Interactions of 15N-Sulfadiazine and Soil Components As Evidenced by 15N-CPMAS NMR.

The extensive use of sulfonamides (SNs) in animal husbandry has led to an unintentional widespread occurrence in several environmental compartments. The implementation of regulations and management recommendations to reduce the potential risk of development of antibiotic resistances necessitates detailed knowledge on their fate in soil. We present results from two independent incubation studies of 15N-labeled sulfadiazines (SDZ) which focused on identifying binding types in bound residues. In the first study 15N-amino labeled SDZ was incubated with two previously isolated humic acids in the presence and absence of Trametes versicolor laccase, while in the second study 15N-double-labeled SDZ was incubated with a typical agricultural Luvisol and the humic acid fraction isolated after sequential extraction of the soil. The freeze-dried humic acid fractions of both studies were then analyzed by 15N-CPMAS NMR and compared with the 15N-spectra of synthesized model compounds. In both studies amide bonds and Michael adducts were identified, while formation of imine bonds could be excluded. In the humic acid study, where less harsh extraction methods were applied, possible formation of H-bridging and sequestration were additionally detected.

Antiplasmodial and Cytotoxic Triterpenoids from the Bark of the Cameroonian Medicinal Plant Entandrophragma congoënse.

Eight new triterpenoids, prototiamins A-G (1-6, 9) and seco-tiaminic acid A (10), were isolated along with four known compounds from the bark of Entandrophragma congoënse. Their structures were elucidated by means of HRMS and different NMR techniques and chemical transformations. Assignments of relative and absolute configurations for the new compounds were achieved using NOESY experiments and by chemical modification including the advanced Mosher's method. Additionally, the structure and relative configuration of compound 3 were confirmed by single-crystal X-ray diffraction analysis. Compounds 1, 3, and 5 displayed significant in vitro antiplasmodial activity against the erythrocytic stages of chloroquine-sensitive Plasmodium falciparum strain NF54. Prototiamin C (3) was the most potent of the compounds isolated, with an IC50 value of 0.44 µM. All compounds tested showed low cytotoxicity for the L6 rat skeletal myoblast cell line.

Hexacyclopeptides secreted by an endophytic fungus Fusarium solani N06 act as crosstalk molecules in Narcissus tazetta.

The basis of chemical crosstalk in plants and associated endophytes lies in certain so-called communication molecules that are responsible for plant-microbe and microbe-microbe interactions. Consequently, elucidating the factors that affect the nature, distribution, and amount of these molecules and how they impact the interaction among endophytes and associated organisms is essential to understand the true potential of endophytes. In the present study, we report the discovery of nine hexacyclopeptides from an endophytic fungus, Fusarium solani, isolated from the bulb of Narcissus tazetta, and their selective accumulation by an endophytic bacterium, Achromobacter xylosoxidans isolated from the same tissue. We used matrix-assisted laser desorption ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) to firstly identify and visualize the spatial distribution of the hexacyclopeptides produced by endophytic F. solani. After culture condition optimization, their sequence was identified to be cyclo((Hyp or Dhp)-Xle-Xle-(Ala or Val)-Thr-Xle) (Dhp: dehydroproline) by the characteristic a, b, or y ions using liquid chromatography tandem mass spectrometry (LC-HRMS(n)). These hexacyclopeptides were confirmed to be fungal biosynthetic products by deuterium labeling experiments. Finally, in order to understand the plausible ecological relevance of one or more of the discovered hexacyclopeptides within the contexts of microbial "neighbor communication," we devised a dual-culture setup to visualize using MALDI-imaging-HRMS how the hexacyclopeptides released by the endophytic fungus are accumulated by another endophytic bacterium, A. xylosoxidans, isolated from the same bulb tissue. This work exemplifies the relevance of cyclopeptides in endophyte-endophyte interspecies neighbor communication occurring in nature. Such communication strategies are evolved by coexisting endophytes to survive and function in their distinct ecological niches.

Antibacterial secondary metabolites from an endophytic fungus, Eupenicillium sp. LG41.

Two new compounds containing the decalin moiety, eupenicinicols A and B (1 and 2), two new sirenin derivatives, eupenicisirenins A and B (3 and 4), and four known compounds, (2S)-butylitaconic acid (5), (2S)-hexylitaconic acid (6), xanthomegnin (7), and viridicatumtoxin (8), were isolated from an endophytic fungus, Eupenicillium sp. LG41, harbored in the roots of the Chinese medicinal plant Xanthium sibiricum. Their structures were confirmed through combined spectroscopic analysis (NMR and HRMS(n)), and their absolute configurations were deduced by ECD calculations or optical rotation data. Since the endophytic fungus was isolated from the roots, the antibacterial efficacies of the compounds 1-6 were investigated against Bacillus subtilis and Acinetobacter sp. BD4, which typically inhabit soil, as well as the clinically important Staphylococcus aureus and Escherichia coli. (2S)-Butylitaconic acid (5) and (2S)-hexylitaconic acid (6) exhibited pronounced efficacy against Acinetobacter sp., corroborating the notion that root-endophytes provide chemical defense to the host plants. Compound 2 was highly active against the clinically relevant S. aureus. By comparing 1 with 2, it was revealed that altering the substitution at C-11 could drastically increase the antibacterial efficacy of 1. Our study reveals plausible ecological roles of the endophyte and its potential pharmaceutical use as a source of antibacterial compounds.

Endophytes are hidden producers of maytansine in Putterlickia roots.

Several recent studies have lent evidence to the fact that certain so-called plant metabolites are actually biosynthesized by associated microorganisms. In this work, we show that the original source organism(s) responsible for the biosynthesis of the important anticancer and cytotoxic compound maytansine is the endophytic bacterial community harbored specifically within the roots of Putterlickia verrucosa and P. retrospinosa plants. Evaluation of the root endophytic community by chemical characterization of their fermentation products using HPLC-HRMS(n), along with a selective microbiological assay using the maytansine-sensitive type strain Hamigera avellanea revealed the endophytic production of maytansine. This was further confirmed by the presence of AHBA synthase genes in the root endophytic communities. Finally, MALDI-imaging-HRMS was used to demonstrate that maytansine produced by the endophytes is typically accumulated mainly in the root cortex of both plants. Our study, thus, reveals that maytansine is actually a biosynthetic product of root-associated endophytic microorganisms. The knowledge gained from this study provides fundamental insights on the biosynthesis of so-called plant metabolites by endophytes residing in distinct ecological niches.

Quorum quenching is an antivirulence strategy employed by endophytic bacteria.

Bacteria predominantly use quorum sensing to regulate a plethora of physiological activities such as cell-cell crosstalk, mutualism, virulence, competence, biofilm formation, and antibiotic resistance. In this study, we investigated how certain potent endophytic bacteria harbored in Cannabis sativa L. plants use quorum quenching as an antivirulence strategy to disrupt the cell-to-cell quorum sensing signals in the biosensor strain, Chromobacterium violaceum. We used a combination of high-performance liquid chromatography high-resolution mass spectrometry (HPLC-ESI-HRMS(n)) and matrix-assisted laser desorption ionization imaging high-resolution mass spectrometry (MALDI-imaging-HRMS) to first quantify and visualize the spatial distribution of the quorum sensing molecules in the biosensor strain, C. violaceum. We then showed, both quantitatively and visually in high spatial resolution, how selected endophytic bacteria of C. sativa can selectively and differentially quench the quorum sensing molecules of C. violaceum. This study provides fundamental insights into the antivirulence strategies used by endophytes in order to survive in their ecological niches. Such defense mechanisms are evolved in order to thwart the plethora of pathogens invading associated host plants in a manner that prevents the pathogens from developing resistance against the plant/endophyte bioactive secondary metabolites. This work also provides evidence towards utilizing endophytes as tools for biological control of bacterial phytopathogens. In continuation, such insights would even afford new concepts and strategies in the future for combating drug resistant bacteria by quorum-inhibiting clinical therapies.

Determination of anxiolytic veterinary drugs from biological fertilizer blood meal using liquid chromatography high-resolution mass spectrometry.

A liquid environment-friendly agricultural material originating from animal blood, blood meal, was employed to detect anxiolytic veterinary drugs using a combination of liquid-liquid extraction (LLE) and positive electrospray ionization Orbitrap mass spectrometry. Every positive ion of the analytes was consistent with [M+H](+) , and the accurate mass analysis and mass spectral filtration with a 2-ppm mass tolerance window were applied to identify and quantitate the analytes and metabolites. The developed LLE method was validated with the lowest calibrated level, linearity (r(2) ), recovery, repeatability and the within-laboratory reproducibility, which were in the ranges of 0.3-1 µg/L, 0.9963-0.9995, 48.3-117.5%, 1.1-12.6% and 2.3-15.7%, respectively. The LLE method was compared with a solid-phase extraction (SPE) method; however, its recoveries were <70% for most of the analytes despite good repeatability of 1.2-7.4%. The analytes and the ascertained acepromazine, azaperone and xylazine metabolites were monitored in four actual liquid blood meal samples, and none of the targeted compounds were observed.

A mechanistic study on the Hooker oxidation: synthesis of novel indane carboxylic acid derivatives from lapachol.

The Hooker oxidation is one of the most intriguing transformations wherein lapachol (1) is readily converted to norlapachol (2) in very good yield. This one-pot reaction involves a very intricate mechanism in which the alkyl side chain of lapachol is shortened by one carbon unit. Previous studies have unequivocally established the involvement of an indane carboxylic acid derivative 3, as a key intermediate (Hooker intermediate), and its simultaneous conversion to norlapachol (2) via the oxidative cleavage of vicinol diol and subsequent intramolecular aldol reaction of the resulting keto acid. However, the formation of the key Hooker intermediate 3 from lapachol (1) remains ambiguous. The present study has thrown some light on the formation of the key intermediate 3 from lapachol (1) via benzilic acid rearrangement of the corresponding labile o-diquinone intermediate 8 derived from lapachol. The involvement of o-diquinone intermediate 8 in the Hooker oxidation has been further established by trapping of this labile intermediate as the corresponding phenazine derivative 9. The involvement of benzilic acid rearrangement as a key step in the Hooker oxidation is further shown with a variety of o-quinones prepared from lapachol (1).

HR-MALDI-MS imaging assisted screening of ß-carboline alkaloids discovered from Mycena metata.

Fruiting bodies of Mycena metata were screened for the presence of new secondary metabolites by means of HPLC-UV, LC-HR-ESIMS, and high-resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (HR-MALDI-MS imaging). Thus, a new ß-carboline alkaloid, 6-hydroxymetatacarboline D (1d), was isolated from fruiting bodies of M. metata. 6-Hydroxymetatacarboline D consists of a highly substituted ß-carboline skeleton, which is likely to be derived biosynthetically from l-tryptophan, 2-oxoglutaric acid, l-threonine, and l-proline. The structure of the alkaloid was established by 2D NMR spectroscopic methods and HR-ESIMS. Moreover, by extensive application of LC-HR-ESIMS, LC-HR-ESIMS/MS, and LC-HR-ESIMS(3) techniques we were able to elucidate the structures of a number of accompanying ß-carboline alkaloids, 1a-1c, 1e-1i, and 2a-2g, structurally closely related to 6-hydroxymetatacarboline D, which are present in M. metata in minor amounts. The absolute configuration of the stereogenic centers of the ß-carboline alkaloids was determined by GC-MS comparison with authentic synthetic samples after hydrolytic cleavage and derivatization of the resulting amino acids.

Antibacterial and antiplasmodial constituents of Beilschmiedia cryptocaryoides.

Four new beilschmiedic acid derivatives, cryptobeilic acids A-D (1-4), and tsangibeilin B (5) have been isolated from the bark of Beilschmiedia cryptocaryoides collected from Madagascar. Their structures were elucidated using detailed spectroscopic and spectrometric methods. Cryptobeilic acid A (1) and tsangibeilin B (5) structures were confirmed by single-crystal X-ray diffraction analysis. Compounds 1-5 displayed moderate antibacterial activity against Escherichia coli 6r3, Acinetobacter calcoaceticus DSM 586, and Pseudonamas stutzeri A1501, with the minimum inhibitory concentrations ranging from 10 to 50 µM, respectively. In addition, the compounds exhibited antiplasmodial activity against erythrocytic stages of chloroquine-resistant Plasmodium falciparum strain NF54 and weak cytotoxicity against L6 cell lines.

Enantioselective degradation of the chiral fungicides metalaxyl and furalaxyl by Brevibacillus brevis.

For almost four decades, the chiral fungicides metalaxyl and furalaxyl have been in use in plant protection on a global scale. Both substances are distributed as racemic mixtures, yet the desirable interference in nucleic acid synthesis of harmful fungi only occurs by the (-)-R-enantiomer. As enantioselective degradation in Scheyern (Germany) and Yaoundé (Cameroon) soils has been documented, the influence of 50 isolated microorganisms on the R/S ratio was investigated. A high-pressure liquid chromatography method with a chiral column to separate enantiomers of metalaxyl and furalaxyl, and subsequent detection by tandem mass spectrometry, was employed. Only one of these microorganisms, a strain of Brevibacillus brevis, showed an enantioselective degradation pattern in liquid culture; the respective (-)-R-enantiomers were preferably degraded. Moreover, (-)-R-furalaxyl was degraded faster in cultures supplemented simultaneously with both fungicides of the same concentration.

Urinary excretion of morphine and biosynthetic precursors in mice.

It has been firmly established that humans excrete a small but steady amount of the isoquinoline alkaloid morphine in their urine. It is unclear whether it is of dietary or endogenous origin. There is no doubt that a simple isoquinoline alkaloid, tetrahydropapaveroline (THP), is found in human and rodent brain as well as in human urine. This suggests a potential biogenetic relationship between both alkaloids. Unlabeled THP or [1,3,4-D(3)]-THP was injected intraperitoneally into mice and the urine was analyzed. This potential precursor was extensively metabolized (96%). Among the metabolites found was the phenol-coupled product salutaridine, the known morphine precursor in the opium poppy plant. Synthetic [7D]-salutaridinol, the biosynthetic reduction product of salutaridine, injected intraperitoneally into live animals led to the formation of [7D]-thebaine, which was excreted in urine. [N-CD(3)]-thebaine was also administered and yielded [N-CD(3)]-morphine and the congeners [N-CD(3)]-codeine and [N-CD(3)]-oripavine in urine. These results show for the first time that live animals have the biosynthetic capability to convert a normal constituent of rodents, THP, to morphine. Morphine and its precursors are normally not found in tissues or organs, presumably due to metabolic breakdown. Hence, only that portion of the isoquinoline alkaloids excreted in urine unmetabolized can be detected. Analysis of urine by high resolution-mass spectrometry proved to be a powerful method for tracking endogenous morphine and its biosynthetic precursors.

Evaluation of sorption-desorption processes for metalaxyl in natural and artificial soils.

The main process controlling soil-pesticide interaction is the sorption-desorption as influenced by active soil surfaces. The sorption phenomena can influence translocation, volatility, persistence and bioactivity of a pesticide in soil. The present investigation was conducted on natural and artificial soils in order to enumerate the effect of soil components such as montmorillonite and ferrihydrite on the sorption behaviour of the fungicide metalaxyl and if sorption-desorption of the chiral pesticide affects the enantiomeric ratio. The sorption-desorption characteristics of metalaxyl were investigated by batch equilibration technique in a natural soil, two artificial soils, and in pure montmorillonite and ferrihydrite. After extraction, pesticide residues were analyzed by conventional and chiral chromatography using tandem mass spectrometry. A KdSorp (2.3-6.5) suggests low level sorption of metalaxyl with an appreciable risk of run-off and leaching. Thus, metalaxyl poses a threat to surface and ground water contamination. Furthermore, desorption tests revealed a hysteretic effect (H ≤ 0.8) in natural and artificial soils. Significant amount of metalaxyl was found tightly bound to the adsorbents without desorbing readily after desorption cycle. Desorption of 22-56% of the total amount of the retained metalaxyl was determined. This study reveals that an artificial soil derived from different soil constituents can be used to assess their influence on sorption/desorption processes. The present investigation showed that both montmorillonite and ferrihydrite play a significant role in the sorption of metalaxyl. The sorption doesn't influence the enantiomeric ratio of racemic metalaxyl.

Persistence of the fluoroquinolone antibiotic difloxacin in soil and lacking effects on nitrogen turnover.

The environmental risks caused by the use of fluoroquinolone antibiotics in human therapeutics and animal husbandry are associated with their persistence and (bio)accessibility in soil. To assess these aspects, we administered difloxacin to pigs and applied the contaminated manure to soil. We then evaluated the dissipation and sequestration of difloxacin in soil in the absence and presence of plants within a laboratory trial, a mesocosm trial, and a field trial. A sequential extraction yielded antibiotic fractions of differing binding strength. We also assessed the antibiotic's effects on nitrogen turnover in soil (potential nitrification and denitrification). Difloxacin was hardly (bio)accessible and was very persistent under all conditions studied (dissipation half-life in bulk soil, >217 d), rapidly forming nonextractable residues. Although varying environmental conditions did not affect persistence, dissipation was accelerated in soil surrounding plant roots. Effects on nitrogen turnover were limited due to the compound's strong binding and small (bio)accessibility despite its persistence.

Spatially Resolved Investigation of Herbicide-Safener Interaction in Maize (Zea mays L.) by MALDI-Imaging Mass Spectrometry.

Monitoring agrochemical distribution within plant tissues delivers significant insights into the adsorption, distribution, metabolism, and elimination of agrochemicals. Detection and imaging of the safener cyprosulfamide (CSA) and the herbicide thiencarbazone-methyl (TCM) after micro-droplet application on the surface of maize leaves (Zea mays L.) have been achieved using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). The agrochemicals were deposited onto the adaxial surface of maize leaves on growing plants, and their uptake, distribution, and metabolism were investigated at four timepoints (3 h, 24 h, 4 days, and 7 days) to assess the influence of CSA treatment on TCM metabolism. MALDI MSI visualized significant changes for the metabolism of TCM after 24 h. Although TCM metabolism was detected neither in the control without the safener nor in the approach with CSA on the second leaf, the co-application on the same leaf showed significant metabolism of the herbicide by detecting the metabolite N-demethylated TCM. These findings suggest that safener protection against herbicide injury is a rapid process in which CSA and TCM need to be present in the same tissues. This study showcases the use of MALDI MSI to visualize and analyze indirect interactions of two substances in planta.

Antimicrobial isopropenyl-dihydrofuranoisoflavones from Crotalaria lachnophora.

Two new isopropenyl-dihydrofuranoisoflavones exhibiting antimicrobial properties have been isolated along with eight known compounds from the Cameroonian medicinal plant Crotalaria lachnophora. The structures of the new compounds were elucidated by 1D and 2D NMR spectroscopy and high-resolution mass spectrometry as 7,2',4'-trihydroxy-5''-isopropenyl-4'',5''-dihydrofurano[2'',3'':5,6]isoflavone (1) and 4,8-dihydroxy-2-isopropenyl-2,3-dihydro-5H-[1]benzofuro[2,3-b]furo[3,2-g]chromen-5-one (2). The CH(2)Cl(2)/MeOH (1:1) extract and the compounds isolated were subjected to in vitro antimicrobial assays against a panel of pathogenic microorganisms, including Gram-positive and Gram-negative bacteria and fungi. The new compounds, named lachnoisoflavones A (1) and B (2), showed moderate inhibitory activities against Escherichia coli and Klebsiella pneumoniae.

Identification of NTBC metabolites in urine from patients with hereditary tyrosinemia type 1 using two different mass spectrometric platforms: triple stage quadrupole and LTQ-Orbitrap.

The objective of our work was to identify known and unknown metabolites of the drug NTBC (2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione) in urine from patients during the treatment of hereditary tyrosinemia type 1 (HT-1) disease, a severe inborn error of tyrosine metabolism. Two different mass spectrometric techniques, a triple stage quadrupole and an LTQ-Orbitrap (Fourier transform mass spectrometry (FTMS)), were used for the identification and the structural elucidation of the detected metabolites. Initially, the mass spectrometric (MS) approach consisted of the precursor ion scan detection of the selected product ions, followed by the corresponding collision-induced dissociation (CID) fragmentation analysis (MS(2)) for the targeted selected reaction monitoring (SRM) mode. Subsequently, accurate and high-resolution full scan and MS/MS measurements were performed on the possible metabolites using the LTQ-Orbitrap. Final confirmation of the identified metabolites was achieved by measuring commercially supplied or laboratory-synthesized standards. Altogether six metabolites, including NTBC itself, were extracted, detected and identified. In addition, two new NTBC metabolites were unambiguously identified as amino acid conjugates, namely glycine-NTBC and beta-alanine-NTBC. These identifications were based on their characteristics of chromatographic retention times, protonated molecular ions, elemental compositions, product ions (using CID and higher-energy C-trap dissociation (HCD) techniques) and synthesized references. The applied MS strategy, based on two different MS platforms (LC/MS/MS and FTMS), allowed the rapid identification analysis of the drug metabolites from human extracts and could be used for pharmaceutical research and drug development.

Semi-synthesis of dihydrochalcone derivatives and their in vitro antimicrobial activities.

We describe the semi-synthesis of dihydrochalcone derivatives and their IN VITRO antimicrobial activities. These compounds were prepared by modifying two naturally occurring antimicrobial dihydrochalcones, erioschalcones A and B, reported by us earlier. The structures of the compounds were assigned on the basis of spectroscopic evidence and by comparing their physical and spectroscopic data with those reported in the literature. All the compounds were subjected to IN VITRO antimicrobial assays against a panel of pathogenic microorganisms, including gram-positive and gram-negative bacteria, and fungi. The antimicrobial efficacies of this class of compounds were established by correlating the activity profile of each compound with its structure and by comparing the activities of all the compounds with each other based on their structure. This should enable the development of other derivatives of the dihydrochalcone family that would serve as more potent antimicrobial agents against specific pathogens.

Metabolism and excretion kinetics of 14C-labeled and non-labeled difloxacin in pigs after oral administration, and antimicrobial activity of manure containing difloxacin and its metabolites.

Fluoroquinolones are amongst the most important antibiotics used in veterinary medicine. On this account the behavior of difloxacin (DIF) and its metabolites was investigated by administering the (14)C-labeled and non-labeled veterinary drug to fattening pigs. The excretion kinetics were determined after daily collection of manure. Sarafloxacin (SAR) was found to be the major metabolite, three further trace metabolites were also recovered, applying high-resolution (HR) mass spectrometric technique. The identification of DIF and SAR was confirmed by comparison with the spectroscopic and chromatographic data of the authentic references. The identification of the three trace metabolites was performed by HR-MS/MS. Only 8.1% of the administered radioactivity remained in the pig after 10 days and DIF accounted for 95.9% of the radioactivity excreted. More than 99% of the labeled compounds were detected and identified in the manure. The mean recoveries for all single electrolytes were 94%. Linearity was established over concentration range 10-10,000 microg/kg manure with a correlation coefficient 0.99. By using in vitro antimicrobial activity tests against a group of standard pathogenic control strains, the results showed that the residual antibiotic concentrations in the manure of pigs are high enough to exhibit antibacterial activity.

Multi-mycotoxin analysis in complex biological matrices using LC-ESI/MS: experimental study using triple stage quadrupole and LTQ-Orbitrap.

In the present study, we report the application of LC-MS based on two different LC-MS systems to mycotoxin analysis. The mycotoxins were extracted with an ACN/water/acetic acid mixture and directly injected into a LC-MS/MS system without any dilution procedure. First, a sensitive and reliable HPLC-ESI-MS/MS method using selected reaction monitoring on a triple quadrupole mass spectrometer (TSQ Quantum Ultra AM) has been developed for determining 32 mycotoxins in crude extracts of wheat and maize. This method was operated both in positive and in negative ionization modes in two separate chromatographic runs. The method was validated by studies of spiked recoveries, linearity, matrix effect, intra-assay precision and sensitivity. Further, we have developed and evaluated a method based on accurate mass measurements of extracted target ions in full scan mode using micro-LC-LTQ-Orbitrap as a tool for fast quantitative analysis. Both instruments exhibited very high sensitivity and repeatability in positive ionization mode. Coupling of micro-LC to Orbitrap technology was not applicable to the negatively ionizable compounds. The LC triple quadrupole MS method has proved to be stable in quantitation, as it is with respect to the matrix effects of grain samples.