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Sterols are produced via complex, multistep biosynthetic pathways involving similar enzymatic conversions in plants, animals and fungi, yielding a variety of sterol metabolites with slightly different chemical properties to exert diverse and specific functions. A tremendously diverse landscape of sterols, and sterol-derived compounds, can be found across the plant kingdom, determining a wide spectrum of functions. Resolving the underlying biosynthetic pathways is thus instrumental to understanding the function and use of these molecules. In only a few plants, sterol biosynthesis has been studied using mutants. In non-model species a pharmacological approach is required. However, this relies on only a few inhibitors. Here, we probed a collection of inhibitors of mammalian cholesterol biosynthesis to identify new inhibitors of plant sterol biosynthesis. We show that imidazole-type fungicides, bifonazole, clotrimazole and econazole inhibit the obtusifoliol 14α-demethylase CYP51 in plants. Moreover, we found that the selective estrogen receptor modulator, clomiphene, inhibits sterol biosynthesis in part by inhibiting the plant-specific cyclopropyl-cycloisomerase CPI1. These results demonstrate that rescreening of inhibitors animal sterol biosynthesis is an easy approach for identifying novel inhibitors of plant sterol biosynthesis. These molecules expand the toolkit for studying and manipulating sterol biosynthesis in the plant kingdom., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Comprehensive data on the occurrence of sterols in plant oils is currently hardly available since only a few sterols are obtainable as standard compounds. Accordingly, many peaks are rarely labeled in gas chromatograms due to missing or outdated information. This lack of information hampers the progress in sterol research. For this reason, gas chromatography with mass spectrometry in selected ion monitoring mode (GC/MS-SIM) was used to create a database that summarizes the occurrence and semi-quantitative levels of 150 sterols with 27-32 carbon atoms and 0-4 double bonds in 66 different vegetable oils and eight other matrices. The highest number of sterols was detected in rice bran and tamanu oil (40 sterols), eggplant (39 sterols), moringa, chili seed, and amaranth oil (37 sterols). Several sterols were detected in >60 of the 74 matrices. This detailed information in the database will serve users working in food authentication and the biosynthesis of sterols., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Gas chromatography with mass spectrometry (GC/MS) and fractionation steps were used to determine the sterol patterns of red goji berries in detail. Twenty-five sterols were detected in fresh berries of two species (Lycium barbarum and L. chinense) from bushes grown in the botanical garden of the University of Hohenheim, and 20 sterols were identified. The rarely occurring campesta-5,24(25)-dienol, β-sitosterol, Δ5-avenasterol, campesterol, and cycloartenol represented >60 % of the total sterol content. Maturity and drying of fresh red goji berries caused small changes but did not affect the characteristic sterol pattern. This was confirmed by analyzing various commercial dried red goji berry samples from different sources. Separated flesh and seed samples revealed pronounced differences in the sterol pattern. A new method of merging GC/MS chromatograms showed that ∼75 % of the sterols were present in seeds and ∼25 % in flesh. The unique sterol profile may be exploited to authenticate red goji berries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

4-Methylsterols (4-M-sterols) and 4,4-dimethylsterols (4,4-D-sterols) are a group of underexplored minor sterols that occur in almost all living organisms. Here, we developed a strategy for the determination of the biochemical precursors of the predominant 4-desmethylsterols in edible oils. Due to their low contribution to the sterol content in the samples, a solid phase extraction (SPE) method was developed for the enrichment of 4-M- and 4,4-D-sterols in the hexane extracts of saponified oils. In a two-fold SPE procedure, the bulk of 4,4-D-sterols was collected in one fraction. The residual sample was subjected to a second SPE step which targeted all 4-M-sterols and low shares of 4,4-D-sterols in one fraction and the predominant 4-desmethylsterols in another one. After silylation of the SPE fractions, gas chromatography with mass spectrometry (GC/MS) was used to analyze 4,4-D- and 4-M-sterols. The results were used to define eight subgroups whose characteristic structural features could be linked with the presence of specific m/z values. These m/z values were measured sensitively by GC/MS operated in selected ion monitoring (SIM) mode. Application of the GC/MS method to eighteen edible oils enabled the detection of 55 mostly very low abundant 4-M- and 4,4-D-sterols. Twenty-four of the 4-M- and 4,4-D-sterols could be assigned and the remaining 31 unknown sterols could be traced back to their basic structures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Countercurrent chromatography (CCC) is a preparative instrumental method where both the mobile and stationary phases are liquids and which are predominantly used for the isolation of natural products. In this study, we widened the scope of CCC by using it as an instrumental method for the direct enrichment of the free sterol fraction from plant oils to which they contribute with ~ 1%. For the enrichment of sterols in a narrow band, we employed the so-called co-current CCC (ccCCC) mode in which both liquid phases of the solvent system (here: n-hexane/ethanol/methanol/water (34:11:12:2, v/v/v/v)) are moved at different flow rates in the same direction. Different from previous applications of ccCCC, the lower and predominant "stationary" phase (LP s ) was pumped twice as fast as the mobile upper phase (UP m ). This novel reversed ccCCC mode improved the performance but also required a higher demand of LP s compared to UP m . Therefore, the exact phase composition of UP m and LP s was determined by gas chromatography and Karl Fischer titration. This step enabled the direct preparation of LP s which considerably reduced the waste of solvents. Internal standards (phenyl-substituted fatty acid alkyl esters) were synthesised and utilised to frame the free sterol fraction. This approach allowed a fractionation of free sterols based on the UV signal and compensated run-to-run variations. The reversed ccCCC method was then applied to the sample preparation of five vegetable oils. In addition to free sterols, free tocochromanols (tocopherols, vitamin E) were also eluted in the same fraction as free sterols., (© 2023. The Author(s).)

4,4-Dimethyl-substituted sterols are bioactive minor sterols of most animal fats and plant oils, but higher shares are present in lanolin (wool grease). Here, the isolation of the 4,4-dimethyl-substituted sterols dihydrolanosterol and lanosterol from lanolin by countercurrent chromatography (CCC) is described. An initial examination of the hexane extract of saponified lanolin showed the presence of relatively high portions of fatty alcohols which were known to co-elute with the target analytes in CCC. Hence, fatty alcohols were precipitated by urea complexation. Unexpectedly, 4,4-dimethyl-substituted sterols were also found in the crystalline fraction, while cholesterol and other desmethylsterols were detected in the liquid phase. Urea complexation represented a useful preparative method for the separation of desmethylsterols and 4,4-dimethyl-substituted sterols from lanolin. Shake flask experiments of 4,4-dimethyl-substituted sterols and fatty alcohols with 14 biphasic solvent systems indicated suitable partition coefficients (K values) with n-hexane/ethanol/water (12:8:1, v/v/v) and n-hexane/benzotrifluoride/acetonitrile (20:7:13, v/v/v). After initial tests with conventional CCC, the application of CCC in heart-cut recycling mode provided 4,4-dimethyl-substituted sterols with purities of 99 % (dihydrolanosterol) and 95 % (lanosterol)., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Phytosterols were analyzed in 34 different quinoa accessions, which were obtained from the same field trial. Twenty different sterols were detected, and 17 could be structurally assigned by means of gas chromatography with mass spectrometry. Sterols were quantitated in selected ion monitoring mode (GC/MS-SIM) with the novel internal standard 3- O - tert -butyldimethylsilyl-cholestanol (cholestanyl-TBDMS). GC/MS-SIM response factors of minor sterols were determined after enrichment by countercurrent chromatography. The total sterol contents varied from 120 to 180 mg/100 g of seeds, which is higher than has been described in quinoa before. This was due to the fact that Δ7-sterols (e.g., Δ7-sitosterol, spinasterol, and Δ7-avenasterol) were quantitated for the first time in quinoa and contributed ∼64% to the total sterol content. Clustering allowed distributing of the 34 different quinoa accessions into four distinct groups on the basis of the different sterol patterns.

Sterols are a highly complex group of lipophilic compounds present in the unsaponifiable matter of virtually all living organisms. In this study, we developed a novel gas chromatography with mass spectrometry selected ion monitoring (GC/MS-SIM) method for the comprehensive analysis of sterols after saponification and silylation. A new referencing system was introduced by means of a series of saturated fatty acid pyrrolidides (FAPs) as internal standards. Linked with retention time locking (RTL), the resulting FAP retention indices (RI FAP ) of the sterols could be determined with high precision. The GC/MS-SIM method was based on the parallel measurement of 17 SIM ions in four time windows. This set included eight molecular ions and seven diagnostic fragment ions of silylated sterols as well as two abundant ions of FAPs. Altogether, twenty molecular ions of C 27 - to C 31 -sterols with 0-3 double bonds were included in the final method. Screening of four common vegetable oils (sunflower oil, hemp oil, rapeseed oil, and corn oil) enabled the detection of 30 different sterols and triterpenes most of which could be identified., (© 2021. The Author(s).)

Halogenated natural products (HNPs) and persistent organic pollutants (POPs) were quantified in South African sardines ( Sardinops sagax ) from one site in the South Atlantic Ocean and one in the Indian Ocean. At both sites, HNPs [2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1), mixed halogenated compound 1 (MHC-1), 2,4,6-tribromoanisole (2,4,6-TBA), 2'-MeO-BDE 68 (BC-2), and 6-MeO-BDE 47 (BC-3)] were 1 order of magnitude higher concentrated than anthropogenic POPs [mainly polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT), ∼3 ng/g lipids]. MHC-1 and Q1 were the major HNPs in the samples from both sites, contributing with up to 49 and 52 ng/g lipids, respectively. The same 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane ( p , p '-DDE)/PCB ratio suggested that the major POPs were evenly distributed at both sites. Different ratios of Q1/MHC-1 in the samples from the Indian (∼2:1) and South Atlantic (∼1:1) Oceans indicated that the occurrence of HNPs in seafood is difficult to predict and should be investigated more in detail. The PCB levels in sardines were found to pose no risk to human consumers, whereas HNPs could not be evaluated because of the lack of toxicological data.

Previous studies have demonstrated that various tricarboxylic acid (TCA) cycle genes, particularly the succinate dehydrogenase genes (sdhCAB), are upregulated in Staphylococcus aureus biofilms. To better study the role of this enzyme complex, an sdhCAB deletion mutant (Deltasdh) was constructed. Compared to the wild type (wt) the mutant was impaired in planktonic growth under aerobic conditions, excreted acetic acid could not be reused and accumulated continuously, succinate was excreted and found in the culture supernatant, and metabolome analysis with cells grown in chemically defined medium revealed reduced uptake/metabolism of some amino acids from the growth medium. Moreover, the mutant was able to counteract the steadily decreasing extracellular pH by increased urease activity. The addition of fumarate to the growth medium restored the wt phenotype. The mutant showed a small-colony variant (SCV)-like phenotype, a slight increase in resistance to various aminoglycoside antibiotics, and decreased pigmentation. The decreased growth under aerobic conditions is due to the interruption of the TCA cycle (indicated by the accumulation of succinate and acetic acid) with the consequence that many fewer reduction equivalents (NADH and FADH2) can fuel the respiratory chain. The results indicate that the TCA cycle is required for acetate and amino acid catabolism; its upregulation under biofilm conditions is advantageous under such nutrient- and oxygen-limited conditions.

The cytoplasmic histidine sensor kinase NreB of Staphylococcus carnosus responds to O(2) and controls together with the response regulator NreC the expression of genes of nitrate/nitrite respiration. nreBC homologous genes were found in Staphylococcus strains and Bacillus clausii, and a modified form was found in some Lactobacillus strains. NreB contains a sensory domain with similarity to heme B binding PAS domains. Anaerobically prepared NreB of S. carnosus exhibited a (diamagnetic) [4Fe-4S](2+) cluster when assessed by Mossbauer spectroscopy. Upon reaction with air, the cluster was degraded with a half-life of approximately 2.5 min. No significant amounts of Mossbauer or EPR detectable intermediates were found during the decay, but magnetic Mossbauer spectra revealed formation of diamagnetic [2Fe-2S](2+) clusters. After extended exposure to air, NreB was devoid of a FeS cluster. Photoreduction with deazaflavin produced small amounts of [4Fe-4S](+), which were degraded subsequently. The magnetically perturbed Mossbauer spectrum of the [4Fe-4S](2+) cluster corroborated the S = 0 spin state and revealed uniform electric field gradient tensors of the iron sites, suggesting full delocalization of the valence electrons and binding of each of the Fe ions by four S ligands, including the ligand to the protein. Mutation of each of the four Cys residues inactivated NreB function in vivo in accordance with their role as ligands. [4Fe-4S](2+) cluster-containing NreB had high kinase activity. Exposure to air decreased the kinase activity and content of the [4Fe-4S](2+) cluster with similar half-lives. We conclude that the sensory domain of NreB represents a new type of PAS domain containing a [4Fe-4S](2+) cluster for sensing and function.

Here, we investigate the functionality of the oxygen-responsive nitrogen regulation system NreABC in the human pathogen Staphylococcus aureus and evaluate its role in anaerobic gene regulation and virulence factor expression. Deletion of nreABC resulted in severe impairment of dissimilatory nitrate and nitrite reduction and led to a small-colony phenotype in the presence of nitrate during anaerobic growth. For characterization of the NreABC regulon, comparative DNA microarray and proteomic analyses between the wild type and nreABC mutant were performed under anoxic conditions in the absence and presence of nitrate. A reduced expression of virulence factors was not observed in the mutant. However, both the transcription of genes involved in nitrate and nitrite reduction and the accumulation of corresponding proteins were highly decreased in the nreABC mutant, which was unable to utilize nitrate as a respiratory oxidant and, hence, was forced to use fermentative pathways. These data were corroborated by the quantification of the extracellular metabolites lactate and acetate. Using an Escherichia coli-compatible two-plasmid system, the activation of the promoters of the nitrate and nitrite reductase operons and of the putative nitrate/nitrite transporter gene narK by NreBC was confirmed. Overall, our data indicate that NreABC is very likely a specific regulation system that is essential for the transcriptional activation of genes involved in dissimilatory reduction and transport of nitrate and nitrite. The study underscores the importance of NreABC as a fitness factor for S. aureus in anoxic environments.

Several environmental stresses have been demonstrated to increase polysaccharide intercellular adhesin (PIA) synthesis and biofilm formation by the human pathogens Staphylococcus aureus and Staphylococcus epidermidis. In this study we characterized an adaptive response of S. aureus SA113 to nitrite-induced stress and show that it involves concomitant impairment of PIA synthesis and biofilm formation. Transcriptional analysis provided evidence that nitrite, either as the endogenous product of respiratory nitrate reduction or after external addition, causes repression of the icaADBC gene cluster, mediated likely by IcaR. Comparative microarray analysis revealed a global change in gene expression during growth in the presence of 5 mM sodium nitrite and indicated a response to oxidative and nitrosative stress. Many nitrite-induced genes are involved in DNA repair, detoxification of reactive oxygen and nitrogen species, and iron homeostasis. Moreover, preformed biofilms could be eradicated by the addition of nitrite, likely the result of the formation of toxic acidified nitrite derivatives. Nitrite-mediated inhibition of S. aureus biofilm formation was abrogated by the addition of nitric oxide (NO) scavengers, suggesting that NO is directly or indirectly involved. Nitrite also repressed biofilm formation of S. epidermidis RP62A.

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens and frequently coinfect the lungs of cystic fibrosis patients. P. aeruginosa secretes an arsenal of small respiratory inhibitors, like pyocyanin, hydrogen cyanide, or quinoline N-oxides, that may act against the commensal flora as well as host cells. Here, we show that with respect to their susceptibility to these respiratory inhibitors, staphylococcal species can be divided into two groups: the sensitive group, comprised of pathogenic species such as S. aureus and S. epidermidis, and the resistant group, represented by nonpathogenic species such as S. carnosus, S. piscifermentans, and S. gallinarum. The resistance in the latter group of species was due to cydAB genes that encode a pyocyanin- and cyanide-insensitive cytochrome bd quinol oxidase. By exchanging cydB in S. aureus with the S. carnosus-specific cydB, we could demonstrate that CydB determines resistance. The resistant or sensitive phenotype was based on structural alterations in CydB, which is part of CydAB, the cytochrome bd quinol oxidase. CydB represents a prime example of both microevolution and the asymmetric pattern of evolutionary change.