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Plasma contains metabolites with diverse physicochemical properties, ranging from highly polar to highly apolar, and concentrations spanning at least nine orders of magnitude. Plasma metabolome analysis is valuable for monitoring health and evaluating medical interventions but is challenging due to the metabolome’s diversity and complexity. This study aims to develop and validate targeted LC-MS/MS methods for quantifying 235 mammalian metabolites from 17 compound classes in porcine plasma without prior derivatization. Utilizing reversed-phase and hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry, each analyte is identified and quantified using two selected reaction monitoring (SRM) transitions. Fast polarity switching and scheduled SRM enhance the metabolome coverage and throughput, enabling the analysis of one sample in about 40 min. A simple “dilute and shoot” sample preparation protocol was employed, with samples injected at two dilution levels to align metabolite concentrations within calibration curve ranges. Validation in porcine plasma included assessments of carryover, linearity, detection and quantification limits, repeatability and recovery. The method was further applied to plasma samples from various animal species, demonstrating its applicability to human and animal studies. This study establishes two robust LC-MS/MS methods for comprehensive porcine plasma metabolome quantification, advancing large-scale targeted metabolomics in biomedical research.

A convenient total synthesis of the imidazo[1,2-f]phenanthridine-type Amaryllidaceae alkaloid zephycandidine A (3) was developed, which further allowed us to perform modifications of substituents on benzenoid ring A and imidazole ring D. The biological activities of all synthesized compounds were evaluated, and it was reported that activities against cancer cells of the parent alkaloid were poorly reproducible, while the closely related analogue THK-121 (11) showed a strong inhibitory effect on proliferation. Additionally, our novel analogue significantly induced cell death via the intrinsic apoptosis pathway, evident by the loss of mitochondrial membrane potential, increased mitochondrial oxidative stress, and disrupted mitochondrial structure in the same cells. At the same time, healthy cells were less affected by the treatment with THK-121 (11), indicating a potential therapeutic margin.

A significant amount of agri-food by-products is generated by large food industry production lines. Aligned with the principles of a circular economy, this project aims to recycle and valorize legumes, such as beans, green beans and soy by-products characterized by different heat treatments, maturation stages and cultivation methods. The valorization of food waste involved the development of an Ultrasound-Assisted Extraction (UAE) method to isolate polyphenols. Analytical techniques, including UHPLC-DAD-ESI-MSn, were used to identify polyphenols in legume, green bean and soy extracts obtained through UAE. Additionally, UV-Vis spectrophotometric assays measured the Total Phenolic Content (TPC) and Total Antioxidant Status (TAS), while the Kjeldahl method was employed to assess the protein content in each UAE extract. The analyses revealed a variety of valuable polyphenols in legume, green bean and soy by-products. For instance, bean by-products contain feruloyl glucaric acid derivatives, green beans by-products have different types of flavonols such as quercetin-3-O-glucuronide, and soy by-products are rich in isoflavones. These findings demonstrate the potential for formulating nutraceuticals from these by-products’ extracts.

Spilanthol is a major N-alkylamide constituent of Acmella oleracea (L.) R.K. Jansen with diverse pharmacological properties. We recently showed the applicability of NADES (natural deep eutectic solvents) for the green extraction of spilanthol. However, the purification of targets from NADES poses a challenging step due to their non-volatility. A simple green method to retrieve spilanthol with minimal instrumental effort was devised, fractioning NADES (choline chloride/methylurea, choline chloride/1,2-propanediol, choline chloride/citric acid) and dry ethanolic extracts by SPE on C18 material, eluting merely with ethanolic solutions. The relative distribution of spilanthol and organic adulteration in SPE fractions were detected by HPLC-DAD, followed by scale-up, quantification and purity determination in an NMR-based approach. Isocratic elution with 52% ethanol (v/v) proved suitable in all experiments. The three purest 10 mL fractions combined yielded 12.21 mg spilanthol at 71.65% purity from NADES extract ChCl/P (choline chloride/1,2-propanediol, molar ratio 1:2, +20% m/m water). Ethanolic extract samples showed purities ranging from 77.27 to 80.27% in combined raw fractions. For all samples, purity increased by removing non-soluble substances from organic solutions. Pooled NADES extract fractions showed 89.71% in final samples, ethanolic extracts 87.25 to 91.93%. The highest purities of individual fractions per extract were 89.23 to 94.15%. This cheap and simple purification process is promising to acquire spilanthol for research purposes or as a sample preparation step before HPLC on a semi-preparative to preparative scale, as the substance is highly priced and scarcely available on the market. Organic solvents can be reused, and preliminary scale-up possibilities are shown.

In Germany, there is currently no official guideline for the submission of placentas for histopathological examination. Placentas are sent for histological examination by obstetricians according to locally defined indications, which leads to different practices in different centers. In this study, two cohorts of placentas were compared to assess the clinical relevance of placental examination. One cohort consisted of placentas with a clinical indication for histologic examination and the other of placentas with a clinically healthy pregnancy and a healthy infant. In this study, a placenta request form based on established international guidelines was used. Placentas from singleton and twin pregnancies with and without clinical indications were histopathologically examined. Clinical information was extracted from the request form and later correlated with histological findings. A total of 236 placentas were examined, including 127 (53.8%) with clinical indications and 109 (46.2%) without. The concordance between submission reasons and histopathological findings was higher in singleton pregnancies with clinical indications (90.9%) compared to twin pregnancies (62.97%). Placentas from singleton and twin pregnancies with clinical indications exhibited significantly more pathological findings than their respective healthy control groups. Histopathological examination of the placenta can confirm or reveal placenta pathologies and therefore improve the care of the mother, child and future pregnancies.

The successful substitution of complex physiological fluids, such as human saliva, remains a major challenge in drug development. Although there are a large number of saliva substitutes on the market, their efficacy is often inadequate due to short residence time in the mouth, unpleasant mouthfeel, or insufficient protection of the teeth. Therefore, systems need to be identified that mimic the functions of saliva, in particular the salivary mucin MUC5B and the unique physiological properties of saliva. To this end, plant extracts known to contain hydrocolloid polysaccharides and to have mucus-forming properties were studied to evaluate their suitability as saliva substitutes. The aqueous plant extracts of Calendula officinalis, Fucus sp. thalli, and lichenan from Lichen islandicus were examined for composition using a range of techniques, including GC-MS, NMR, SEC, assessment of pH, osmolality, buffering capacity, viscoelasticity, viscoelastic interactions with human saliva, hydrocolloid network formation, and in vitro cell adhesion. For this purpose, a physiologically adapted adhesive test was developed using human buccal epithelial cells. The results show that lichenan is the most promising candidate to mimic the properties of MUC5B. By adjusting the pH, osmolality, and buffering capacity with K2HPO4, it was shown that lichenan exhibited high cell adhesion, with a maximum detachment force that was comparable to that of unstimulated whole mouth saliva.

Fusarium verticillioides produces fumonisins, which are mycotoxins inhibiting sphingolipid biosynthesis in humans, animals, and other eukaryotes. Fumonisins are presumed virulence factors of plant pathogens, but may also play a role in interactions between competing fungi. We observed higher resistance to added fumonisin B1 (FB1) in fumonisin-producing Fusarium verticillioides than in nonproducing F. graminearum, and likewise between isolates of Aspergillus and Alternaria differing in production of sphinganine-analog toxins. It has been reported that in F. verticillioides, ceramide synthase encoded in the fumonisin biosynthetic gene cluster is responsible for self-resistance. We reinvestigated the role of FUM17 and FUM18 by generating a double mutant strain in a fum1 background. Nearly unchanged resistance to added FB1 was observed compared to the parental fum1 strain. A recently developed fumonisin-sensitive baker’s yeast strain allowed for the testing of candidate ceramide synthases by heterologous expression. The overexpression of the yeast LAC1 gene, but not LAG1, increased fumonisin resistance. High-level resistance was conferred by FUM18, but not by FUM17. Likewise, strong resistance to FB1 was caused by overexpression of the presumed F. verticillioides “housekeeping” ceramide synthases CER1, CER2, and CER3, located outside the fumonisin cluster, indicating that F. verticillioides possesses a redundant set of insensitive targets as a self-resistance mechanism.

Clickable chemical tools are essential for studying the localization and role of biomolecules in living cells. For this purpose, alkyne-based close analogs of the respective biomolecules are of outstanding interest. Here, in the field of phytosterols, we present the first alkyne derivative of sitosterol, which fulfills the crucial requirements for such a chemical tool as follows: very similar in size and lipophilicity to the plant phytosterols, and correct absolute configuration at C-24. The alkyne sitosterol FB-DJ-1 was synthesized, starting from stigmasterol, which comprised nine steps, utilizing a novel alkyne activation method, a Johnson–Claisen rearrangement for the stereoselective construction of a branched sterol side chain, and a Bestmann–Ohira reaction for the generation of the alkyne moiety.

Trichothecenes produced by Fusarium species are commonly detected in oats. However, the ratios of the concentrations of free trichothecenes and their conjugates and how they are impacted by different interacting environmental conditions are not well documented. This study aims to examine the effect of water activity (0.95 and 0.98 aw) and temperature (20 and 25 °C) stress on the production of T-2 and HT-2 toxins, deoxynivalenol and their conjugates, as well as diacetoxyscirpenol (DAS). Multiple mycotoxins were detected using liquid chromatography–tandem mass spectrometry from 64 contaminated oat samples. The highest concentrations of HT-2-glucoside (HT-2-Glc) were observed at 0.98 aw and 20 °C, and were higher than other type A trichothecenes in the natural oats’ treatments. However, no statistical differences were found between the mean concentrations of HT-2-Glc and HT-2 toxins in all storage conditions analysed. DAS concentrations were generally low and highest at 0.95 aw and 20 °C, while deoxynivalenol-3-glucoside levels were highest at 0.98 aw and 20 °C in the naturally contaminated oats. Emerging mycotoxins such as beauvericin, moniliformin, and enniatins mostly increased with a rise in water activity and temperature in the naturally contaminated oats treatment. This study reinforces the importance of storage aw and temperature conditions in the high risk of free and modified toxin contamination of small cereal grains.

Fusarium is a genus that mostly consists of plant pathogenic fungi which are able to produce a broad range of toxic secondary metabolites. In this study, we focus on a type A trichothecene-producing isolate (15-39) of Fusarium sporotrichioides from Lower Austria. We assessed the secondary metabolite profile and optimized the toxin production conditions on autoclaved rice and found that in addition to large amounts of T-2 and HT-2 toxins, this strain was able to produce HT-2-glucoside. The optimal conditions for the production of T-2 toxin, HT-2 toxin, and HT-2-glucoside on autoclaved rice were incubation at 12 °C under constant light for four weeks, darkness at 30 °C for two weeks, and constant light for three weeks at 20 °C, respectively. The HT-2-glucoside was purified, and the structure elucidation by NMR revealed a mixture of two alpha-glucosides, presumably HT-2-3-O-alpha-glucoside and HT-2-4-O-alpha-glucoside. The efforts to separate the two compounds by HPLC were unsuccessful. No hydrolysis was observed with two the alpha-glucosidases or with human salivary amylase and Saccharomyces cerevisiae maltase. We propose that the two HT-2-alpha-glucosides are not formed by a glucosyltransferase as they are in plants, but by a trans-glycosylating alpha-glucosidase expressed by the fungus on the starch-containing rice medium.

With a growing focus on green chemistry, the extraction of natural products with natural deep eutectic solvents (NADES), which are eutectic mixtures of hydrogen bond donors and acceptors, has become an ever-expanding field of research. However, the use of NADES for the extraction of spilanthol from Acmella oleracea (L.) R.K.Jansen has not yet been investigated. Therefore, in this study, 20 choline chloride-based NADES, and for comparison, ethanol, were used as green extraction agents for spilanthol from Acmella oleracea flower heads. The effects of time, water addition, and temperature on NADES extractions were investigated and analysed by HPLC-DAD quantification. Additionally, UHPLC-DAD-ESI-MSn results for dichloromethane extracts, as well as the isolation of spilanthol and other main constituents as reference compounds, are reported. The best green extraction results were achieved by choline chloride (ChCl) with 1,2-propanediol (P, 1:2 molar ratio, +20% water) at 244.58 µg/mL, comparable to yields with ethanol (245.93 µg/mL). Methylurea (MeU, 1:2, +20% water) also showed promising results as a hydrogen bond donor in combination with choline chloride (208.12 µg/mL). In further experiments with NADES ChCl/P (1:2) and ChCl/MeU (1:2), extraction time had the least effect on spilanthol extraction with NADES, while yield decreased with water addition over 20% and increased with extraction temperature up to 80 °C. NADES are promising extraction agents for the extraction of spilanthol, and these findings could lead to applicable extracts for medicinal purposes, due to their non-toxic constituents.

Propolis, a naturally sticky substance used by bees to secure their hives and protect the colony from pathogens, presents a fascinating challenge. Despite its adhesive nature, honeybees adeptly handle propolis with their mandibles. Previous research has shown a combination of an anti-adhesive fluid layer and scale-like microstructures on the inner surface of bee mandibles. Our aim was to deepen our understanding of how surface energy and microstructure influence the reduction in adhesion for challenging substances like propolis. To achieve this, we devised surfaces inspired by the intricate microstructure of bee mandibles, employing diverse techniques including roughening steel surfaces, creating lacquer structures using Bénard cells, and moulding resin surfaces with hexagonal patterns. These approaches generated patterns that mimicked the bee mandible structure to varying degrees. Subsequently, we assessed the adhesion of propolis on these bioinspired structured substrates. Our findings revealed that on rough steel and resin surfaces structured with hexagonal dimples, propolis adhesion was significantly reduced by over 40% compared to unstructured control surfaces. However, in the case of the lacquer surface patterned with Bénard cells, we did not observe a significant reduction in adhesion.

Estradiol methyl ether (EDME) has recently been described by us as a very potent and subtype-specific inhibitor of the lysosomal cation channel TRPML1. Following the principle of bioisosteres, we worked out efficient synthetic approaches to ring-A aza-analogs of EDME, namely a methoxypyridine and a methoxypyrimidine analog. Both target compounds were obtained in good overall yields in six and eight steps starting from 19-nortestosterone via the oxidative cleavage of ring A followed over several intermediates and with the use of well-selected protective groups by re-cyclization to provide the desired hetero-analogs. The methoxypyridine analog largely retained its TRPML1-inhibitory activity, whereas the methoxypyrimidine analog significantly lost activity.

The heartwood of the Swiss Stone Pine, Pinus cembra L., has been scarcely investigated for secondary metabolites for a long period of time. Considering age and relative simplicity of heartwood investigations dating back to the 1940s to 1960s, we conducted the first investigation of P. cembra heartwood by HPLC, using UHPLC-DAD-ESI-MSn and HPLC-DAD techniques in combination with isolation and NMR spectroscopy, with focus on stilbenes, bibenzyls and flavonoids. Analytical problems in the HPLC analysis of Pinus stilbenes and flavonoids on reversed stationary phases were also challenged, by comparing HPLC on pentafluorophenyl (PFP) and C18 stationary phases. Seven flavonoids (1, 2, 3, 7, 8, 11, 12), four stilbenes (4, 6, 10, 13), two bibenzyls (5, 9), three fatty acids (14, 16, 17) and one diterpenic acid (15) were detected in an ethanolic extract of Pinus cembra heartwood. HPLC comparison of reversed stationary phases in HPLC showed that the antifungal, antibacterial and chemosensitizing dihydropinosylvin monomethyl ether (9) and pinosylvin monomethyl ether (10) can be separated on PFP, but not on C18 material, when eluting with a screening gradient of 20–100% acetonitrile. Flavonoid separation showed additional benefits of combining analyses on different stationary phases, as flavonoids 7 and 8 could only be separated on one of two C18 stationary phases. Earlier phytochemical results for heartwood investigations were shown to be mostly correct, yet expandable. Substances 5 to 12 were found in alignment with these references, proving remarkable phytochemical analyses at the time. Evidence for the described presence of pinobanksin could not be found. Substances 1 to 4 and 13 have to our knowledge not yet been described for P. cembra.

Astragalus glycyphyllos (Fabaceae) is used in the traditional medicine of many countries against hepatic and cardiac disorders. The plant contains mainly flavonoids and saponins. From a defatted methanol extract from its overground parts, a new triterpenoid saponin, 3-O-[α-L-rhamnopyranosyl-(1→2)]-β-D-xylopyranosyl]-24-O-α-L-arabinopyranosyl-3β,6α,16β,24(R),25-pentahydroxy-20R-cycloartane, together with the rare saponin astrachrysoside A, were isolated using various chromatography methods. The compounds were identified via extensive high resolution electrospray ionisation mass spectrometry (HRESIMS) and NMR analyses. Both saponins were examined for their possible antioxidant and neuroprotective activity in three different in vitro models. Rat brain synaptosomes, mitochondria, and microsomes were isolated via centrifugation using Percoll gradient. They were treated with the compounds in three different concentrations alone, and in combination with 6-hydroxydopamine or tert-butyl hydroperoxide as toxic agents. It was found that the compounds had statistically significant dose-dependent in vitro protective activity on the sub-cellular fractions. The compounds exhibited a weak inhibitory effect on the enzyme activity of human recombinant monoamine oxidase type B (hMAO-B), compared to selegiline.

Concern about the global emergence of multidrug-resistant fungal pathogens led us to explore the use of combination therapy to combat azole resistance in Candida auris. Clorgyline had previously been shown to be a multi-target inhibitor of Cdr1 and Mdr1 efflux pumps of Candida albicans and Candida glabrata. A screen for antifungal sensitizers among synthetic analogs of Clorgyline detected interactions with the C. auris efflux pump azole substrates Posaconazole and Voriconazole. Of six Clorgyline analogs, M19 and M25 were identified as potential sensitizers of azole resistance. M19 and M25 were found to act synergistically with azoles against resistant C. auris clade I isolates and recombinant Saccharomyces cerevisiae strains overexpressing C. auris efflux pumps. Nile Red assays with the recombinant strains showed M19 and M25 inhibited the activity of Cdr1 and Mdr1 efflux pumps that are known to play key roles in azole resistance in C. auris clades I, III, and IV. While Clorgyline, M19 and M25 uncoupled the Oligomycin-sensitive ATPase activity of Cdr1 from C. albicans and C. auris, their mode of action is yet to be fully elucidated. The experimental combinations described herein provides a starting point to combat azole resistance dominated by overexpression of CauCdr1 in C. auris clades I and IV and CauMdr1 in C. auris clade III.

Predicting the chemical stability of yet-to-be-discovered materials is an important aspect of the discovery and development of virtual materials. The conventional approach for computing the enthalpy of formation based on ab initio methods is time consuming and computationally demanding. In this regard, alternative machine learning approaches are proposed to predict the formation energies of different classes of materials with decent accuracy. In this paper, one such machine learning approach, a novel two-step method that predicts the formation energy of ternary compounds, is presented. In the first step, with a classifier, we determine the accuracy of heuristically calculated formation energies in order to increase the size of the training dataset for the second step. The second step is a regression model that predicts the formation energy of the ternary compounds. The first step leads to at least a 100% increase in the size of the dataset with respect to the data available in the Materials Project database. The results from the regression model match those from the existing state-of-the-art prediction models. In addition, we propose a slightly modified version of the Adam optimizer, namely centered Adam, and report the results from testing the centered Adam optimizer.

Data on the evolution of blood metabolites and metabolic markers in neonatal piglets are scarce, although this information is vital to detect physiological aberrations from normal development. We aimed to characterize age- and nutrition-related changes in the plasma metabolome and serum biochemistry of suckling and newly weaned piglets and assess metabolite patterns as physiological markers for the two phases. In two replicate batches (n = 10 litters/group), piglets either received sow milk alone or were additionally offered creep feed from day 10 until weaning (day 28). Blood was collected from one piglet/litter on days 7, 14, 21, 28, 31 and 35 of life, totaling five females and five males/group/day. Signature feature ranking identified plasma triglycerides (TG) as discriminative for age and nutrition during the suckling phase. Influential TG 20:4_36:5, TG 17:0_34:2 and TG 18:2_38:6 were higher in creep-fed piglets on days 14, 21 and 28 of life, respectively, compared to only sow milk-fed piglets. Metabolites belonging to pathways within histidine, D-glutamine and D-glutamate metabolism as well as hippuric acid were distinctive for the postweaning compared to the suckling period. In conclusion, plasma lipid profiles especially corresponded to the type of nutrition in the suckling phase and showed a strong weaning effect.

The Galapagos Islands have been declared a World Heritage site due to their unique biodiversity, which makes them a living museum and a natural laboratory for humankind. However, to fulfill the energy needs of its habitants and foreign visitors, the islands have depended on fossil fuel energies that have produced levels of lead and chemical agents that are affecting the islands’ air quality, flora, and fauna. Therefore, zero-carbon initiatives have been created to protect the islands, wherein solar and wind power plants have been studied as reliable alternatives. In this way, Geographical Information Systems based on Multicriteria Decision Methods constitute a methodology that minimizes the destruction and disturbance of nature in order to assess the best location for the implementation of these alternative energy sources. Therefore, by exploring the geographical information along with the Analytical Hierarchical Processes and the Ordered Weighted Average methods, it was possible to identify the potential for solar power plants of 10 MW on each island; likewise, for wind power plants, it was found that the islands possess implementation potential that has been analyzed in the field, showing that the best location is on Baltra Island, but is not limited to it.

Clivia miniata (Amaryllidaceae) is an herbaceous evergreen flowering plant that is endemic to South Africa and Swaziland and belongs to one of the top-10 traded medicinal plants in informal medicine markets in South Africa. The species has been reported as the most important component of a traditional healer’s pallet of healing plants. Eighteen known Amaryllidaceae alkaloids (AAs) of various structural types, and one undescribed alkaloid of homolycorine-type, named clivimine B (3), were isolated from Clivia miniata. The chemical structures of the isolated alkaloids were elucidated by a combination of MS, HRMS, 1D and 2D NMR techniques and by comparison with literature data. Compounds isolated in a sufficient quantity, and not tested previously, were evaluated for their in vitro acetylcholinesterase (AChE; E.C. 3.1.1.7) and butyrylcholinesterase (BuChE; E.C. 3.1.1.8) inhibition activities.

The Annonaceae are an old family of flowering plants belonging to the order Magnoliales, distributed mainly in tropical regions. Numerous Annonaceae species find ethnobotanical use for curing a broad range of diseases, among them cancer and infections by diverse pathogens. Hence, bioactive natural products from Annonaceae have received considerable interest in drug development. Beyond cytotoxic acetogenins, unique aporphine-derived polycyclic aromatic alkaloids are characteristic constituents of Annonaceae. Among them are unique tri- and tetracyclic aromatic alkaloids like azafluorenones, diazafluoranthenes, azaanthracenes, and azaoxoaporphines. The complex substitution pattern of these alkaloids represents a major challenge in structure elucidation of isolated natural products. Based on a broad spectrum of alkaloids available from our previous work, we present a GC-MS protocol for the identification of over 20 polycyclic aromatic alkaloids from Annonaceae. This collection of data will contribute to the future identification of the metabolite patterns of extracts from Annonaceae as an important source of novel bioactive secondary metabolites.

Campylobacter jejuni is a major foodborne pathogen and the leading cause of bacterial gastroenteritis, i.e., campylobacteriosis. Besides searching for novel antimicrobials, identification of new targets for their action is becoming increasingly important. Rhodiola rosea has long been used in traditional medicine. Ethanolic extracts from the roots and rhizomes of the plant contain a wide range of bioactive compounds with various pharmacological activities. In this study, cultivated plant materials have been used, i.e., “Mattmark” and “Rosavine”. Through optimized protocols, we obtained fractions of the initial ethanolic extracts rich in most important bioactive compounds from R. rosea, including salidroside, rosavins, proanthocyanidins (PACs), and flavonoids. The antimicrobial activity in relation to the chemical composition of the extracts and their fractions was studied with an emphasis on C. jejuni AI-2-mediated intercellular signaling. At concentration 15.625 mg/L, bioluminescence reduction rates varied from 27% to 72%, and the membrane remained intact. Fractions rich in PACs had the strongest antimicrobial effect against C. jejuni, with the lowest minimal inhibitory concentrations (MICs) (M F3 40%: 62.5 mg/L; R F3 40%: 250 mg/L) and the highest intercellular signaling reduction rates (M F3 40%: 72%; R F3 40%: 65%). On the other hand, fractions without PACs were less effective (MICs: M F5 PVP: 250 mg/L; R F5 PVP: 1000 mg/L and bioluminescence reduction rates: M F5 PVP: 27%; R F5 PVP: 43%). Additionally, fractions rich in flavonoids had strong antimicrobial activity (MICs: M F4 70%: 125 mg/L; R F4 70%: 250 mg/L and bioluminescence reduction rates: M F4 70%: 68%; R F4 70%: 50%). We conclude that PACs and flavonoids are crucial compound groups responsible for the antimicrobial activity of R. rosea roots and rhizomes in C. jejuni.

The food industry is constantly struggling with one of the most prevalent biofilm-forming and food-borne pathogenic bacteria, Campylobacter jejuni. Different approaches are used to control biofilms in the food production chain, but none is fully effective. In this study, we aim to produce and determine the chemical profile of essential oils (EOs), ethanolic extracts of flowers prior to distillation (EFs), and ethanolic extracts of post-distillation waste material (EWMs) from Lavandula × intermedia ‘Bila’, ‘Budrovka’ St Nicholas and ‘Budrovka’, which were further used to reduce C. jejuni intercellular signaling, adhesion, and biofilm formation, as well as to test their antioxidant activity. Glycosides of hydroxycinnamic acids were the major constituents of both types of lavandin ethanolic extract, while linalool, linalyl acetate, 1,8-cineol, and camphor were the major compounds found in lavandin EOs. Tested EOs showed the best antibacterial activity with a minimal inhibitory concentration of 0.25 mg/mL. Lavandin EFs proved more effective in reducing C. jejuni intercellular signaling and adhesion compared to lavandin EOs and EWMs, while lavandin EOs showed a slightly better effect against biofilm formation. Interestingly, the best antioxidant activity was determined for lavandin EWMs. A positive and moderate correlation was found between the reduction of C. jejuni intercellular signaling and adhesion, as well as between adhesion and biofilm formation. These findings mean novel bacterial targets are of interest for biofilm control with alternative natural agents throughout the whole food production chain.

The coordinate system of the Austrian cadastre is physically realised through control points provided by the national institution for surveying. Due to historical development over the centuries and changes in measurement technologies, inhomogeneities can occur within the local control point network. These inhomogeneities affect the derived boundary point coordinates. When staking out boundary points in an area with inhomogeneous control points, deviations from the boundary marks in the field can occur that exceed the accuracy requirements of the ordinance for surveying. Examples show that a suitable approach to tackle this issue has to be selected on a case-based strategy. Different situations might require different approaches. This needs to be considered in the legal framework to enable cadastral experts to select the optimal approach.

Species from the genus Globularia L. have been used as healing agents for various ailments, with utilization of Globularia alypum L. being most frequently reported. The aim of this study was to evaluate the antidiabetic, antioxidant, anti-inflammatory, antibacterial and anticancer potential of G. alypum and three related species, G. punctata Lapeyr., G. cordifolia L. and G. meridionalis (Podp.) O.Schwarz, in relation to their phytochemical compositions. Globularin and verbascoside were identified using LC-PDA-ESI-MSn as the major metabolites of G. alypum with known biological activities. G. alypum demonstrated the greatest α-glucosidase inhibitory activity and DPPH radical scavenging activity (IC50 = 17.25 μg/mL), while its anti-inflammatory activity was not significantly different from those of related species. All investigated species showed considerable antibacterial activity against methicillin-resistant Staphylococcus aureus in the broth microdilution method (MIC = 1.42–3.79 mg/mL). G. punctata also showed antibacterial activities against Escherichia coli (MIC = 1.42 mg/mL), Bacillus subtilis (MIC = 1.89 mg/mL), B. cereus (MIC = 2.84 mg/mL) and Enterococcus faecalis (MBC = 5.68 mg/mL). G. punctata, G. cordifolia and G. meridionalis showed greater anticancer potential than G. alypum. Obtained results indicate investigated Globularia species could serve as sources of diverse bioactive molecules, with G. punctata having the greatest antibacterial potential.

The evaluation of structural response constitutes a fundamental task in the design of ground-excited structures. In this context, the Monte Carlo simulation is a powerful tool to estimate the response statistics of nonlinear systems, which cannot be represented analytically. Unfortunately, the number of samples which is required for estimations with high confidence increases disproportionally to obtain a reliable estimation of low-probability events. As a consequence, the Monte Carlo simulation becomes a non-realizable task from a computational perspective. We show that the application of machine learning algorithms significantly lowers the computational burden of the Monte Carlo method. We use artificial neural networks to predict structural response behavior using supervised learning. However, one shortcoming of supervised learning is the inability of a sufficiently accurate prediction when extrapolating to data the neural network has not seen yet. In this paper, neural networks predict the response of structures subjected to non-stationary ground excitations. In doing so, we propose a novel selection process for the training data to provide the required samples to reliably predict rare events. We, finally, prove that the new strategy results in a significant improvement of the prediction of the response statistics in the tail end of the distribution.

Flavonoids are widely occurring secondary plant constituents, and are abundant in vegetable and fruit diets as well as herbal medicines. Therapeutic treatment options for bacterial infections are limited due to the spread of antimicrobial resistances. Hence, in a number of studies during the last few years, different classes of plant secondary metabolites as resistance-modifying agents have been carried out. In this review, we present the role of flavonoids as inhibitors of bacterial efflux pumps. Active compounds could be identified in the subclasses of chalcones, flavan-3-ols, flavanones, flavones, flavonols, flavonolignans and isoflavones; by far the majority of compounds were aglycones, although some glycosides like kaempferol glycosides with p-coumaroyl acylation showed remarkable results. Staphylococcus aureus NorA pump was the focus of many studies, followed by mycobacteria, whereas Gram-negative bacteria are still under-investigated.

The aliphatic heterocycles piperidine and morpholine are core structures of well-known antifungals such as fenpropidin and fenpropimorph, commonly used as agrofungicides, and the related morpholine amorolfine is approved for the treatment of dermal mycoses in humans. Inspired by these lead structures, we describe here the synthesis and biological evaluation of 4-aminopiperidines as a novel chemotype of antifungals with remarkable antifungal activity. A library of more than 30 4-aminopiperidines was synthesized, starting from N-substituted 4-piperidone derivatives by reductive amination with appropriate amines using sodium triacetoxyborohydride. Antifungal activity was determined on the model strain Yarrowia lipolytica, and some compounds showed interesting growth-inhibiting activity. These compounds were tested on 20 clinically relevant fungal isolates (Aspergillus spp., Candida spp., Mucormycetes) by standardized microbroth dilution assays. Two of the six compounds, 1-benzyl-N-dodecylpiperidin-4-amine and N-dodecyl-1-phenethylpiperidin-4-amine, were identified as promising candidates for further development based on their in vitro antifungal activity against Candida spp. and Aspergillus spp. Antifungal activity was determined for 18 Aspergillus spp. and 19 Candida spp., and their impact on ergosterol and cholesterol biosynthesis was determined. Toxicity was determined on HL-60, HUVEC, and MCF10A cells, and in the alternative in vivo model Galleria mellonella. Analysis of sterol patterns after incubation gave valuable insights into the putative molecular mechanism of action, indicating inhibition of the enzymes sterol C14-reductase and sterol C8-isomerase in fungal ergosterol biosynthesis.

The recently issued EN ISO 52016-1 technical standard provides a new simplified dynamic method for the building energy performance assessment. Since an extensive validation of the EN ISO 52016-1 hourly method is still missing, the present work investigates the effect of the main modelling assumptions—related to the heat balance on the outdoor and the indoor envelope surfaces—on the building thermal behaviour. The model validation was carried out by assessing the accuracy variation consequent to the application of the EN ISO 52016-1 modelling assumptions to a detailed dynamic calculation tool (EnergyPlus). To guarantee a general validity of the outcomes, two buildings, two levels of thermal insulation, and two Italian climatic zones were considered, for a total of eight case studies. To explore different applications of the standard method, the analysis was performed both under a free-floating condition—to evaluate the accuracy of the model in predicting the indoor operative temperatures—and to assess the annual energy needs for space heating and cooling. Results show that the assumptions related to the definition of the external convective and the shortwave (solar) radiation heat transfer lead to non-negligible inaccuracies in the EN ISO 52016-1 hourly model.

Antibiotic resistance is a growing problem and may become the next major global health crisis if no timely actions are taken. Mycobacterial infections are widespread and, due to antibiotic resistance, also hard to treat and a major cause of mortality. Natural compounds have the potential to increase antibiotic effectiveness due to their resistance modulatory and antimicrobial effects. In this study, Peucedanum ostruthium extracts, fractions, and isolated compounds were investigated regarding their antimicrobial and resistance-modulatory effects as well as efflux pump inhibition in Mycobacterium smegmatis. P. ostruthium extracts were found to have anti-mycobacterial potential and resistance modulating effects on ethidium bromide activity. The major antibacterial effect was attributed to ostruthin, and we found that the more lipophilic the substrate, the greater the antimicrobial effect. Imperatorin caused potent modulatory effects by interfering with the action of the major LfrA efflux pump in M. smegmatis. The plant P. ostruthuim has a complex effect on M. smegmatis, including antibacterial, efflux pump inhibition, resistance modulation, and membrane permeabilization, and its major constituents, ostruthin and imperatorin, have a distinct role in these effects. This makes P. ostruthium and its coumarins promising therapeutics to consider in the fight against drug-resistant mycobacteria.

Background: In Down syndrome (DS), adaptive behaviour often shows a “surplus effect” (i.e., higher adaptive abilities than expected from cognitive skills). As inclusive schooling has become mandatory in Italy, we studied the impact of school inclusion on the surplus effect of adaptive behaviour in adult DS, considering potential confounding factors such as parental education. Methods: All consecutive DS individuals from three different sites were queried prospectively regarding type of schooling (inclusive and non-inclusive). Demographic data were documented; cognitive abilities and adaptive behaviour were assessed (Coloured Progressive Matrices and Vineland Adaptive Behaviour Scales). The aim was to establish the presence of a surplus effect in adaptive behaviour, primarily in the overall level and secondarily in the main domains and subdomains. A multivariable-adjusted logistic regression model was used for the association of schooling, and parental education. Results: The majority (65%) showed a surplus effect in adaptive behaviour and had attended inclusive schools (85%). Higher adaptive skills as well as early and longer functional treatment programmes were more readily available for younger individuals. In the group of inclusive schooling, the surplus effect on overall adaptive behaviour was present in 70% as opposed to 38% in the group without inclusive schooling, significant when adjusted for gender and maternal education. This was also observed in socialisation, written, and community, and after adjustment in playing and leisure time. Conclusions: Adaptive behaviour showed a surplus effect in the majority of DS adults, even more so after inclusive schooling. Younger adults showed higher adaptive skills. Moreover, female gender and higher maternal educational level significantly enhanced this surplus effect.

Meiotic recombination is the main tool used by breeders to generate biodiversity, allowing genetic reshuffling at each generation. It enables the accumulation of favorable alleles while purging deleterious mutations. However, this mechanism is highly regulated with the formation of one to rarely more than three crossovers, which are not randomly distributed. In this study, we showed that it is possible to modify these controls in oilseed rape (Brassica napus, AACC, 2n = 4x = 38) and that it is linked to AAC allotriploidy and not to polyploidy per se. To that purpose, we compared the frequency and the distribution of crossovers along A chromosomes from hybrids carrying exactly the same A nucleotide sequence, but presenting three different ploidy levels: AA, AAC and AACC. Genetic maps established with 202 SNPs anchored on reference genomes revealed that the crossover rate is 3.6-fold higher in the AAC allotriploid hybrids compared to AA and AACC hybrids. Using a higher SNP density, we demonstrated that smaller and numerous introgressions of B. rapa were present in AAC hybrids compared to AACC allotetraploid hybrids, with 7.6 Mb vs. 16.9 Mb on average and 21 B. rapa regions per plant vs. nine regions, respectively. Therefore, this boost of recombination is highly efficient to reduce the size of QTL carried in cold regions of the oilseed rape genome, as exemplified here for a QTL conferring blackleg resistance.

Ergosterol has traditionally been used as a proxy to estimate fungal biomass as it is almost exclusively found in fungal lipid membranes. Ergosterol determination has been mostly used for fungal samples from terrestrial, freshwater, salt marsh- and mangrove-dominated environments or to describe fungal degradation of plant matter. In the open ocean, however, the expected concentrations of ergosterol are orders of magnitude lower than in terrestrial or macrophyte-dominated coastal systems. Consequently, the fungal biomass in the open ocean remains largely unknown. Recent evidence based on microscopy and -omics techniques suggests, however, that fungi contribute substantially to the microbial biomass in the oceanic water column, highlighting the need to accurately determine fungal biomass in the open ocean. We performed ergosterol extractions of an oceanic fungal isolate (Rhodotorula sphaerocarpa) with biomass concentrations varying over nine orders of magnitude. While after the initial chloroform-methanol extraction ~87% of the ergosterol was recovered, a second extraction recovered an additional ~10%. Testing this extraction method on samples collected from the open Atlantic Ocean, we successfully determined ergosterol concentrations as low as 0.12 pM. Thus, this highly sensitive method is well suited for measuring fungal biomass from open ocean waters, including deep-sea environments.

Reynoutria japonica Houtt. is a critical invasive alien plant in Europe and North America with a drastic impact on native flora. However, R. japonica has medicinal potential, especially as a source of stilbenes. In order to explore the potential of simple extractions of R. japonica, we conducted qualitative and quantitative analyses of fresh R. japonica rhizome infusion, decoction, and macerates with ethanol by UHPLC-DAD-ESI-MSn and UHPLC-DAD, with a focus on major constituent groups of stilbenes and anthranoids. Since R. japonica rhizome extracts showed antimicrobial potential in the past, we also evaluated the antimycobacterial effect of raw R. japonica extracts for the first time against Mycobacterium smegmatis. Of thirty-four characterized substances, six were stilbenes and twelve anthranoids. The main constituents, four trans-stilbenes and eight anthranoids, were quantified in a validated UHPLC-DAD method. The 38% ethanol macerate showed high stilbene (155.078 mg/100 g fluid extract) and low anthranoid content (5.420 mg/100 g fluid extract), while decoction showed the highest anthranoids. Antimycobacterial testing gave good results for all macerates (MIC 256 µg/mL) and trans-resveratrol (64 µg/mL). Extraction and enrichment of stilbenes from fresh plant material by simple extraction methods with food-grade solvents might encourage consideration of wild harvest of rhizomes over classic means of eradication of R. japonica.

The Fusarium mycotoxin deoxynivalenol (DON) is a common contaminant of cereals and is often co-occurring with its modified forms DON-3-glucoside (D3G), 3-acetyl-DON (3ADON) or 15-acetyl-DON (15ADON). A stable-isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method for their determination in cereals was developed and validated for maize. Therefore, 13C-labelled D3G was enzymatically produced using 13C-DON and [13C6Glc]-sucrose and used as an internal standard (IS) for D3G, while uniformly 13C labelled IS was used for the other mycotoxins. Baseline separation was achieved for the critical peak pair DON/D3G, while 3ADON/15ADON could not be fully baseline separated after testing various reversed phase, fluorinated phase and chiral LC columns. After grinding, weighing and extracting the cereal samples, the raw extract was centrifuged and a mixture of the four 13C-labelled ISs was added directly in a microinsert vial. The subsequent analytical run took 7 min, followed by negative electrospray ionization and selected reaction monitoring on a triple quadrupole MS. Maize was used as a complex cereal model matrix for validation. The use of the IS corrected the occurring matrix effects efficiently from 76 to 98% for D3G, from 86 to 103% for DON, from 68 to 100% for 15ADON and from 63 to 96% for 3ADON.

The emerging mycotoxin fusaproliferin is produced by Fusarium proliferatum and other related Fusarium species. Several fungi from other taxonomic groups were also reported to produce fusaproliferin or the deacetylated derivative, known as siccanol or terpestacin. Here, we describe the identification and functional characterization of the Fusarium proliferatum genes encoding the fusaproliferin biosynthetic enzymes: a terpenoid synthase, two cytochrome P450s, a FAD-oxidase and an acetyltransferase. With the exception of one gene encoding a CYP450 (FUP2, FPRN_05484), knock-out mutants of the candidate genes could be generated, and the production of fusaproliferin and intermediates was tested by LC-MS/MS. Inactivation of the FUP1 (FPRN_05485) terpenoid synthase gene led to complete loss of fusaproliferin production. Disruption of a putative FAD-oxidase (FUP4, FPRN_05486) did not only affect oxidation of preterpestacin III to terpestacin, but also of new side products (11-oxo-preterpstacin and terpestacin aldehyde). In the knock-out strains lacking the predicted acetyltransferase (FUP5, FPRN_05487) fusaproliferin was no longer formed, but terpestacin was found at elevated levels. A model for the biosynthesis of fusaproliferin and of novel derivatives found in mutants is presented.

With the rise of long-read sequencers and long-range technologies, delivering high-quality plant genome assemblies is no longer reserved to large consortia. Not only sequencing techniques, but also computer algorithms have reached a point where the reconstruction of assemblies at the chromosome scale is now feasible at the laboratory scale. Current technologies, in particular long-range technologies, are numerous, and selecting the most promising one for the genome of interest is crucial to obtain optimal results. In this study, we resequenced the genome of the yellow sarson, Brassica rapa cv. Z1, using the Oxford Nanopore PromethION sequencer and assembled the sequenced data using current assemblers. To reconstruct complete chromosomes, we used and compared three long-range scaffolding techniques, optical mapping, Omni-C, and Pore-C sequencing libraries, commercialized by Bionano Genomics, Dovetail Genomics, and Oxford Nanopore Technologies, respectively, or a combination of the three, in order to evaluate the capability of each technology.

This work presents an updated and more complete version of the transcriptome of a long-distance migrant, the Northern Wheatear (Oenanthe oenanthe). The improved transcriptome was produced from the independent mRNA sequencing of adipose tissue, brain, intestines, liver, skin, and muscle tissues sampled during the autumnal migratory season. This new transcriptome has better sequencing coverage and is more representative of the species’ migratory phenotype. We assembled 20,248 transcripts grouped into 16,430 genes, from which 78% were successfully annotated. All the standard assembly quality parameters were improved in the second transcriptome version.

This study aimed to investigate the effects of different levels of soil- and plant-associated bacterial diversity on the rates of litter decomposition, and bacterial community dynamics during its early phases. We performed an incubation experiment where soil bacterial diversity (but not abundance) was manipulated by autoclaving and reinoculation. Natural or autoclaved maize leaves were applied to the soils and incubated for 6 weeks. Bacterial diversity was assessed before and during litter decomposition using 16S rRNA gene metabarcoding. We found a positive correlation between litter decomposition rates and soil bacterial diversity. The soil with the highest bacterial diversity was dominated by oligotrophic bacteria including Acidobacteria, Nitrospiraceae, and Gaiellaceae, and its community composition did not change during the incubation. In the less diverse soils, those taxa were absent but were replaced by copiotrophic bacteria, such as Caulobacteraceae and Beijerinckiaceae, until the end of the incubation period. SourceTracker analysis revealed that litter-associated bacteria, such as Beijerinckiaceae, only became part of the bacterial communities in the less diverse soils. This suggests a pivotal role of oligotrophic bacteria during the early phases of litter decomposition and the predominance of copiotrophic bacteria at low diversity.

The β-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of β-carboline and related scaffolds aimed at learning about structure–activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors.

Information on spectral emissivity (SE) is vital when retrieving and evaluating land surface temperature (LST) estimates from remotely sensed observations. SE measurements often come from spectral libraries based upon laboratory spectroscopic measurements, with uncertainties typically derived from repeated measurements. To go further, we organised a “round-robin” inter-comparison exercise involving SE measurements of three samples collected at seven different international laboratories. The samples were distilled water, which has a uniformly high spectral emissivity, and two artificial samples (aluminium and gold sheets laminated in polyethylene), with variable emissivities and largely specular and Lambertian characteristics. Large differences were observed between some measurements, with standard deviations over 2.5–14 μm of 0.092, 0.054 and 0.028 emissivity units (15.98%, 7.56% and 2.92%) for the laminated aluminium sheet, laminated gold sheet and distilled water respectively. Wavelength shifts of up to 0.09 μm were evident between spectra from different laboratories for the specular sample, attributed to system design interacting with the angular behaviour of emissivity. We quantified the impact of these SE differences on satellite LST estimation and found that emissivity differences resulted in LSTs differing by at least 3.5 K for each artificial sample and by more than 2.5 K for the distilled water. Our findings suggest that variations between SE measurements derived via laboratory setups may be larger than previously assumed and provide a greater contribution to LST uncertainty than thought. The study highlights the need for the infrared spectroscopy community to work towards standardized and interlaboratory comparable results.

The use of machine learning to estimate joint angles from inertial sensors is a promising approach to in-field motion analysis. In this context, the simplification of the measurements by using a small number of sensors is of great interest. Neural networks have the opportunity to estimate joint angles from a sparse dataset, which enables the reduction of sensors necessary for the determination of all three-dimensional lower limb joint angles. Additionally, the dimensions of the problem can be simplified using principal component analysis. Training a long short-term memory neural network on the prediction of 3D lower limb joint angles based on inertial data showed that three sensors placed on the pelvis and both shanks are sufficient. The application of principal component analysis to the data of five sensors did not reveal improved results. The use of longer motion sequences compared to time-normalised gait cycles seems to be advantageous for the prediction accuracy, which bridges the gap to real-time applications of long short-term memory neural networks in the future.

Carvotacetones (1–7) isolated from Sphaeranthus africanus were screened for their antimycobacterial and efflux pump (EP) inhibitory potential against the mycobacterial model strains Mycobacterium smegmatis mc2 155, Mycobacterium aurum ATCC 23366, and Mycobacterium bovis BCG ATCC 35734. The minimum inhibitory concentrations (MICs) of the carvotacetones were detected through high-throughput spot culture growth inhibition (HT-SPOTi) and microbroth dilution assays. In order to assess the potential of the compounds 1 and 6 to accumulate ethidium bromide (EtBr) in M. smegmatis and M. aurum, a microtiter plate-based fluorometric assay was used to determine efflux activity. Compounds 1 and 6 were analyzed for their modulating effects on the MIC of EtBr and the antibiotic rifampicin (RIF) against M. smegmatis. Carvotacetones 1 and 6 had potent antibacterial effects on M. aurum and M. bovis BCG (MIC ≤ 31.25 mg/L) and could successfully enhance EtBr activity against M. smegmatis. Compound 1 appeared as the most efficient agent for impairing the efflux mechanism in M. smegmatis. Both compounds 1 and 6 were highly effective against M. aurum and M. bovis BCG. In particular, compound 1 was identified as a valuable candidate for inhibiting mycobacterial efflux mechanisms and as a promising adjuvant in the therapy of tuberculosis or other non-tubercular mycobacterial infections.

Climate change is primarily manifested by elevated temperature and carbon dioxide (CO2) levels and is projected to provide suitable cultivation grounds for pests and pathogens in the otherwise unsuitable regions. The impacts of climate change have been predicted in many parts of the world, which could threaten global food safety and food security. The aim of the present work was therefore to examine the interacting effects of water activity (aw) (0.92, 0.95, 0.98 aw), CO2 (400, 800, 1200 ppm) and temperature (30, 35 °C and 30, 33 °C for Fusarium verticillioides and F. graminearum, respectively) on fungal growth and mycotoxin production of acclimatised isolates of F. verticillioides and F. graminearum isolated from maize. To determine fungal growth, the colony diameters were measured on days 1, 3, 5, and 7. The mycotoxins produced were quantified using a quadrupole-time-of-flight mass spectrometer (QTOF-MS) combined with ultra-high-performance liquid chromatography (UHPLC) system. For F. verticillioides, the optimum conditions for growth of fumonisin B1 (FB1), and fumonisin B2 (FB2) were 30 °C + 0.98 aw + 400 ppm CO2. These conditions were also optimum for F. graminearum growth, and zearalenone (ZEA) and deoxynivalenol (DON) production. Since 30 °C and 400 ppm CO2 were the baseline treatments, it was hence concluded that the elevated temperature and CO2 levels tested did not seem to significantly impact fungal growth and mycotoxin production of acclimatised Fusarium isolates. To the best of our knowledge thus far, the present work described for the first time the effects of simulated climate change conditions on fungal growth and mycotoxin production of acclimatised isolates of F. verticillioides and F. graminearum.

Enzyme-catalyzed chemical reactions produce heat. We developed an enclosed, capillary-perfused nanocalorimeter platform for thermometric enzyme-linked immunosorbent assay (TELISA). We used catalase as enzymes to model the thermal characteristics of the micromachined calorimeter. Model-assisted signal analysis was used to calibrate the nanocalorimeter and to determine reagent diffusion, enzyme kinetics, and enzyme concentration. The model-simulated signal closely followed the experimental signal after selecting for the enzyme turnover rate (kcat) and the inactivation factor (InF), using a known label enzyme amount (Ea). Over four discrete runs (n = 4), the minimized model root mean square error (RMSE) returned 1.80 ± 0.54 fmol for the 1.5 fmol experiments, and 1.04 ± 0.37 fmol for the 1 fmol experiments. Determination of enzyme parameters through calibration is a necessary step to track changing enzyme kinetic characteristics and improves on previous methods to determine label enzyme amounts on the calorimeter platform. The results obtained using model-system signal analysis for calibration led to significantly improved nanocalorimeter platform performance.

The culinary herb Satureja montana, known as winter savory, is an ingredient of traditional dishes known in different parts of the world. As an ingredient of foods it has the potential to improve their safety. In this study, the herb’s activity was investigated against Campylobacter jejuni, the leading cause of the most prevalent bacterial gastroenteritis worldwide. The ethanolic extract and essential oil of the herb were chemically characterized and six pure compounds—carvacrol, thymol, thymoquinone, p-cymene, γ-terpinene, and rosmarinic acid—were chosen for further analysis. The antimicrobial activity of the ethanolic extract (MIC 250 mg/L) was 4-fold higher compared to the essential oil. Carvacrol, thymol and thymoquinone had the strongest antimicrobial effect (MIC 31.25 mg/L) and a strong synergistic activity between carvacrol and thymol was determined (FICi 0.2). Strong inhibitory effect on C. jejuni efflux pumps (2-fold inhibition) and disruption of membrane integrity (> 80% disruption) of the herb were determined as modes of action. For resistance against the herb, C. jejuni need efflux pumps, although increased resistance against this herb does not co-occur with increased efflux pump activity, as for antibiotics. This study shows the potential of a common culinary herb for the reduction of the food pathogen C. jejuni without increasing resistance.

Inhibitors of enzymes in essential cellular pathways are potent probes to decipher intricate physiological functions of biomolecules. The analysis of Arabidopsis thaliana sterol profiles upon treatment with a series of azasterols reveals a specific in vivo inhibition of SMT2, a plant sterol-C-methyltransferase acting as a branch point between the campesterol and sitosterol biosynthetic segments in the pathway. Side chain azasteroids that modify sitosterol homeostasis help to refine its particular function in plant development.

Recently, deoxynivalenol-3-sulfate (DON-3-sulfate) was proposed as a major DON metabolite in poultry. In the present work, the first LC-MS/MS based method for determination of DON-3-sulfate, deepoxy-DON-3-sulfate (DOM-3-sulfate), DON, DOM, DON sulfonates 1, 2, 3, and DOM sulfonate 2 in excreta samples of chickens and turkeys was developed and validated. To this end, DOM-3-sulfate was chemically synthesized and characterized by NMR and LC-HR-MS/MS measurements. Application of the method to excreta and chyme samples of four feeding trials with turkeys, chickens, pullets, and roosters confirmed DON-3-sulfate as the major DON metabolite in all poultry species studied. Analogously to DON-3-sulfate, DOM-3-sulfate was formed after oral administration of DOM both in turkeys and in chickens. In addition, pullets and roosters metabolized DON into DOM-3-sulfate. In vitro transcription/translation assays revealed DOM-3-sulfate to be 2000 times less toxic on the ribosome than DON. Biological recoveries of DON and DOM orally administered to broiler chickens, turkeys, and pullets were 74%–106% (chickens), 51%–72% (roosters), and 131%–151% (pullets). In pullets, DON-3-sulfate concentrations increased from jejunum chyme samples to excreta samples by a factor of 60. This result, put into context with earlier studies, indicates fast and efficient absorption of DON between crop and jejunum, conversion to DON-3-sulfate in intestinal mucosa, liver, and possibly kidney, and rapid elimination into excreta via bile and urine.

Deoxynivalenol (DON) is a protein synthesis inhibitor produced by the Fusarium species, which frequently contaminates grains used for human or animal consumption. We treated a wheat suspension culture with DON or one of its acetylated derivatives, 3-acetyl-DON (3-ADON), 15-acetyl-DON (15-ADON) and 3,15-diacetyl-DON (3,15-diADON), and monitored the metabolization over a course of 96 h. Supernatant and cell extract samples were analyzed using a tailored LC-MS/MS method for the quantification of DON metabolites. We report the formation of tentatively identified DON-15-O-β-D-glucoside (D15G) and of 15-acetyl-DON-3-sulfate (15-ADON3S) as novel deoxynivalenol metabolites in wheat. Furthermore, we found that the recently identified 15-acetyl-DON-3-O-β-D-glucoside (15-ADON3G) is the major metabolite produced after 15-ADON challenge. 3-ADON treatment led to a higher intracellular content of toxic metabolites after six hours compared to all other treatments. 3-ADON was exclusively metabolized into DON before phase II reactions occurred. In contrast, we found that 15-ADON was directly converted into 15-ADON3G and 15-ADON3S in addition to metabolization into deoxynivalenol-3-O-β-D-glucoside (D3G). This study highlights significant differences in the metabolization of DON and its acetylated derivatives.

In the scientific field, there is a progressive awareness about the potential implications of food processing on mycotoxins especially concerning thermal treatments. High temperatures may cause, in fact, transformation or degradation of these compounds. This work is aimed to study the fate of mycotoxins during bakery processing, focusing on deoxynivalenol (DON) and deoxynivalenol-3-glucoside (DON3Glc), along the chain of industrial rusk production. Starting from naturally contaminated bran, we studied how concentrations of DON and DON3Glc are influenced by modifying ingredients and operative conditions. The experiments were performed using statistical Design of Experiment (DoE) schemes to synergistically explore the relationship between mycotoxin reduction and the indicated processing transformation parameters. All samples collected during pilot plant experiments were analyzed with an LC-MS/MS multimycotoxin method. The obtained model shows a good fitting, giving back relevant information in terms of optimization of the industrial production process, in particular suggesting that time and temperature in baking and toasting steps are highly relevant for minimizing mycotoxin level in rusks. A reduction up to 30% for DON and DON3Glc content in the finished product was observed within an acceptable technological range.