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Translocation of metabolites between different plant species provides important hints in understanding the fate of bioactive root exudates. In the present study, targeted and untargeted mass spectrometry-based metabolomics was applied to elucidate the transfer of bioactive compounds between rye and several crops and weed species. Our results demonstrated that benzoxazinoids (BXs) synthesized by rye were taken up by roots of neighbouring plant species and translocated into their shoots. Furthermore, we showed that roots of rye plants took up compounds originating from neighbouring plants. Among the compounds taken up by rye roots, wogonin was detected in the rye shoot, which indicated a root-to-shoot translocation of this compound. Elucidating the transfer of bioactive compounds between plants is essential for understanding plant–plant interactions, developing natural pesticides and understanding their modes of action.

Plants have evolved advanced chemical defense mechanisms, including root exudation, which enable them to respond to changes occurring in their surroundings rapidly. Yet, it remains unresolved how root exudation affects belowground plant-plant interactions. The objective of this study was to elucidate the fate of benzoxazinoids (BXs) exuded from the roots of rye (Secale cereale L.) plants grown with hairy vetch (Vicia villosa). A rapid method that allows nondestructive and reproducible chemical profiling of the root exudates was developed. Targeted chemical analysis with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was performed to investigate the changes in the composition and concentration of BXs in the rye plant, and its root exudate in response to cocultivation with hairy vetch. Furthermore, hairy vetch plants were screened for the possible uptake of BXs from the rhizosphere and their translocation to the shoot. Rye significantly increased the production and root exudation of BXs, in particular 2-β-d-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-glc) and 2-β-d-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-glc), in response to cocultivation with hairy vetch. DIBOA-glc and DIMBOA-glc were absorbed by the roots of the cocultivated hairy vetch plants and translocated to the shoots. These findings will strongly improve our understanding of the exudation of BXs from the rye plant and their role in interaction with other plant species.

Introduction: Benzoxazinoids (BXs) are plant phytochemicals that have both defensive properties in plants and therapeutic effects in humans. The presence of BXs has been largely studied in the Poaceae family (monocots). To study the presence or absence of BXs in dicotyledons and monocotyledons outside the Poaceae family, parts of 24 plant species at several growth stages were selected for analysis, some of which were already known to contain BXs.Objectives: To devise a step-wise mass spectrometry-based approach for confirming the presence of BXs in plant samples, and to use the method to explore the status of BXs in selected plant species.Experimental: Plant samples were extracted using accelerated solvent extraction and analyzed using triple-quadrupole liquid chromatography-mass spectrometry.Results: The use of different columns, double mass transitions, and ion ratios proved to be a robust tool for confirming the presence of BXs in different plant species. By this method, the presence of BXs was confirmed in three of the 24 species. Double-hexose forms of BXs, which have not been reported before in dicotyledons, were confirmed to be present in the dicotyledon plants Acanthus mollis and Lamium galeobdolon, and the presence of BXs in the seeds of Consolida orientalis is reported for the first time here. High concentrations of BXs were found in the aerial parts of Acanthus mollis and Lamium galeobdolon, at 20 and 32 μmol/g plant dry weight, respectively.Conclusions: The stepwise approach described in this work confirmed the presence of BXs in new samples.

Rapid nucleic-acid based tests that can be performed by non-professionals outside laboratory settings could help the containment of the pandemic SARS-CoV-2 virus and may potentially prevent further widespread lockdowns. Here, we present a novel compact portable detection instrument (the Egoo Health System) for extraction-free detection of SARS-CoV-2 using isothermal Reverse Transcription Strand Invasion Based Amplification (RT-SIBA). The SARS-CoV-2 RT-SIBA assay can be performed directly on crude oropharyngeal swabs without nucleic acid extraction with a reaction time of 30 minutes. The Egoo health system uses a capsule system, which is automatically sealed tight in the Egoo instrument after applying the sample, resulting in a closed system optimal for molecular isothermal amplification. The performance of the Egoo Health System is comparable to the PCR instrument with an analytical sensitivity of 25 viral RNA copies per SARS-CoV-2 RT-SIBA reaction and a clinical sensitivity and specificity between 87.0-98.4% and 96.6-98.2% respectively.

Nepenthesins are categorized under the subfamily of the nepenthesin-like plant aspartic proteases (PAPs) that form a distinct group of atypical PAPs. This study describes the effect of nepenthesin 1 (HvNEP-1) protease from barley (Hordeum vulgare L.) on fungal histidine acid phosphatase (HAP) phytase activity. Signal peptide lacking HvNEP-1 was expressed in Pichia pastoris and biochemically characterized. Recombinant HvNEP-1 (rHvNEP-1) strongly inhibited the activity of Aspergillus and Fusarium phytases, which are enzymes that release inorganic phosphorous from phytic acid. Moreover, rHvNEP-1 suppressed in vitro fungal growth and strongly reduced the production of mycotoxin, 15-acetyldeoxynivalenol (15-ADON), from Fusarium graminearum. The quantitative PCR analysis of trichothecene biosynthesis genes (TRI) confirmed that rHvNEP-1 strongly repressed the expression of TRI4, TRI5, TRI6, and TRI12 in F. graminearum. The co-incubation of rHvNEP-1 with recombinant F. graminearum (rFgPHY1) and Fusarium culmorum (FcPHY1) phytases induced substantial degradation of both Fusarium phytases, indicating that HvNEP-1-mediated proteolysis of the fungal phytases contributes to the HvNEP-1-based suppression of Fusarium.

During the current COVID-19 pandemic, different methods have been used to evaluate patients suspected with infection of SARS-CoV-2. In this study, we evaluate the longevity of saliva and dry swab samples to retain SARS-CoV-2 for storage and transport at different environmental settings. Our results show that at ambient temperature of 20°C, SARS-CoV-2 RNA remains stable for up to 9 days giving a long span of time for transport and storage without compromising clinical results. Additionally, this study demonstrates that sali­­­va and dry swabs specimens can also be stored at -20°C and +4°C for up to 26 days without affecting RT-qPCR results. Our data is relevant for low-and middle-income countries, which have limited access to rapid refrigerated transport and storage of samples representing an economical alternative. Finally, our study demonstrates that dry swabs provide clear advantages over using transport medium.

Phytochemicals or phenolic compounds are important natural bioactive molecules that plants accumulate in response to environmental conditions and may exert benef icial effects on health by protecting humans against many diseases. The aim of this work was to analyze the influence of biotic and abiotic stress on the accumulation of flavonoids and phenolic acids on leaves of two cultivars of Amaranthus hypochondriacus, which are differentiated by the colour of their leaves (red or green). Phenolic compounds were extracted using the accelerated solvent extraction (ASE) method and their identif ication was carried out by LC-MS analysis. Rutin was the main flavonoid in amaranth leaves; the highest concentrations were found in green leaves when plants were subjected to stress (9715 μg g-1). Phenolic acids were minor compounds; ferulic acid was only present in red leaves (0.5 μg g-1) and p-coumaric acid only in green leaves (0.7 μg g-1). Our results indicate that leaves from A. hypochondriacus, the main species that produce edible seeds, are a good source of phytochemical compounds and their accumulation could be driven by the growing conditions.

Vaccines such as Vaccinia or BCG have non-specific effects conferring protection against other diseases than their target infection, which are likely partly mediated through induction of innate immune memory (trained immunity). MVA85A, a recombinant strain of modified Vaccinia Ankara (MVA), has been suggested as an alternative vaccine against tuberculosis, but its capacity to induce positive or negative non-specific immune effects has not been studied. This study assesses whether Vaccinia and MVA are able to induce trained innate immunity in monocytes. Human primary monocytes were primed in an in vitro model with Vaccinia or MVA for 1 day, after which the stimulus was washed off and the cells were rechallenged with unrelated microbial ligands after 1 week. Heterologous cytokine responses were assessed and the capacity of MVA to induce epigenetic changes at the level of cytokine genes was investigated using chromatin immunoprecipitation and pharmacological inhibitors. Monocytes trained with Vaccinia showed significantly increased IL-6 and TNF-α production to stimulation with non-related stimuli, compared to non-trained monocytes. In contrast, monocytes primed with MVA showed significant decreased heterologous IL-6 and TNF-α responses, an effect which was abrogated by the addition of a histone methyltransferase inhibitor. No effects on H3K4me3 were observed after priming with MVA. It can be thus concluded that Vaccinia induces trained immunity in vitro, whereas MVA induces innate immune tolerance. This suggests the induction of trained immunity as an immunological mechanism involved in the non-specific effects of Vaccinia vaccination and points to a possible explanation for the lack of effect of MVA85A against tuberculosis.

Glucosinolates are biologically active secondary metabolites in Brassicaceae plants that play a critical role in positive and negative interactions between plants and root-associated microbial communities. The aim of this study was to develop a reversed-phase liquid chromatography method to quantify and identify intact glucosinolates in the root of Arabidopsis thaliana (Arabidopsis) grown in non-sterile natural soil by using liquid chromatography-hybrid triple quadruple-linear ion trap (LC-QqQ(LIT)) mass spectrometry. The Synergi Fusion C18-based column was found to be effective for sufficient retention and separation of nine intact glucosinolates without the need for time-consuming desulfation or ion-pairing steps. Method validation results showed satisfactory inter-day and intra-day precision for all glucosinolates except for 4-hydroxyglucobrassicin. Good inter-day and intra-day accuracy and recovery results were observed for glucoiberin, gluconapin, glucobrassicin, 4-methoxyglucobrassicin and neoglucobrassicin. However, for 4-hydroxyglucobrassicin, glucoraphanin and glucoerucin corrections to quantification results might be necessary since the recovery and accuracy results were not optimal. Matrix effects were shown to have a negligible effect on the ionization of all target analytes. The established liquid chromatography&ndash, tandem mass spectrometry (LC-MS/MS) method was applied to quantify target intact glucosinolates in Arabidopsis root crude extract of four different wild-type accessions where differences in terms of concentration and composition of intact glucosinolates were observed. Employment of sensitive and selective precursor ion survey scan of m/z 97 in combination with the information-dependent acquisition (IDA) of the enhanced product ion (EPI) dependent scan (Prec97-IDA-EPI) using LC-QqQ(LIT) provided high confidence in structural characterization of diverse intact glucosinolate profiles in complex Arabidopsis root crude extract.

Honey bees are important pollinators and are subject to numerous stressors, such as changing floral resources, parasites, and agrochemical exposure. Pesticide exposure has been linked to the decline in the global honey bee population. We have limited knowledge of the metabolic pathways and synergistic effects of xenobiotics in bees. Quercetin is one of the most abundant phytochemicals in plants and is therefore abundant in the honey bee diet. Quercetin can upregulate the detoxification system in honey bees; however, it is still unknown to what extent quercetin ingestion can reduce the content of absorbed pesticides. In this study, we investigated the effect of dietary quercetin on the contents of three pesticides in honey bees: imidacloprid (insecticide), tebuconazole (fungicide), and tau-fluvalinate (insecticide and acaricide). Bees were divided into two main groups and fed either quercetin-sucrose paste or only sucrose for 72 h. Thereafter, they were orally exposed to ∼10 ng/bee imidacloprid or contact-exposed to ∼ 0.9 μg/bee tau-fluvalinate or ∼ 5.2 μg/bee tebuconazole. After 1 h of oral exposure or 24 h of contact exposure, the bees were anaesthetised with CO2, sacrificed by freezing, and extracted with a validated QuEChERS method. Subsequently, the concentrations of the three pesticides and quercetin in the bees were determined with a triple quadrupole tandem mass spectrometer coupled to an HPLC system. No significant effect on the concentration of tebuconazole or tau-fluvalinate was observed in bees fed quercetin. Intake of quercetin led to a reduction in the concentration of imidacloprid in honey bees. Quercetin-rich plants may be exploited in future beekeeping.

The honey bee pollen/nectar diet is rich in bioactive phytochemicals and recent studies have demonstrated the potential of phytochemicals to influence honey bee disease resistance. To unravel the role of dietary phytochemicals in honey bee health it is essential to understand phytochemical uptake, bioavailability, and metabolism but presently limited knowledge exists. With this study we aim to build a knowledge foundation. For 5 days, we continuously fed honey bees on eight individual phytochemicals and measured the concentrations in whole and dissected bees by HPLC-MS/MS. Ample phytochemical metabolization was observed, and only 6-30% of the consumed quantities were recovered. Clear differences in metabolization rates were evident, with atropine, aucubin, and triptolide displaying significantly slower metabolism. Phytochemical gut uptake was also demonstrated, and oral bioavailability was 4-31%, with the highest percentages observed for amygdalin, triptolide, and aucubin. We conclude that differences in the chemical properties and structure impact phytochemical uptake and metabolism.

Fusarium oxysporum is a destructive root-infecting plant pathogen that causes significant yield losses in many economically important crop species. Hence, a deeper understanding of pathogen infection strategies is needed. With liquid chromatography-tandem mass spectrometry and gas chromatography-time of flight mass spectrometry platforms, we analyzed the metabolic changes in a time-course experiment with Arabidopsis accessions either resistant (Col-0) or susceptible (Ler-0) to isolates of Fusarium oxysporum forma specialis matthioli infection. We showed a concurrent effect of Fusarium-derived polyols and the mycotoxin beauvericin in the suppression of the immune response of susceptible hosts. A significant increase in oxidized glutathione in the resistant host was probably associated with effective reactive oxygen species-mediated resistance responses. Through a combination of targeted and untargeted metabolomics, we demonstrated the concurrent action of several Arabidopsis defense systems as well as the concurrent action of several virulence systems in the fungal attack of susceptible Arabidopsis.

Honey bees regularly ingest quercetin, one of the most abundant phytochemicals in plants. Past research suggests that honey and pollen feeding upregulates the bees’ detoxification system and that dietary quercetin reduces the toxicity of imidacloprid and tau-fluvalinate [1,2].The aim of this study was to investigate the effect of dietary quercetin on the concentration of three pesticides present in honey bees. Honey bees (n = 600) were divided in five groups and fed either quercetin-sucrose paste or only sucrose for 72 h before being exposed to the neonicotinoid imidacloprid (oral exposure), the fungicide tebuconazole or the acaricide tau-fluvalinate (contact exposure). Bees were anesthetized with CO2, frozen, and extracted with a validated QuEChERS method. Finally, the concentrations of all three above-mentioned pesticides were determined by LC-MSMS. Quantification results of the three pesticides in honey bee samples [Fig. 1] displayed that there was a significant difference in concentration of imidacloprid (P = 2 ͯ 10-5) and tau-fluvalinate (P = 3 ͯ 10-2) between the bees that were fed quercetin-sucrose paste and bees that were fed only sucrose. However, there was no significant difference in the level of tebuconazole in the group of bees that were fed quercetin-sucrose paste and bees that were fed only sucrose (P = 4.2 ͯ 10-1). The results of this study demonstrate that honey bee intake of quercetin leads to a reduction in the concentration of imidacloprid and tau-fluvalinate by up regulating the detoxification systems in bees. Hence, quercetin may be a promising bioactive compound in chemo-ecological aspects to counteract the hazard of pesticides. Honey bees regularly ingest quercetin, one of the most abundant phytochemicals in plants. Past research suggests that honey and pollen feeding upregulates the bees’ detoxification system and that dietary quercetin reduces the toxicity of imidacloprid and tau-fluvalinate [1,2].The aim of this study was to investigate the effect of dietary quercetin on the concentration of three pesticides present in honey bees. Honey bees (n = 600) were divided in five groups and fed either quercetin-sucrose paste or only sucrose for 72 h before being exposed to the neonicotinoid imidacloprid (oral exposure), the fungicide tebuconazole or the acaricide tau-fluvalinate (contact exposure). Bees were anesthetized with CO2, frozen, and extracted with a validated QuEChERS method. Finally, the concentrations of all three above-mentioned pesticides were determined by LC-MSMS. Quantification results of the three pesticides in honey bee samples [Fig. 1] displayed that there was a significant difference in concentration of imidacloprid (P = 2 ͯ 10-5) and tau-fluvalinate (P = 3 ͯ 10-2) between the bees that were fed quercetin-sucrose paste and bees that were fed only sucrose. However, there was no significant difference in the level of tebuconazole in the group of bees that were fed quercetin-sucrose paste and bees that were fed only sucrose (P = 4.2 ͯ 10-1). The results of this study demonstrate that honey bee intake of quercetin leads to a reduction in the concentration of imidacloprid and tau-fluvalinate by up regulating the detoxification systems in bees. Hence, quercetin may be a promising bioactive compound in chemo-ecological aspects to counteract the hazard of pesticides.

The presence of bioactive benzoxazinoids (BXs) in mature whole grain and experimental bread products was recently shown. The aim of this study was to reveal the level of BXs in a broad range of commercial cereal products and discuss these findings in relation to ingredients and preparation processes. The concentrations of 11 BX compounds were determined by liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS) in 25 supermarket-available cereal food products. The total BX content varied from below the limit of detection to >500 μg/g dry matter (DM) with some degree of variation among different lots. The products W (Rugsandwich), H (Gulerodsrugbrod), L (Morke Multikerneboller) and AA (Solsikkeklapper), which were all rich in rye-based ingredients, had a total BX concentration >300 μg/g DM. Double hexose derivatives of 2-hydroxy-1,4-benzoxazin-3-one (HBOA), 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and 2-β- d -glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-glc) were the major BXs in most products. Further studies into the health effects of BXs should be conducted, and future exploitation of these compounds seems possible.

OBJECTIVES: Prostate cancer is a common cancer diagnosis among men, but the role of diet and food components remains controversial. Benzoxazinoids (BXs) is a potent group of bioactive compounds found in rye foods that seem to have the potential to inhibit prostate cancer growth in vitro but studies in humans are lacking. We therefore aimed to investigate the concentrations of different BXs and their metabolites after whole grain rye consumption in comparison to refined wheat and to correlate these compounds to PSA levels in men with indolent prostate cancer. METHODS: In a randomized controlled crossover study, men with prostate cancer (n = 24) consumed 485 gram of whole grain/bran rye and refined wheat foods for six weeks, with two weeks of washout in-between. The diets were isocaloric and contained the same amount of fiber by addition of cellulose in the refined wheat intervention. BXs in plasma samples were analyzed with HPLC- QTRAP/MS-ESI. RESULTS: Seventeen men finished the trial and were included in data analysis. For most BXs, the concentrations were significantly higher after the whole grain rye compared to the refined wheat intervention. The BXs or BXs metabolites HBOA-glc, HHPAA, HBOA-glcA, HPAA-glcA (∼19.4–0.4 nmol/L in plasma after interventions) were significantly inversely correlated to PSA level. CONCLUSIONS: In men with indolent prostate cancer, most BXs were significantly higher after an intervention with whole grain rye compared to refined wheat. The inverse correlation between four BXs or BXs metabolites and PSA may indicate on an inhibitory effect on prostate cancer cell growth. This could open for new preventive food-based strategies, but results would need to be replicated. FUNDING SOURCES: Independent Research Fund Denmark - Technology and Production Formas Swedish Research Council.

Pesticide metabolite identification is gaining increased attention because of the interest in potential metabolite toxicity. Azoxystrobin is one of the most prevalent pesticide residues in foods in Europe. The majority of azoxystrobin metabolites have been identified using radiolabelled standards, which are either expensive or not readily available. Thus, alternative approaches for metabolite identification are desirable. Here, an LC-MS/MS method for quantifying azoxystrobin and identifying its metabolites using quadrupole-linear ion trap mass spectrometry is reported. Seven metabolites of azoxystrobin were identified 2 and 4 weeks after spraying lettuce with azoxystrobin. Among them, two metabolites are reported for the first time. The hydrolysis, reduction, hydroxylation, photoisomerisation and hydrolytic cleavage of ether bonds are identified as biotransformation processes involved in azoxystrobin metabolism in lettuce.

Benzoxanoids (BXs) were identified for the first time in young cereal plants in 1957 and in medicinal plants in 1969. In 2008 our lab discovered that BXs are present in mature cereal grains and in food/feed based on cereal whole grains [1]. We showed that BXs are taken up in pigs, rats and humans [2]–[4], and that they appear in prostate tissue of humans consuming rye bread [5]. The aim of this study was to develop a method for identification and quantification of human phase 1 and phase 2 metabolites in plasma. The method included an initial protein precipitation step of the plasma with the purpose of reducing the matrix interference. The chromatographic analysis of BXs and their metabolites in the remaining extract was performed in LC-MS/MS. Pure standards were unavailable for most of the phase 2 metabolites. Phase 2 metabolites are present in much higher amounts in urine than in plasma. Thus urine metabolites of BXs for which structures could be assigned using data dependent acquisition were isolated and used for verification of MS/MS signals in plasma extracts. The isolation of approx 10 µg of each compound was done with consecutive HLB and mixed mode SPE’s (Oasis, Waters) exploiting the variety of polarity and acidity of the metabolites. Glucuronide and sulphate metabolites of paracetamol were used as standards for semi-quantification due to their structural similarity and commercial availability. Benzoxanoids (BXs) were identified for the first time in young cereal plants in 1957 and in medicinal plants in 1969. In 2008 our lab discovered that BXs are present in mature cereal grains and in food/feed based on cereal whole grains [1]. We showed that BXs are taken up in pigs, rats and humans [2]–[4], and that they appear in prostate tissue of humans consuming rye bread [5]. The aim of this study was to develop a method for identification and quantification of human phase 1 and phase 2 metabolites in plasma. The method included an initial protein precipitation step of the plasma with the purpose of reducing the matrix interference. The chromatographic analysis of BXs and their metabolites in the remaining extract was performed in LC-MS/MS. Pure standards were unavailable for most of the phase 2 metabolites. Phase 2 metabolites are present in much higher amounts in urine than in plasma. Thus urine metabolites of BXs for which structures could be assigned using data dependent acquisition were isolated and used for verification of MS/MS signals in plasma extracts. The isolation of approx 10 µg of each compound was done with consecutive HLB and mixed mode SPE’s (Oasis, Waters) exploiting the variety of polarity and acidity of the metabolites. Glucuronide and sulphate metabolites of paracetamol were used as standards for semi-quantification due to their structural similarity and commercial availability.

Xenobiotic detoxification in plant as well as in animals has mostly been studied in relationship to the deactivation of the toxic residues of the compound that, surely for azoxystrobin, is represented by its &beta, methoxyacrylate portion. In maize roots treated for 96 h with azoxystrobin, the fungicide accumulated over time and detoxification compounds or conjugates appeared timewise. The main detoxified compound was the methyl ester hydrolysis product (azoxystrobin free acid, 390.14 m/z) thought to be inactive followed by the glutathione conjugated compounds identified as glutathione conjugate (711.21 m/z) and its derivative lacking the glycine residue from the GSH (654.19 m/z). The glycosylated form of azoxystrobin was also found (552.19 m/z) in a minor amount. The identification of these analytes was done by differential untargeted metabolomics analysis using Progenesis QI for label free spectral counting quantification and MS/MS confirmation of the compounds was carried out by either Data Independent Acquisition (DIA) and Data Dependent Acquisition (DDA) using high resolution LC-MS methods. Neutral loss scanning and comparison with MS/MS spectra of azoxystrobin by DDA and MSe confirmed the structures of these new azoxystrobin GSH conjugates.

Fusarium head blight (FHB) is a devastating disease of wheat (Triticum aestivum L.) caused by a mycotoxigenic fungus Fusarium graminearum resulting in significantly decreased yields and accumulation of toxic trichothecenes in grains. We tested 7 major secondary metabolites from wheat for their effect on trichothecene production in liquid cultures of F. graminearum producing trichothecene 15-acetyldeoxynivalenol (15-ADON). 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) benzoxazinoid completely abolished toxin production without any apparent effect on fungal growth. DIMBOA strongly affected the expression of Tri6, encoding a major transcriptional regulator of several genes of the trichothecene biosynthesis pathway. DIMBOA also repressed expression of Tri5, encoding trichodiene synthase, the first enzyme in the trichothecene biosynthesis pathway. Thus, DIMBOA could play an important role against the accumulation of trichothecenes in wheat grain. Breeding or engineering of wheat with increased levels of benzoxazinoids could provide varieties with increased resistance against trichothecene contamination of grain and lower susceptibility to FHB.

Fusarium head blight (FHB) causes substantial losses of yield and quality in grains, both in the field and in post-harvest storage. To date, adequate natural genetic resistance is not available for the control of FHB. This study reports the cloning and overexpression of a barley (Hordeum vulgare L.) antifungal gene, nepenthesin 1 (HvNEP-1), in the endosperm of barley grains. Transgenic barley lines overexpressing HvNEP-1 substantially reduced FHB severity and disease progression after inoculation with Fusarium graminearum or Fusarium culmorum. The transgenic barley also showed reduced accumulation of the mycotoxin deoxynivalenol (DON) in grain, far below the minimum value allowable for food. Semi-field evaluation of four HvNEP-1 transgenic lines revealed substantial reduction of FHB severity and progression as compared with the control H. vulgare cultivar Golden promise (GP) plants. Our study demonstrated the utility of HvNEP-1 for the control of FHB in barley, and possibly other grains such as wheat and maize.

Disease progression of human immunodeficiency virus type 1 (HIV-1) is delayed by HIV type 2 (HIV-2) in individuals with dual HIV-1/HIV-2 infection. The protective mechanisms, however, are still to be revealed. In the current study we examined type-specific and cross-reactive antibody-dependent cellular cytotoxicity (ADCC) in HIV-1 and HIV-2 monoinfection or dual infection. Of note, intertype cross-reactive antibodies that mediated HIV-1 envelope glycoprotein (Env)–targeted ADCC were frequently identified in HIV-2–infected individuals. Furthermore, the magnitude of HIV-1 cross-reactive ADCC activity during HIV-2 infections depended on the HIV-1 Env origin and was associated with the duration of infection. These results suggest that preexisting antibodies against HIV-2, which mediate intertype ADCC, might contribute to control of HIV-1 during dual infection., Antibody-dependent cellular cytotoxicity (ADCC) was analyzed in human immunodeficiency virus (HIV) 1 and 2 infections. Cross-reactive antibodies that mediated HIV-1 envelope glycoprotein–targeted ADCC were identified in HIV-2–infected individuals, suggesting that preexisting HIV-2 antibodies might help control HIV-1 during dual infection.

Secondary metabolites secreted by microbes and plants act as mediators in plant-microbe interactions including nutrient uptake. However, until now very little is known about their role in nutrient assimilation, particularly amino acids, which are important compounds due to their high N content. Here we show that the addition of flavonoid secondary metabolites, derived from clover, to soil changed the bacterial diversity, enhanced the flux of asparagine, and increased the pools of glutamine/glutamate in the soil. This indicates that flavonoids are functionally important qualitative and quantitative components of clover root exudates. Furthermore, the addition of microbial secondary metabolites negatively affected clover uptake of asparagine and plant performance, which demonstrates that microbial competition for nutrients may have multiple physiological targets in the plant. Finally, the detection of intact asparagine in clover roots confirms that amino acid uptake is significant to the plant in agricultural soil. In conclusion, amino-acid flow in the clover rhizosphere can be modified by the effects of clover-derived flavonoids on the bacterial community structure, which affects the flux and pools of amino acids; microbial secondary metabolites, which reduce clover uptake of asparagine; and direct recapture of amino acids by clover.

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/ binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Macrophages play important roles during human immunodeficiency virus (HIV) infection, reflected by changes in macrophage-activation biomarker soluble CD163 (sCD163). Here, we present data on the novel macrophage-activation biomarker soluble mannose receptor/CD206 (sCD206) in HIV infection. We investigated sCD206 blood levels at baseline and follow-up with/without antiretroviral therapy (ART), in 212 patients with HIV type 1 (HIV-1), HIV type 2 (HIV-2), or dual infection. At baseline, there was no difference in sCD206 level between HIV types, and sCD206 was unchanged at follow-up without ART. However, in contrast to sCD163, sCD206 levels decreased significantly for both HIV-1 and HIV-2, but not for HIV-1/2 patients, during ART. Further investigations are needed to establish sCD206 as a biomarker in HIV infection.

Hairy roots induced by Agrobacterium rhizogenes are well established models to study the metabolism of xenobiotics in plants for phytoremediation purposes. However, the model requires special skills and resources for growing and is a time-consuming process. The roots induction process alters the genetic construct of a plant and is known to express genes that are normally absent from the non-transgenic plants. In this study, we propose and establish a non-transgenic maize root model to study xenobiotic metabolism in plants for phytoremediation purpose using azoxystrobin as a xenobiotic compound. Maize roots were grown aseptically in Murashige and Skoog medium for two weeks and were incubated in 100 μM azoxystrobin solution. Azoxystrobin was taken up by the roots to the highest concentration within 15 min of treatment and its phase I metabolites were also detected at the same time. Conjugated metabolites of azoxystrobin were detected and their identities were confirmed by enzymatic and mass spectrometric methods. Further, azoxystrobin metabolites identified in maize root culture were compared against azoxystrobin metabolites in azoxystrobin sprayed lettuce grown in green house. A very close similarity between metabolites identified in maize root culture and lettuce plant was obtained. The results from this study establish that non-transgenic maize roots can be used for xenobiotic metabolism studies instead of genetically transformed hairy roots due to the ease of growing and handling.

Benzoxazinoids (BXs) are a group of wholegrain phytochemicals with potential pharmacological properties; however, limited information exists on their absorption, metabolism, and excretion in humans. The aim of this study was to investigate the dose-dependent uptake and excretion of dietary BXs in a healthy population. Blood and urine were collected from 19 healthy participants from a crossover study after a washout, a LOW BX diet or HIGH BX diet, and analysed for 12 BXs and 4 phenoxazinone derivatives. We found that the plasma BX level peaked approximately 3 h after food intake, whereas BXs in urine were present even at 36 h after consuming a meal. No phenoxazinone derivatives could be detected in either plasma or urine. The dominant BX metabolite in both plasma and urine was 2-β-d-glucopyranosyloxy-1,4-benzoxazin-3-one (HBOA-Glc), even though 2-β-d-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one (DIBOA-Glc) was the major component in the diet. The dietary BX treatment correlated well with the plasma and urine levels, illustrating strong dose-dependent BX absorption, which also had a rapid washout, especially from the plasma compartment.

SCOPE: To examine potentially immunomodulating effects of dietary benzoxazinoids (BXs), present in cereal grains.METHODS AND RESULTS: Nineteen healthy volunteers were randomly distributed into two groups, who received diets with high or low content of BXs for three weeks. After a week's wash-out, the groups switched diets. Peripheral blood mononuclear cells (PBMCs) were stimulated with Porphyromonas gingivalis, Escherichia coli lipopolysaccharide (LPS), or tetanus toxoid (TT). PBMCs from a healthy donor received the same stimuli in presence of serum from each participant receiving BXs. The production of monokines, T-cell cytokines and T-helper cell proliferation were assessed. A three-week diet with high BX content enhanced IL-1β responses against LPS and P. gingivalis, as well as TNF-α response against P. gingivalis, after 24 hours of stimulation. Moreover, IL-6 was found to be increased after 7 days of stimulation with LPS. No effect was observed on T-cell cytokines or proliferation. BX levels in serum after a single meal did not modify cytokine responses.CONCLUSIONS: High dietary intake of BXs enhances bacteria-induced production of pro-inflammatory monokines by PBMCs, but not T-cell responses; presumably due to intrinsic changes within PBMCs, built up over three weeks of BX-rich diet, rather than to immediate effects of BXs contained in serum. This article is protected by copyright. All rights reserved.

Weaver ants (genus Oecophylla) are tropical carnivorous ant species living in high numbers in the canopies of trees. The ants excrete copious amounts of fecal matter on leaf surfaces, and these feces may provide nutrients to host trees. This hypothesis is supported by studies of ant-plant interactions involving other ant species that have demonstrated the transfer of nutrients from ants to plants. In this 7-months study, a GC–MS-based metabolomics approach along with an analysis of total nitrogen and carbon levels was used to study metabolic changes in ant-hosting Coffea arabica plants compared with control plants. The results showed elevated levels of total nitrogen, amino acids, fatty acids, caffeine, and secondary metabolites of the phenylpropanoid pathway in leaves from ant-hosting plants. Minor effects were observed for sugars, whereas little or no effect was observed for organic acids, despite the fact that lower levels of total carbon were found in ant-hosting plants. The increased levels of total nitrogen, amino acids, fatty acids and caffeine and the decreased total carbon were consistent with changes observed in plants grown with an increased supply of nitrogen-containing nutrients. The up-regulation of the phenylpropanoid pathway could indicate biotic stress and/or nutrient deficiency. However, because nutrient deficiency was contradicted by the remaining results, this pathway up-regulation was ascribed to biotic stress caused by the physical presence of the weaver ants.

Lettuce is an important part of the diet in Europe. The permitted levels of pesticides in lettuce are strictly regulated and there is growing urge among food safety authorities to analyse pesticide metabolites as well. Azoxystrobin is one of pesticides that is frequently detected in lettuce. Although there are several analytical methods for the determination of azoxystrobin in lettuce, a sensitive method for the determination of its metabolites in lettuce is lacking. This study aimed at developing an extraction and LC-MS/MS method for the simultaneous determination of azoxystrobin, and its metabolites azoxystrobin free acid and 2-hydroxybenzonitrile in lettuce. Accelerated solvent extraction, QuEChERS extraction, and shaking extraction were compared using various solvents. The final method consisted of shaking freeze-dried sample in 0.1% formic acid in 80% aqueous acetonitrile. The selected method was validated by spiking each analyte at 125 ng/g and 500 ng/g. The method resulted in acceptable recovery for 2-hydroxybenzonitrile, azoxystrobin free acid, and azoxystrobin, with a RSD of

Benzoxazinoids are secondary metabolites with plant defense properties and possible health-promoting effects in humans. In this study, the transcriptional activity of ScBx genes (ScBx1-ScBx5; ScBx6-like), involved in benzoxazinoid biosynthesis, was analyzed during germination and early seedling development in rye. Our results showed that ScBx genes had highest levels of expression at 24-30 h after germination, followed by a decrease at later stages. For ScBx1-ScBx5 genes expression was higher in shoots compared with root tissues and vice versa for ScBx6-like gene transcripts. Moreover, methylated forms of benzoxazinoids accumulated in roots rather than in shoots during seedling development, in particular reaching high levels of HMBOA-glc in roots. Chemical profiles of benzoxazinoid accumulation in the developing seedling reflected the combined effects of de novo biosynthesis of the compounds as well as the turnover of compounds either pre-stored in the embryo or de novo biosynthesized. Bioinformatic analysis, together with the differential distribution of ScBx6-like transcripts in root and shoot tissues, suggested the presence of a ZmBx6 homolog encoding a 2-oxoglutarate dependent dehydrogenase in rye. The ScBx6-like cDNA was expressed in E. coli for functional characterization in vitro. LC-MS/MS analysis showed that the purified enzyme was responsible for the oxidation of DIBOA-glc into TRIBOA-glc, strongly suggesting the ScBX6-like enzyme in rye to be a functional ortholog of maize ZmBX6.

Benzoxazinoids are highly studied compounds due to their biological activity and presence in several cereals. They include compound classes such as hydroxamic acids and lactams and usually occur as inactive glucosides in unstressed plants. Injury to the plant causes enzymatic hydrolysis of the inactive glucosides to the biologically active hydroxamic acid and lactam aglucones. The hydroxamic acids further undergo spontaneous hydrolysis to benzoxazolinones in aqueous solution. Extraction methods that do not cause immediate inactivation of enzymes result in accumulation of aglucones in samples. Using HPLC-MS to profile benzoxazinoids in maize seedlings subjected to several sample preparation techniques, we have found that hydroxamic acid aglucones and benzoxazolinones are present in uninjured maize seedlings, but that the benxozazinoid profile varies depending on sample treatment, potentially underrepresenting the glucoside content and overrepresenting the aglucone and benzoxazolinone content.

Our knowledge of the binding sites for neutralizing Abs (NAb) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). In this article, we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and simian immunodeficiency virus (SIV) Envs. Heterologous NAb titers, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of Ab-binding reactivity revealed preferential recognition of the C1, C2, V2, V3, and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1.

Background Adductor canal blockade (ACB) has been demonstrated to be effective in the treatment of post-operative pain after major knee surgery. We hypothesised that the ACB would reduce pain and analgesic requirements after minor arthroscopic knee surgery. Methods Seventy-two patients scheduled for minor knee surgery were enrolled in this placebo-controlled, blinded trial. The patients were randomised to receive an ACB with either 30 ml ropivacaine 7.5 mg/ml (n = 36) or saline (n = 35) in addition to a basic analgesic regimen with paracetamol and ibuprofen. Primary outcome measure was pain during standing at 2 h after surgery. Secondary outcomes were pain at rest, while standing and after a 5-m walk; opioid consumption and opioid-related side effects 0–24 h after surgery. Results Pain scores {median [interquartile range (IQR)]}, regarding primary outcome were 15 (0–26) mm in the ropivacaine vs. 17 (5–28) mm in the control group, 95% confidence interval (CI) (−10 to 4) mm, P = 0.41. Ketobemidone consumption 0–2 h post-operatively [median (IQR)] was lower in the ropivacaine vs. the control group: 0.0 (0.0–2.5) mg vs. 2.5 (0.0–5.0) mg, 95% CI: −2.5 to 0 mg, P = 0.01. No differences were observed for any other outcome. Conclusion No significant analgesic effect of the ACB could be detected after minor arthroscopic knee surgery with a basic analgesic regimen with acetaminophen and ibuprofen, except from a minor reduction in immediate requirements for supplemental opioids. Clinicaltrials.gov Identifier: NCT01254825.

Benzoxazinoids are important phytochemicals found in wheat and rye that are associated with plant resistance against pathogens, and recent studies have emphasized the potential health-promoting role of these compounds i.e. anti-cancer, anti-allergy and anti-inflammation. Accordingly, an understanding of their distribution in seeds and the effect of different processing techniques on their transformation will be helpful in identifying the mechanisms of their production and distribution and will support the on-going efforts to utilize these compounds in health-promoting food products.The analysis of seed fractions obtained from the milling of wheat and rye showed significantly higher concentrations of these bioactive compounds in the germ than in the other fractions, i.e. the bran and endosperm. Di-hexoses of 2,4-dihydroxy-1, 4-benzoxazin-3-one (DIBOA-glc-hexose) and 2-hydroxy-1, 4-benzoxazin-3-one (HBOA-glc-hexose) were the predominant compounds found in the different wheat and rye seed fractions followed by DIBOA-glc and DIBOA.The soaking and boiling of three rye-based breakfast cereals resulted in considerable changes in the benzoxazinoid contents. The soaking of pearled rye promoted the conversion of DIBOA-glc-hexose into DIBOA-glc. When these cereals were boiled, the increase in the DIBOA-glc content was much lower than that observed for soaking. For rye flakes, the pattern of these benzoxazinoids was different from that in pearled rye seeds. A considerable amount of the benzoxazinoids was also leached into the water during soaking or boiling. This study contributes to the understanding of the underlying processes involved in the biochemical changes of benzoxazinoids and will be the basis for future studies on other food-processing techniques.

In line of the development of a food composition database for Bangladesh, 10 minor indigenous fruits were analysed for their nutrient composition comprising ascorbic acid, carotenoids and mineral values. Nutrient data obtained have been compared with published data reported in different literatures, book and United States Department of Agriculture-National Nutrient Database for Standard Reference. Ascorbic acid was highest in Wood apple and lowest in Roselle. Monkey jack contained the highest amount of carotenoids, zinc and copper. Content of calcium, magnesium and phosphorous were found highest in Antidesma velutinum. Potassium was the highest in Wood apple followed by in Moneky jack. It was noted that most of the minor fruits have much higher amount of ascorbic acid than the national fruit – Jack fruit ripe, the king fruit – Mango ripe of Bangladesh and exotic fruits – Apple and Grapes. The nutrient values of these minor fruits would make awareness among the people for their mass consumption for healthy life and to grow more minor fruit trees from extinction in order to maintain biodiversity.

Background We have previously reported patch test reactivity to nickel sulphate in a cohort of unselected infants tested repeatedly at 3–18 months of age. A reproducible positive reaction at 12 and 18 months was selected as a sign of nickel sensitivity provided a patch test with an empty Finn chamber was negative. A reproducible positive reaction was seen in 8.6% of the infants. The objective of this study is to follow-up on infants with alleged nickel sensitivity. Methods A total of 562 infants were included in the cohort and patch tested with nickel sulphate (ICDRG guidelines). The 26 children with a positive patch test reaction to nickel sulphate at 12 and 18 months were offered repeated patch tests at 3 and 6 yr. Results Among the 21 children tested at both 12 months, 18 months and at 3 and 6 yr only 2 of 21 had reproducible nickel reactions (one clinically relevant), 13 of 21 were negative and 6 of 21 were negative at 3 or 6 yr. Conclusions Only 9.5% of the children had reproducible nickel sulphate reactivity, while 62% became negative. The results are noteworthy and can be interpreted in different ways: Repeated nickel patch tests did not cause patch test sensitization. The test reactions in infancy are probably of irritant or non-specific nature. Hence, nickel patch tests should only be performed in small children if there is a clinical suspicion of nickel-induced allergic contact dermatitis.

This study was conducted to answer the question of whether clover can absorb asparagine in the presence and absence of inorganic nitrogen, as well as to determine the resulting concentration of post-uptake compounds closely involved in asparagine metabolism. Clover was grown at two asparagine concentrations (10 μM and 1 mM) supplied in both the absence and presence of ammonium nitrate. Using dual-labeled 13C15N-asparagine, the uptake rate was analyzed via bulk 15N and 13C excess and the detection of intact 13C15N-asparagine in white clover. The results from the two methods indicated greater utilization of 13C15N-asparagine in the 10 μM treatment than in the 1 mM treatment. The 13C15N-asparagine uptake rate was higher when 13C15N-asparagine was provided alone than when it was supplemented with inorganic nitrogen. Up to nine times lower uptake rates were obtained when intact 13C15N-asparagine was measured than when bulk 15N and 13C excess were analyzed. The labeled amino acids that are closely related to 13C15N-asparagine metabolism (aspartic acid, glutamic acid and glutamine) were detected in clover roots and shoots. Using two different methods, white clover’s potential to absorb intact asparagine, even in the presence of inorganic nitrogen, was confirmed. The dual-methodology approach employed in this study demonstrates how the post-uptake metabolism can affect quantification of amino acid uptake.

Estimating the potential for direct plant acquisition of organic N, in particular amino acids, requires assessment of their turnover times in soil. It is well known from 14C studies that mineralization of amino acids occurs within hours, but mineralization to 14CO2 does not indicate the rate of disappearance of the intact amino acid or the possible formation of metabolites during amino acid dissipation. We here used compound-specific isotope analysis with metabolite tracking to investigate the dissipation rate of universally labeled intact 13C15N-asparagine at two concentrations and the subsequent formation of compounds closely related to 13C15N-asparagine metabolism over 3 h in agricultural soil. 13C15N-asparagine was rapidly dissipated with half-lives of 55 and 231 min for the low and high 13C15N-asparagine concentrations used. Universally labeled aspartic acid and interestingly also universally labeled glutamic acid were detected in soil. This highlights the fast turnover of amino acid in soil and that the estimation of concentration of the formed compounds is important when evaluating plant available organic N. Efficiency of the compound-specific analysis showed to be a powerful technique in following the pathway of amino acids in soil.

Fusarium infection in wheat causes Fusarium head blight, resulting in yield losses and contamination of grains with trichothecenes. Some plant secondary metabolites inhibit accumulation of trichothecenes. Eighteen Fusarium infected wheat cultivars were harvested at five time points and analyzed for the trichothecene deoxynivalenol (DON) and 38 wheat secondary metabolites (benzoxazinoids, phenolic acids, carotenoids, and flavonoids). Multivariate analysis showed that harvest time strongly impacted the content of secondary metabolites, more distinctly for winter wheat than spring wheat. The benzoxazinoid 2-β-glucopyranoside-2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-glc), α-tocopherol, and the flavonoids homoorientin and orientin were identified as potential inhibitors of DON accumulation. Several phenolic acids, lutein and β-carotene also affected DON accumulation, but the effect varied for the two wheat types. The results could form a basis for choosing wheat cultivars using metabolite profiling as a marker for selecting wheat cultivars with improved resistance against Fusarium head blight and accumulation of trichothecene toxins in wheat heads.

Certain isoflavones are plant growth inhibitors, and biochanin A is a major isoflavone in clover species used for weed management. The effect of biochanin A on the monocot weed species Echinochloa crus-galli L. and Lolium perenne L. and dicot species Silene noctiflora L., Geranium molle L., and Amaranthus caudatus L. was evaluated in agar medium bioassays. S. noctiflora and G. molle root growth was progressively inhibited with increasing concentrations of biochanin A, whereas the monocot species were unaffected. With regard to the dicot species, S. noctiflora (EC(50) = 35.80 μM and EC(25) = 5.20 μM) was more susceptible than G. molle (EC(50), EC(25)400 μM). S. noctiflora, G. molle, and E. crus-galli root and shoot samples, representing a susceptible, a less susceptible, and a nonsusceptible species, respectively, were analyzed by LC-MS to quantify biochanin A and its transformation products. Biochanin A and its known transformation products genistein, dihydrobiochanin A, pratensein, and p-coumaric acid were quantified. Sissotrin was identified and quantified while assigning unknown peaks. The treated root samples contained more biochanin A, genistein, pratensein, and dihydrobiochanin A than shoot samples.

In a cultivation experiment 18 different Amaranthus genotypes were cultivated in parallel in Argentina, Mexico, Spain and two different locations in the Czech Republic. The ripe seeds were analysed for their content of 11 polyphenols and the variations among genotype, species and location were analysed by principal component analysis (PCA). The flavonoid, rutin, exhibited large variations with varying environmental conditions whereas the flavonoid, nicotiflorin, was affected less. Amaranthus hypochondriacus displayed the most stable content of polyphenols with a high end content of flavonoids. The variations between location/environmental condition were primarily described by the variations in the content of p-coumaric acid and protocatechuic acid in the seed samples.

Bread is consumed in large quantities all over the world, and rye bread is especially popular throughout the Nordic countries. Wholemeal bread is highly recommended as a basic ingredient in daily food, because wholemeal food products generally promote good health due to their vitamin, mineral and fibre content. The literature suggests that wholemeal products have other health-promoting effects even if the ingredients responsible have not been identified. Benzoxazinoids are a group of natural products that have not previously been reported in mature grains. Here, we report for the first time the identity and quantity of 10 compounds of the benzoxazinoid family in mature grains, hydrothermally processed grains of durum wheat ( Triticum durum , cv. Kamut), a commercial variety of rye ( Secale cereale cv. Picasso) and an old Nordic rye landrace ( S. cereale , Svedjerug), as well as in bread baked with flour milled from those grains. Concentrations of the 10 benzoxanoids were determined using LC–MS/MS and ranged from 0 to 348 nmol g −1 for conventional flour, to 772–1177 nmol g −1 in bread baked with flour from hydrothermally processed grains and to 3116–5570 nmol g −1 in flour from hydrothermally processed grains. Benzoxazinoids possess documented physiological effects, and research into the importance of these compounds in the daily diet is therefore needed. Ongoing studies in our lab on the uptake and transformation of benzoxazinoids in mammals will be reported in the near future.

The proximate composition, phenolic acid, and flavonoid characterization of two commercial and eight wild Opuntia spp., as well two processed products, were analyzed. Commercial varieties were obtained from a local market; wild varieties were collected from the Central and High Plateau zones of Mexico, and Opuntia tablets were purchased at a natural commodities store. Spines from fresh cladodes were removed and clean cladodes were freeze-dried and milled to obtain nopal powder. Differences in proximate composition were determined: wild nopal blanco collected from the High Plateau had the highest protein content, while tapon-II had the highest fiber content. Nopal tablets had low protein and carbohydrate content but had the highest ash content. The wild morado, tempranillo, blanco, and cristalino varieties had the highest total phenolic acid content, while the commercial varieties had the highest total flavonoid contents. Very low amounts of flavonoids were found in nopal tablets. LC-MS/MS was used to identify six phenolic acids (gallic, coumaric, 3,4-dihydroxybenzoic, 4-hydroxybenzoic, ferulic, and salicylic acid), and only tapon-II contained all six. The five flavonoids (iso-quercitrin, isorhamnetin-3-O-glucoside, nicotiflorin, rutin, and narcissin) were found in all varieties; nicotiflorin was predominant, followed by narcissin.

The demand for food is increasing, not only to meet food security for growing populations, but also to provide more nutritious food, rich in good quality proteins and nutraceutical compounds. The amaranth ( Amaranthus hypochondriacus ) plant, in addition to its high nutritive and nutraceutical characteristics, has excellent agronomic features. The objective of the present study was to analyze some physical and proximal-nutritional properties of amaranth seeds obtained from different varieties grown in arid zones and characterize their phenolic acids and flavonoids. Two commercial (Tulyehualco and Nutrisol) and two new (DGETA and Gabriela) varieties of A. hypochondriacus were grown at the Mexican Highlands zone. Tulyehualco and DGETA varieties had higher seed yield of 1475 and 1422 kg ha −1 , respectively, comparable to corn and soybean production in agricultural areas. Gabriela had the highest protein content of 17.3%, but all varieties had an adequate balance of essential amino acids. Polyphenols as rutin (4.0–10.2 μg g −1 flour) and nicotiflorin (7.2–4.8 μg g −1 flour) were detected. Amaranth can be cultivated in arid zones where commercial crops cannot be grown; the seeds besides their well known nutritive characteristics could be a source of phenolic compounds of high antioxidant properties.

This study evaluates the applicability of the liquid handling workstation Tecan Evo Freedom 200 and 200 μL Media Scout RoboColumns to dynamic chromatographic operations such as frontal analysis (breakthrough) and elution experiments in a high throughput screening mode. Breakthrough experiments were conducted using BSA as a model protein and the stationary phases Poros 50 D, Q Ceramic HyperD and DEAE Sepharose FF. The obtained dynamic capacities at 10 and 50 % breakthrough matched well with reference data. Elution experiments were performed applying three protein mixtures: a) lipolase and BSA, b) human growth hormone and a process-related impurity, and c) an insulin analogue and a process-related impurity. The resins used resembled a variety of different ion exchange resins. In all cases, the resulting elution curves matched well with reference measurements performed on an AKTA explorer system at 1 mL or 2 mL scale.

Fusarium Head Blight (FHB) is a destructive disease that affects the grain yield and quality of cereals. The relationship between the natural defense chemicals benzoxazinoids and the FHB resistance of field grown winter wheat varieties was investigated. FHB resistance was assessed by the inoculation of wheat ears with mixtures of Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, and Microdochium nivale. The benzoxazinoids detected in the highest concentration were 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (3.7–9.4 μmol/kg DW) and 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HMBOA, 2.0–11 μmol/kg DW). The cultivars most susceptible to FHB were cvs. Hanseat, Asketis, and Ritmo, while cvs. Petrus, Terra, and Hattrick showed high resistance. 2-O-β- d -Glucopyranosyloxy-4,7-dimethoxy-(2H)-1,4-benzoxazin-3(4H)-one (HDMBOA-glc) and 2-O-β- d -glucopyranosyloxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HMBOA-glc) were detected. HMBOA-glc was found in higher concentrations than 2-O-β- d -glucopyranosyloxy-2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA-glc). Principal component analyses demonstrated correlation between the susceptibility to FHB and the concentrations of DIMBOA-glc, HMBOA-glc, HMBOA, 2-O-β- d -glucopyranosyloxy-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA-glc), 2-O-β- d -glucopyranosyloxy-1,4-benzoxazin-3(4H)-one, and 2-O-β- d -glucopyranosyloxy-4-dihydroxy-(2H)-7,8-dimethoxy-1,4-benzoxazin-3(4H)-one (DIM2BOA-glc).

Wheat (Triticum aestivum L.) and other cereals produce allelochemicals as natural defense compounds against weeds, fungi, insects and soil-borne diseases. The main benzoxazinoid allelochemical of wheat is 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), bound as beta-glucoside and released upon plant injury. When leached from wheat to soil, DIMBOA is microbially transformed to 6-methoxy-benzoxazolin-2-one (MBOA). Exploiting benzoxazinoids and their degradation products as substitutes for synthetic pesticides depends on knowledge of transformation pathways and kinetics. In an MBOA degradation experiment at a concentration of 2400 nmol g(-1) soil, the previously identified transformation products 2-amino-7-methoxy-phenoxazin-3-one (AMPO) and 2-acetylamino-7-methoxy-phenoxazin-3-one (AAMPO) were quantified. Three different kinetic models were applied to MBOA transformation kinetics; single first-order (SFO), first-order multi-compartment, and double first-order in parallel. SFO proved to be adequate and was subsequently applied to the transformations of MBOA, AMPO and AAMPO. Degradation endpoints, expressed as degradation time (DT), were calculated for MBOA, AMPO and AAMPO to test whether the maximum values for synthetic pesticides set by the European Commission and the Danish Environmental Protection Agency were exceeded. DT(50) values for MBOA and AMPO were 5.4 d and 321.5 d, respectively, and DT(90) values were 18.1 d and 1068 d, respectively. The DT(50) value for AMPO exceeded the maximum value. The persistence, concentrations and toxicity of metabolites such as AMPO should be considered when breeding cereal crops with increased levels of benzoxazinoids.

The effects of two arbuscular mycorrhizal fungi (AMF) (Glomus mosseae and G. claroideum) and a pathogenic fungus (Pythium ultimum) on the production of eight flavonoids in roots of two white clover (Trifolium repens L.) cultivars were evaluated. Quantification of AM and pathogenic fungi in the roots showed that the AM symbiosis significantly reduced P. ultimum biomass and in some cases prevented infection. The flavonoid productions in clover roots varied depending on the presence of beneficial and/or pathogenic fungi, fungal isolate or plant cultivar. Only plants colonized with G. claroideum showed detectable concentrations of either coumestrol or kaempferol (cultivar-dependant). In addition, inoculation with G. claroideum resulted in significantly higher concentrations of coumestrol in cv. Sonja and medicarpin in cv. Milo. A low production of coumestrol and kaempferol in mycorrhizal plants may be G. mosseae-specific. Only the concentrations of formononetin and daidzein increased in clover roots in response to infection with P. ultimum. These flavonoids are supposedly stress metabolites, synthesized or produced from glycosides in response to pathogen infection. However, the presence of one or both AMF significantly lowered the formononetin and daidzein concentrations, and overruled the inductive effect of P. ultimum. Therefore the antagonistic action of AM against the pathogen must take place through another mechanism.

Isoflavonoids can inhibit the growth of certain plants and are produced by leguminous species such as clover used for cover cropping and weed suppression. Isoflavonoids have, therefore, been suspected of playing a part in chemical weed suppression by legumes. The effects of biochanin A, an isoflavone, and catechin, a phytotoxic flavanol on the growth of Arabidopsis thaliana were studied in a cultivation assay and compared to the effect of the synthetic herbicide glyphosate. A. thaliana was most susceptible to glyphosate (EC50 = 1.9 mg L−1) followed by catechin (EC50 = 85 mg L−1). EC50 could not be determined for biochanin A; a concentration of 900 mg L−1 was needed to produce a small (15 %) but statistically significant decrease in biomass. Metabolic profiling by GC-TOF and multivariate data analysis showed metabolic alterations in A. thaliana at doses insufficient to cause decreased plant growth. For the two phytotoxic compounds over 80 % of the annotated plant metabolites were significantly affected by the dose. Even for biochanin A, 72 % of the metabolites were affected. Furthermore, the accumulation of the flavonoid to which A. thaliana was exposed could be followed, as could the accumulation of shikimic acid due to glyphosate treatment.