based software, D-Tracer, was developed to efficiently extract metabolites with dual-isotope labels. The labeled species were then identified with LiPydomics based on their retention times, collision cross section, and m/z values. Changes in exogenous AA incorporation in the absence and presence of a ferroptosis inducer were elucidated.

Differential Contributions of Distinct Free Radical Peroxidation Mechanisms to the Induction of Ferroptosis.

Ferroptosis is a form of regulated cell death driven by lipid peroxidation of polyunsaturated fatty acids (PUFAs). Lipid peroxidation can propagate through either the hydrogen-atom transfer (HAT) or peroxyl radical addition (PRA) mechanism. However, the contribution of the PRA mechanism to the induction of ferroptosis has not been studied. In this study, we aim to elucidate the relationship between the reactivity and mechanisms of lipid peroxidation and ferroptosis induction. We found that while some peroxidation-reactive lipids, such as 7-dehydrocholesterol, vitamins D3 and A, and coenzyme Q10, suppress ferroptosis, both nonconjugated and conjugated PUFAs enhanced cell death induced by RSL3, a ferroptosis inducer. Importantly, we found that conjugated PUFAs, including conjugated linolenic acid (CLA 18:3) and conjugated linoleic acid (CLA 18:2), can induce or potentiate ferroptosis much more potently than nonconjugated PUFAs. We next sought to elucidate the mechanism underlying the different ferroptosis-inducing potency of conjugated and nonconjugated PUFAs. Lipidomics revealed that conjugated and nonconjugated PUFAs are incorporated into distinct cellular lipid species. The different peroxidation mechanisms predict the formation of higher levels of reactive electrophilic aldehydes from conjugated PUFAs than nonconjugated PUFAs, which was confirmed by aldehyde-trapping and mass spectrometry. RNA sequencing revealed that protein processing in the endoplasmic reticulum and proteasome are among the most significantly upregulated pathways in cells treated with CLA 18:3, suggesting increased ER stress and activation of unfolded protein response. These results suggest that protein damage by lipid electrophiles is a key step in ferroptosis.

Emergence of Dalbavancin, Vancomycin, and Daptomycin Nonsusceptible Staphylococcus aureus in a Patient Treated With Dalbavancin: Case Report and Isolate Characterization.

A patient with end-stage renal disease received 2 doses of dalbavancin for methicillin-resistant Staphylococcus aureus (MRSA) arteriovenous fistula infection and presented 5 weeks later with infective endocarditis secondary to vancomycin, daptomycin, and dalbavancin nonsusceptible MRSA. Resistance was associated with walK and scrA mutations, reduced long-chain lipid content, and reduced membrane fluidity.

Evolution of Enterococcus faecium in Response to a Combination of Daptomycin and Fosfomycin Reveals Distinct and Diverse Adaptive Strategies.

Infections caused by vancomycin-resistant Enterococcus faecium (VREfm) are an important public health threat. VREfm isolates have become increasingly resistant to the front-line antibiotic daptomycin (DAP). As such, the use of DAP combination therapies with other antibiotics like fosfomycin (FOS) has received increased attention. Antibiotic combinations could extend the efficacy of currently available antibiotics and potentially delay the onset of further resistance. We investigated the potential for E. faecium HOU503, a clinical VREfm isolate that is DAP and FOS susceptible, to develop resistance to a DAP-FOS combination. Of particular interest was whether the genetic drivers for DAP-FOS resistance might be epistatic and, thus, potentially decrease the efficacy of a combinatorial approach in either inhibiting VREfm or in delaying the onset of resistance. We show that resistance to DAP-FOS could be achieved by independent mutations to proteins responsible for cell wall synthesis for FOS and in altering membrane dynamics for DAP. However, we did not observe genetic drivers that exhibited substantial cross-drug epistasis that could undermine the DAP-FOS combination. Of interest was that FOS resistance in HOU503 was largely mediated by changes in phosphoenolpyruvate (PEP) flux as a result of mutations in pyruvate kinase (pyk). Increasing PEP flux could be a readily accessible mechanism for FOS resistance in many pathogens. Importantly, we show that HOU503 was able to develop DAP resistance through a variety of biochemical mechanisms and was able to employ different adaptive strategies. Finally, we showed that the addition of FOS can prolong the efficacy of DAP and slow down DAP resistance in vitro.

Discovery of coordinately regulated pathways that provide innate protection against interbacterial antagonism.

Bacterial survival is fraught with antagonism, including that deriving from viruses and competing bacterial cells. It is now appreciated that bacteria mount complex antiviral responses; however, whether a coordinated defense against bacterial threats is undertaken is not well understood. Previously, we showed that Pseudomonas aeruginosa possess a danger-sensing pathway that is a critical fitness determinant during competition against other bacteria. Here, we conducted genome-wide screens in P. aeruginosa that reveal three conserved and widespread interbacterial antagonism resistance clusters (arc1-3). We find that although arc1-3 are coordinately activated by the Gac/Rsm danger-sensing system, they function independently and provide idiosyncratic defense capabilities, distinguishing them from general stress response pathways. Our findings demonstrate that Arc3 family proteins provide specific protection against phospholipase toxins by preventing the accumulation of lysophospholipids in a manner distinct from previously characterized membrane repair systems. These findings liken the response of P. aeruginosa to bacterial threats to that of eukaryotic innate immunity, wherein threat detection leads to the activation of specialized defense systems.

Synergy Between Beta-Lactams and Lipo-, Glyco-, and Lipoglycopeptides, Is Independent of the Seesaw Effect in Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant S. aureus (MRSA) are resistant to beta-lactams, but synergistic activity between beta-lactams and glycopeptides/lipopeptides is common. Many have attributed this synergy to the beta-lactam-glycopeptide seesaw effect; however, this association has not been rigorously tested. The objective of this study was to determine whether the seesaw effect is necessary for synergy and to measure the impact of beta-lactam exposure on lipid metabolism. We selected for three isogenic strains with reduced susceptibility to vancomycin, daptomycin, and dalbavancin by serial passaging the MRSA strain N315. We used whole genome sequencing to identify genetic variants that emerged and tested for synergy between vancomycin, daptomycin, or dalbavancin in combination with 6 beta-lactams with variable affinity for staphylococcal penicillin binding proteins (PBPs), including nafcillin, meropenem, ceftriaxone, ceftaroline, cephalexin, and cefoxitin, using time-kills. We observed that the seesaw effect with each beta-lactam was variable and the emergence of the seesaw effect for a particular beta-lactam was not necessary for synergy between that beta-lactam and vancomycin, daptomycin, or dalbavancin. Synergy was more commonly observed with vancomycin and daptomycin based combinations than dalbavancin in time-kills. Among the beta-lactams, cefoxitin and nafcillin were the most likely to exhibit synergy using the concentrations tested, while cephalexin was the least likely to exhibit synergy. Synergy was more common among the resistant mutants than the parent strain. Interestingly N315-D1 and N315-DAL0.5 both had mutations in vraTSR and walKR despite their differences in the seesaw effect. Lipidomic analysis of all strains exposed to individual beta-lactams at subinhibitory concentrations suggested that in general, the abundance of cardiolipins (CLs) and most free fatty acids (FFAs) positively correlated with the presence of synergistic effects while abundance of phosphatidylglycerols (PGs) and lysylPGs mostly negatively correlated with synergistic effects. In conclusion, the beta-lactam-glycopeptide seesaw effect and beta-lactam-glycopeptide synergy are distinct phenomena. This suggests that the emergence of the seesaw effect may not have clinical importance in terms of predicting synergy. Further work is warranted to characterize strains that don't exhibit beta-lactam synergy to identify which strains should be targeted with combination therapy and which ones cannot and to further investigate the potential role of CLs in mediating synergy.

Development and Application of a Peroxyl Radical Clock Approach for Measuring Both Hydrogen-Atom Transfer and Peroxyl Radical Addition Rate Constants.

The rate-determining step in free radical lipid peroxidation is the propagation of the peroxyl radical, where generally two types of reactions occur: (a) hydrogen-atom transfer (HAT) from a donor to the peroxyl radical; (b) peroxyl radical addition (PRA) to a "C═C" double bond. Peroxyl radical clocks have been used to determine the rate constants of HAT reactions (kH), but no radical clock is available to measure the rate constants of PRA reactions (kadd). In this work, we modified the analytical approach on the linoleate-based peroxyl radical clock to enable the simultaneous measurement of both kH and kadd. Compared to the original approach, this new approach involves the use of a strong reducing agent, LiAlH4, to completely reduce both HAT and PRA-derived products and the relative quantitation of total linoleate oxidation products with or without reduction. The new approach was then applied to measuring the kH and kadd values for several series of organic substrates, including para- and meta-substituted styrenes, substituted conjugated dienes, and cyclic alkenes. Furthermore, the kH and kadd values for a variety of biologically important lipids were determined for the first time, including conjugated fatty acids, sterols, coenzyme Q10, and lipophilic vitamins, such as vitamins D3 and A.

LiPydomics: A Python Package for Comprehensive Prediction of Lipid Collision Cross Sections and Retention Times and Analysis of Ion Mobility-Mass Spectrometry-Based Lipidomics Data.

Comprehensive profiling of lipid species in a biological sample, or lipidomics, is a valuable approach to elucidating disease pathogenesis and identifying biomarkers. Currently, a typical lipidomics experiment may track hundreds to thousands of individual lipid species. However, drawing biological conclusions requires multiple steps of data processing to enrich significantly altered features and confident identification of these features. Existing solutions for these data analysis challenges (i.e., multivariate statistics and lipid identification) involve performing various steps using different software applications, which imposes a practical limitation and potentially a negative impact on reproducibility. Hydrophilic interaction liquid chromatography-ion mobility-mass spectrometry (HILIC-IM-MS) has shown advantages in separating lipids through orthogonal dimensions. However, there are still gaps in the coverage of lipid classes in the literature. To enable reproducible and efficient analysis of HILIC-IM-MS lipidomics data, we developed an open-source Python package, LiPydomics, which enables performing statistical and multivariate analyses ("stats" module), generating informative plots ("plotting" module), identifying lipid species at different confidence levels ("identification" module), and carrying out all functions using a user-friendly text-based interface ("interactive" module). To support lipid identification, we assembled a comprehensive experimental database of m/z and CCS of 45 lipid classes with 23 classes containing HILIC retention times. Prediction models for CCS and HILIC retention time for 22 and 23 lipid classes, respectively, were trained using the large experimental data set, which enabled the generation of a large predicted lipid database with 145,388 entries. Finally, we demonstrated the utility of the Python package using Staphylococcus aureus strains that are resistant to various antimicrobials.

Mass spectrometry-based metabolomics approach to reveal differential compounds in pufferfish soups: Flavor, nutrition, and safety.

Some Eastern Asian countries deem pufferfish - especially its muscle - a culinary delight. Herein, molecular mass fingerprinting of soups prepared by Takifugu flavidus, Takifugu obscurus and Takifugu rubripes was established via matrix-assisted laser desorption/ionization - mass spectrometry (MALDI MS). Soup samples were directly analyzed by MALDI MS to collect mass spectra within 0-700 Da in a quick way, followed by principal component analysis to distinguish the different soups and to find out the distinctive compounds among the soups. High performance liquid chromatography - tandem MS (HPLC-MS/MS) was applied to identify the compounds. Nineteen compounds were identified from the HPLC-MS/MS data by using METLIN database. Through literature mining, we found that these compounds are closely related to the flavor, nutrition, and safety of pufferfish soups. This method can also be used as a facile way to distinguish between different pufferfish fillets when morphological characters have been damaged or destroyed.

Identification of pathogenic bacteria in human blood using IgG-modified Fe3O4 magnetic beads as a sorbent and MALDI-TOF MS for profiling.

A method is described for fast identification of bacteria by combining (a) the enrichment of bacterial cells by using magnetite (Fe3O4) magnetic beads modified with human IgG (IgG@Fe3O4) and (b) MALDI-TOF MS analysis. IgG has affinity to protein A, protein G, protein L and glycans on the surface of bacterial cells, and IgG@Fe3O4. It therefore is applicable to the preconcentration of a range of bacterial species. The feasibility of the method has been demonstrated by collecting six species of pathogenic bacteria (Gram-positives: Staphylococcus aureus and Kocuria rosea; Gram-negatives: Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae and Pseudomonas aeruginosa). Bacteria with concentrations as low as 10 CFU·mL-1 in spiked water samples were extracted by this sorbent with recovery rates of >50%. After enrichment, bacteria on the IgG@Fe3O4 sorbent were further identified by MALDI-TOF MS. Bacteria in concentrations as low as 105 CFU in 100 µL of human whole blood can be identified by the method. Compared to other blood culture based tests, the culture time is shortened by 40% (from ~10 h to ~6 h), and the plate culture procedure (overnight) is avoided. After short blood culture, the enrichment and identification can be finished in one hour. The IgG@Fe3O4 is of practical value in clinical diagnosis and may be combined with other identification methods, e.g. PCR, Raman spectroscopy, infrared spectroscopy, etc. Graphical abstract A non-targeted, fast and sensitive assay for bacterial identification from human blood has been developed based on the enrichment of bacteria by IgG@Fe3O4 and identification by MALDI-TOF MS.

Lanosterol Synthase Pathway Alleviates Lens Opacity in Age-Related Cortical Cataract.

PURPOSE: Lanosterol synthase (LSS) abnormity contributes to lens opacity in rats, mice, dogs, and human congenital cataract development. This study examined whether LSS pathway has a role in different subtypes of age-related cataract (ARC). METHODS: A total of 390 patients with ARC and 88 age-matched non-ARC patients were enrolled in this study. LSS expression was analyzed by western blot and enzyme-linked immunosorbent assay (ELISA). To further examine the function of LSS, we used U18666A, an LSS inhibitor in rat lens culture system. RESULTS: In lens epithelial cells (LECs), LSS expression in LECs increased with opaque degree C II, while it decreased with opaque degree C IV and C V. While in the cortex of age-related cortical cataract (ARCC), LSS expression was negatively related to opaque degree, while lanosterol level was positively correlated to opaque degree. No obvious change in both LSS and lanosterol level was found in either LECs or the cortex of age-related nuclear cataract (ARNC) and age-related posterior subcapsular cataract (ARPSC). In vitro, inhibiting LSS activity induced rat lens opacity and lanosterol effectively delayed the occurrence of lens opacity. CONCLUSIONS: This study indicated that LSS and lanosterol were localized in the lens of human ARC, including ARCC, ARNC, and ARPSC. LSS and lanosterol level are only correlated with opaque degree of ARCC. Furthermore, activated LSS pathway in lens is protective for lens transparency in cortical cataract.

TiO2-Assisted Laser Desorption/Ionization Mass Spectrometry for Rapid Profiling of Candidate Metabolite Biomarkers from Antimicrobial-Resistant Bacteria.

Antimicrobial resistance (AMR) is one of the most serious problems affecting public health and safety. It is crucial to understand antimicrobial resistance from the molecular level. In this work, TiO2-assisted laser desorption/ionization (LDI) mass spectrometry (MS) was used for the fast metabolites analysis from intact bacterial cells to discriminate different strains of bacteria and to detect AMR. With the mass spectra of bacterial metabolites by TiO2-LDI MS, multivariable analysis was performed for bacterial identification to determine distinctive metabolites as the potential biomarkers. The most statistically significant metabolites were screened out by the method and further identified using liquid-chromatography (LC) tandem MS (MS/MS). Robustness of our developed methods in bacterial taxonomy was demonstrated by iterative validation using 48 clinical samples. The strategy was further illustrated with three clinical strains of ESBL (extended-spectrum ß-lactamase-resistant)-positive Escherichia. coli and four clinical strains of ESBL-negative ones. Eleven key metabolites were identified as potential biomarkers of ESBL-positive E. coli. We also implemented the pathway and network analysis on the key metabolites to prove the feasibility of our method in executing metabolomics analysis. Compared to the most prevalent techniques in a metabolomics study, such as LC-MS, gas chromatography MS, and nuclear magnetic resonance spectroscopy, the current method has advantages in its simple sample preparation and short analysis time, thereby fitting especially into clinical usages and fast analyses.

Electrostatic Spray Ionization-Mass Spectrometry for Direct and Fast Wine Characterization.

Due to the globally existed and economically motivated adulteration including mislabeling and/or blending, fast wine characterization is important in wine industry. Herein, we developed an electrostatic spray ionization-mass spectrometry (ESTASI-MS)-based method to distinguish wines. Wine samples were directly analyzed by ESTASI-MS without any pretreatment. Microdroplets of wine were deposited on a plastic plate for analysis. The collection of each mass spectrometric datum can be accomplished in 1-2 min without any need of pretreatment to the sample, followed by principle component analysis to discriminate wines with different labels and vintages. Long-term storage of wine was simulated and characterized by utilizing the method. High-performance liquid chromatography-MS was further applied to identify the distinctive compounds in wines to indicate their difference. We found that the method can offer a strategy for quick wine analysis, which is of practical value in wine industry for wine classification and aging control.

Detection, occurrence and monthly variations of typical lipophilic marine toxins associated with diarrhetic shellfish poisoning in the coastal seawater of Qingdao City, China.

In recent years, related research has mainly examined lipophilic marine toxins (LMTs) in contaminated bivalves or toxic algae, whereas the levels of LMTs in seawater remain largely unexplored. Okadaic acid (OA), yessotoxin (YTX), and pectenotoxin-2 (PTX2) are three typical LMTs produced by certain marine algae that are closely linked to diarrhetic shellfish poisoning. In this study, a new method of solid phase extraction combined with liquid chromatography - electrospray ionization ion trap tandem mass spectrometry was developed to determine the presence of OA, YTX, and PTX2 in seawater simultaneously. Satisfactory sensitivity, repeatability (RSD<25.00%) and recovery (56.25-70.18%) of the method were achieved. Then, the method was applied to determine the amounts of the three toxins in the coastal seawater. OA and PTX2 were detected in all the seawater samples collected from eight locations along the coastline of Qingdao City, China on October 23, 2012, with concentration ranges of OA 4.24-9.64ngL(-1) and PTX2 0.42-0.74ngL(-1). Monthly concentrations of OA and PTX2 in the seawater of four locations were determined over the course of a year, with concentration ranges of OA 1.41-89.52ngL(-1) and PTX2 below detectable limit to 1.70ngL(-1). The peak values of OA and PTX2 in coastal seawater were observed in August and July, respectively. Our results suggest that follow-up research on the fate modeling and risk assessment of LMTs in coastal seawater should be implemented.