Evaluation of sterols as markers of fungal spoilage in red pepper powder.

Red pepper powder (RPP) made from ground dried red pepper (Capsicum annuum L.) is prone to adulteration with fungal-spoiled RPP to gain unfair profits in Korea. This study aimed to investigate the effects of fungal infection on the ergosterol and phytosterol content of RPP and evaluate the potential of the sterol content as a marker for identifying fungal-spoiled RPP. Ergosterol was detected only in fungal-spoiled RPP and not in unspoiled RPP [

Comprehensive gas chromatography with mass spectrometry analysis of sterols in red goji berries (Lycium sp.).

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, ß-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.

FooDOxS: a database of oxidized sterols content in foods.

Dietary oxidized sterols (DOxS) are cholesterol-like molecules known to exert pro-inflammatory, pro-oxidant, and pro-apoptotic effects, among others. We present the FooDOxS database, a comprehensive compilation of DOxS content in over 1680 food items from 120 publications across 25 countries, augmented by data generated by our group. This database reports DOxS content in foods classified under the NOVA and What We Eat in America (WWEIA) systems, allowing a comprehensive and statistically robust summary of DOxS content in foods. Notably, we evaluated the efficacy of using NOVA and WWEIA classifications in capturing DOxS variations across food categories. Our findings provide insights into the strengths and limitations of these classification systems, enhancing their utility for assessing dietary components. This research contributes to the understanding of DOxS in food processing and suggests refinements for classification systems, holding promise for improved food safety and public health assessments.

Relationship between nitrate, heavy metal, and sterols contents in Japanese agricultural soils with risk of groundwater pollution.

In Japanese agricultural lands, nitrate-nitrogen contamination of soil and groundwater often occurs due to the application of livestock excrements and compost. Therefore, rural soils in Japan were sampled and analyzed for nitrate-nitrogen leaching, heavy metal content, and sterols associated with livestock excrement and compost to calculate contamination risk indicators. The results were analyzed using self-organizing maps and cluster analysis. Nitrate-nitrogen content using water extraction was detected in most of the sampled soils. In addition, many samples from areas that were already severely contaminated with nitrate-nitrogen showed particularly high concentrations. Coprostanol, an indicator of fecal contamination, was detected in more than half of the samples. The main source of nitrate-nitrogen contamination in these areas is livestock excrement and compost. Self-organization maps showed that areas with high nitrate-nitrogen contamination also corresponded to areas with high copper and zinc soil contents. The self-organization maps and cluster analysis resulted in five clusters: a nitrate-contaminated group mainly originating from livestock excrement and compost, a heavy metal-contaminated group, a general group, a nitrate-contaminated group mainly originating from chemical fertilizers, and a contaminated group with potentially hazardous substances requiring attention. Authorities and decision-makers can use the results to prioritize areas requiring remediation.

7-Dehydrocholesterol: A sterol shield against an iron sword.

Li et al. and Freitas et al. recently identified 7-dehydrocholesterol (7-DHC), a sterol produced through the cholesterol biosynthetic pathway, as a lipid-soluble antioxidant that protects cells from ferroptosis, a cell death pathway triggered by iron-catalyzed phospholipid peroxidation.1,2.

Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation.

The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.

Pregnenolone and progesterone production from natural sterols using recombinant strain of Mycolicibacterium smegmatis mc2 155 expressing mammalian steroidogenesis system.

BACKGROUND: Pregnenolone and progesterone are the life-important steroid hormones regulating essential vital functions in mammals, and widely used in different fields of medicine. Microbiological production of these compounds from sterols is based on the use of recombinant strains expressing the enzyme system cholesterol hydroxylase/C20-C22 lyase (CH/L) of mammalian steroidogenesis. However, the efficiency of the known recombinant strains is still low. New recombinant strains and combination approaches are now needed to produce these steroid hormones. RESULTS: Based on Mycolicibacterium smegmatis, a recombinant strain was created that expresses the steroidogenesis system (CYP11A1, adrenodoxin reductase, adrenodoxin) of the bovine adrenal cortex. The recombinant strain transformed cholesterol and phytosterol to form progesterone among the metabolites. When 3-methoxymethyl ethers of sterols were applied as bioconversion substrates, the corresponding 3-ethers of pregnenolone and dehydroepiandrosterone (DHEA) were identified as major metabolites. Under optimized conditions, the recombinant strain produced 85.2 ± 4.7 mol % 3-methoxymethyl-pregnenolone within 48 h, while production of 3-substituted DHEA was not detected. After the 3-methoxymethyl function was deprotected by acid hydrolysis, crystalline pregnenolone was isolated in high purity (over 98%, w/w). The structures of steroids were confirmed using TLC, HPLC, MS and 1H- and 13C-NMR analyses. CONCLUSION: The use of mycolicybacteria as a microbial platform for the expression of systems at the initial stage of mammalian steroidogenesis ensures the production of valuable steroid hormones-progesterone and pregnenolone from cholesterol. Selective production of pregnenolone from cholesterol is ensured by the use of 3-substituted cholesterol as a substrate and optimization of the conditions for its bioconversion. The results open the prospects for the generation of the new microbial biocatalysts capable of effectively producing value-added steroid hormones.

Functional Plasticity, Redundancy, and Specificity of Lanosterol 14α-Demethylase in Regulating the Sensitivity to DMIs in Calonectria ilicicola.

Cytochrome P450 sterol 14α-demethylase (CYP51) is a key enzyme involved in the sterol biosynthesis pathway and serves as a target for sterol demethylation inhibitors (DMIs). In this study, the 3D structures of three CPY51 paralogues from Calonectria ilicicola (C. ilicicola) were first modeled by AlphaFold2, and molecular docking results showed that CiCYP51A, CiCYP51B, or CiCYP51C proteins individually possessed two active pockets that interacted with DMIs. Our results showed that the three paralogues play important roles in development, pathogenicity, and sensitivity to DMI fungicides. Specifically, CiCYP51A primarily contributed to cell wall integrity maintenance and tolerance to abiotic stresses, and CiCYP51B was implicated in sexual reproduction and virulence, while CiCYP51C exerted negative regulatory effects on sterol 14α-demethylase activity within the ergosterol biosynthetic pathway, revealing its genus-specific function in C. ilicicola. These findings provide valuable insights into developing rational strategies for controlling soybean red crown rot caused by C. ilicicola.

Sterol Derivatives Specifically Increase Anti-Inflammatory Oxylipin Formation in M2-like Macrophages by LXR-Mediated Induction of 15-LOX.

The understanding of the role of LXR in the regulation of macrophages during inflammation is emerging. Here, we show that LXR agonist T09 specifically increases 15-LOX abundance in primary human M2 macrophages. In time- and dose-dependent incubations with T09, an increase of 3-fold for ALOX15 and up to 15-fold for 15-LOX-derived oxylipins was observed. In addition, LXR activation has no or moderate effects on the abundance of macrophage marker proteins such as TLR2, TLR4, PPARγ, and IL-1RII, as well as surface markers (CD14, CD86, and CD163). Stimulation of M2-like macrophages with FXR and RXR agonists leads to moderate ALOX15 induction, probably due to side activity on LXR. Finally, desmosterol, 24(S),25-Ep cholesterol and 22(R)-OH cholesterol were identified as potent endogenous LXR ligands leading to an ALOX15 induction. LXR-mediated ALOX15 regulation is a new link between the two lipid mediator classes sterols, and oxylipins, possibly being an important tool in inflammatory regulation through anti-inflammatory oxylipins.

The relationship between aflatoxin B1 with the induction of extrinsic/intrinsic pathways of apoptosis and the protective role of taraxasterol in TM3 leydig cell line.

Aflatoxins B1 (AFB1) a dangerous type of aflatoxin, poses a serious threat to human health. Meanwhile, Taraxasterol, a bioactive compound in dandelion, exhibits strong anti-inflammatory and antioxidant activity. Therefore, the aim of this study was to investigate the impact of AFB1 on the intrinsic and extrinsic pathways of apoptosis, as well as evaluate the protective role of taraxasterol in the TM3 Leydig cell line. Cell viability was evaluated using an MTT assay, measuring the effects of 3.6 µM AFB1 and varying concentrations of taraxasterol. Expression levels of Caspase 3,8, and 9 were analyzed with RT-qPCR, and flow cytometry was used to assess cell cycle progression and apoptotic alterations. The findings of this study demonstrated that exposure to 3.6 µM of AFB1 resulted in an upregulation of Caspase 3 and Caspase 9 expression, indicating an activation of apoptotic pathways in TM3 cells. Additionally, the analysis of apoptosis revealed a significant increase in cellular apoptosis at this AFB1 concentration. However, when TM3 cells were exposed to 5 µM of taraxasterol, a downregulation of Caspase 3 and Caspase 9 expression was observed, suggesting a protective effect against apoptosis. Moreover, the apoptotic rate in TM3 cells was reduced in the presence of 5 µM of taraxasterol. Consequently, this study highlights the potential of taraxasterol as a protective agent against AFB1-induced apoptosis and suggest its potential application in regulating cell survival and apoptosis-related processes. Further investigations are necessary to elucidate the underlying mechanisms and evaluate the clinical implications of taraxasterol in the context of fertility disorders and other conditions associated with AFB1 exposure.

Sterols, pleiotropic players in plant-microbe interactions.

Plant-microbe interactions (PMIs) are regulated through a wide range of mechanisms in which sterols from plants and microbes are involved in numerous ways, including recognition, transduction, communication, and/or exchanges between partners. Phytosterol equilibrium is regulated by PMIs through expression of genes involved in phytosterol biosynthesis, together with their accumulation. As such, PMI outcomes also include plasma membrane (PM) functionalization events, in which phytosterols have a central role, and activation of sterol-interacting proteins involved in cell signaling. In spite (or perhaps because) of such multifaceted abilities, an overall mechanism of sterol contribution is difficult to determine. However, promising approaches exploring sterol diversity, their contribution to PMI outcomes, and their localization would help us to decipher their crucial role in PMIs.

Chemical Inhibition of Sterol Biosynthesis.

Cholesterol is an essential molecule of life, and its synthesis can be inhibited by both genetic and nongenetic mechanisms. Hundreds of chemicals that we are exposed to in our daily lives can alter sterol biosynthesis. These also encompass various classes of FDA-approved medications, including (but not limited to) commonly used antipsychotic, antidepressant, antifungal, and cardiovascular medications. These medications can interfere with various enzymes of the post-lanosterol biosynthetic pathway, giving rise to complex biochemical changes throughout the body. The consequences of these short- and long-term homeostatic disruptions are mostly unknown. We performed a comprehensive review of the literature and built a catalogue of chemical agents capable of inhibiting post-lanosterol biosynthesis. This process identified significant gaps in existing knowledge, which fall into two main areas: mechanisms by which sterol biosynthesis is altered and consequences that arise from the inhibitions of the different steps in the sterol biosynthesis pathway. The outcome of our review also reinforced that sterol inhibition is an often-overlooked mechanism that can result in adverse consequences and that there is a need to develop new safety guidelines for the use of (novel and already approved) medications with sterol biosynthesis inhibiting side effects, especially during pregnancy.

Expression of honey bee (Apis mellifera) sterol homeostasis genes in food jelly producing glands of workers.

Adult workers of Western honey bees (Apis mellifera L.) acquire sterols from their pollen diet. These food sterols are transported by the hemolymph to peripheral tissues such as the mandibular and the hypopharyngeal glands in the worker bees' heads that secrete food jelly which is fed to developing larvae. As sterols are obligatory components of biological membranes and essential precursors for molting hormone synthesis in insects, they are indispensable to normal larval development. Thus, the study of sterol delivery to larvae is important for a full understanding of honey bee larval nutrition and development. Whereas hypopharyngeal glands only require sterols for their membrane integrity, mandibular glands add sterols, primarily 24-methylenecholesterol, to its secretion. For this, sterols must be transported through the glandular epithelial cells. We have analyzed for the first time in A. mellifera the expression of genes which are involved in intracellular movement of sterols. Mandibular and hypopharyngeal glands were dissected from newly emerged bees, 6-day-old nurse bees that feed larvae and 26-day-old forager bees. The expression of seven genes involved in intracellular sterol metabolism was measured with quantitative real-time PCR. Relative transcript abundance of sterol metabolism genes was significantly influenced by the age of workers and specific genes but not by gland type. Newly emerged bees had significantly more transcripts for six out of seven genes than older bees indicating that the bulk of the proteins needed for sterol metabolism are produced directly after emergence.

Investigating the Quality and Purity Profiles of Olive Oils from Diverse Regions in Selçuk, Izmir.

The Selçuk district of Izmir is one of the most essential regions in terms of olive oil production. In this study, 60 olive oil samples were obtained from five different locations (ES: Eski Sirince Yolu, KK: Kinali Köprü, AU: Abu Hayat Üst, AA: Abu Hayat Alt, and DB: Degirmen Bogazi) in the Selçuk region of Izmir during two (2019-2020 and 2020-2021) consecutive cropping seasons. Quality indices (free acidity, peroxide value, p-Anisidine value, TOTOX, and spectral absorption at 232 and 270 nm) and the fatty acid, phenolic, and sterol profiles of the samples were determined to analyze the changes in the composition of Selcuk olive oils according to their growing areas. When the quality criteria were analyzed, it was observed that KK had the lowest FFA (0.11% oleic acid, PV (6.66 meq O2/kg), p-ANV (11.95 mmol/kg), TOTOX (25.28), and K232 (1.99) values and K270 had the highest value. During the assessment of phenolic profiles, the ES group exhibited the highest concentration of the phenolic compound p-HPEA-EDA (oleocanthal), with a content of 93.58 mg/kg, equivalent to tyrosol. Upon analyzing the fatty acid and sterol composition, it was noted that AU displayed the highest concentration of oleic acid (71.98%) and ß-sitosterol (87.65%). PCA analysis illustrated the distinct separation of the samples, revealing significant variations in both sterol and fatty acid methyl ester distributions among oils from different regions. Consequently, it was determined that VOOs originating from the Selçuk region exhibit distinct characteristics based on their geographical locations. Hence, this study holds great promise for the region to realize geographically labeled VOOs.

Octadecyl and sulfonyl modification of diatomite synergistically improved the immobilization efficiency of lipase and its application in the synthesis of pine sterol esters.

Phytosterols usually have to be esterified to various phytosterol esters to avoid their disadvantages of unsatisfactory solubility and low bioavailability. The enzymatic synthesis of phytosterol esters in a solvent-free system has advantages in terms of environmental friendliness, sustainability, and selectivity. However, the limitation of the low stability and recyclability of the lipase in the solvent-free system, which often requires a relatively high temperature to induce the viscosity, also increased the industrial production cost. In this context, a low-cost material, namely diatomite, was employed as the support in the immobilization of Candida rugosa lipase (CRL) due to its multiple modification sites. The Fe3 O4 was also then introduced to this system for quick and simple separation via the magnetic field. Moreover, to further enhance the immobilization efficiency of diatomite, a modification strategy which involved the octadecyl and sulfonyl group for regulating the hydrophobicity and interaction between the support and lipase was successfully developed. The optimization of the ratio of the modifiers suggested that the -SO3 H/C18 (1:1.5) performed best with an enzyme loading and enzyme activity of 84.8 mg·g-1 and 54 U·g-1 , respectively. Compared with free CRL, the thermal and storage stability of CRL@OSMD was significantly improved, which lays the foundation for the catalytic synthesis of phytosterol esters in solvent-free systems. Fortunately, a yield of 95.0% was achieved after optimizing the reaction conditions, and a yield of 70.0% can still be maintained after six cycles.

Transgenerational effects of triazole fungicides on gene expression and egg compounds in non-exposed offspring: A case study using Red-Legged Partridges (Alectoris rufa).

Triazole fungicides are widely used to treat cereal seeds before sowing. Granivorous birds like the Red-legged Partridge (Alectoris rufa) have high exposure risk because they ingest treated seeds that remain on the field surface. As triazole fungicides can act as endocrine disruptors, affecting sterol synthesis and reproduction in birds several months after exposure, we hypothesized that these effects could also impact subsequent generations of exposed birds. To test this hypothesis, we exposed adult partridges (F0) to seeds treated at commercial doses with four different formulations containing triazoles as active ingredients (flutriafol, prothioconazole, tebuconazole, and a mixture of the latter two), simulating field exposure during late autumn sowing. During the subsequent reproductive season, two to four months after exposure, we examined compound allocation of steroid hormones, cholesterol, vitamins, and carotenoids in eggs laid by exposed birds (F1), as well as the expression of genes encoding enzymes involved in sterol biosynthesis in one-day-old chicks of this F1. One year later, F1 animals were paired again to investigate the expression of the same genes in the F2 chicks. We found changes in the expression of some genes for all treatments and both generations. Additionally, we observed an increase in estrone levels in eggs from partridges treated with flutriafol compared to controls, a decrease in tocopherol levels in partridges exposed to the mixture of tebuconazole and prothioconazole, and an increase in retinol levels in partridges exposed to prothioconazole. Despite sample size limitations, this study provides novel insights into the mechanisms of action of the previously observed effects of triazole fungicide-treated seeds on avian reproduction with evidence that the effects can persist beyond the exposure windows, affecting unexposed offspring of partridges fed with treated seeds. The results highlight the importance of considering long-term chronic effects when assessing pesticide risks to wild birds.

Resolving phytosterols in microalgae using offline two-dimensional reversed phase liquid chromatography-supercritical fluid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Sterols are unsaponifiable lipids resulting from plant metabolism that exhibit interesting bioactive properties. Microalgae are a major source of specific phytosterols, most of which are still not fully characterized. The similarity in sterol structures and the existence of positional isomers make the separation of phytosterols challenging. A method was developed based on an offline two-dimensional (2D) system, reversed-phase liquid chromatography (RPLC)-supercritical fluid chromatography (SFC)/quadrupole time-of-flight (Q-ToF) mass spectrometry, for the identification of sterols in microalgae. Subsequent positive-mode MS/MS was used to confirm the identified phytosterols. The 2D chromatogram exhibited a pattern related to the positions of the double bonds, which were confirmed by standard injection, enabling structural elucidation. The analysis of the unsaponifiable fraction of two algae, namely Scenedesmus obliquus, a freshwater microalgae, and Padina pavonica, a marine macroalgae, highlighted the ability of the method to distinguish a large number of sterol isomers.

Stable Carbon Isotope Ratios (δ13C Values [‰]) of Individual Sterols in the Oils of C3, C4, and CAM Plants.

The compound-specific determination of δ13C values [‰] by gas chromatography interfaced with isotope ratio mass spectrometry (GC-IRMS) is a powerful analytical method to indicate minute but relevant variations in the 13C/12C ratio of sample compounds. In this study, the δ13C values [‰] of individual sterols were measured in eleven different oils of C3, C4, and CAM plants (n = 33) by GC-IRMS. For this purpose, a suitable acetylation method was developed for sterols. Nine of the eleven phytosterols identified by GC with mass spectrometry (GC/MS) could be measured by GC-IRMS. The δ13C values [‰] of individual sterols and squalene of C3 plant oils were between 3‰ and >16‰ more negative (lighter in carbon) than in C4 and CAM oils. We also showed that the blending of C4 oils into C3 oils (exemplarily conducted with one olive and one corn oil) would be precisely determined by means of the δ13C value [‰] of ß-sitosterol.

Druggable Sterol Metabolizing Enzymes in Infectious Diseases: Cell Targets to Therapeutic Leads.

Sterol biosynthesis via the mevalonate-isoprenoid pathway produces ergosterol (24ß-methyl cholesta-5,7-dienol) necessary for growth in a wide-range of eukaryotic pathogenic organisms in eukaryotes, including the fungi, trypanosomes and amoebae, while their animal hosts synthesize a structurally less complicated product-cholesterol (cholest-5-enol). Because phyla-specific differences in sterol metabolizing enzyme architecture governs the binding and reaction properties of substrates and inhibitors while the order of sterol metabolizing enzymes involved in steroidogenesis determine the positioning of crucial chokepoint enzymes in the biosynthetic pathway, the selectivity and effectiveness of rationally designed ergosterol biosynthesis inhibitors toward ergosterol-dependent infectious diseases varies greatly. Recent research has revealed an evolving toolbox of mechanistically distinct tight-binding inhibitors against two crucial methylation-demethylation biocatalysts-the C24 sterol methyl transferase (absent from humans) and the C14-sterol demethylase (present generally in humans and their eukaryotic pathogens). Importantly for rational drug design and development, the activities of these enzymes can be selectively blocked in ergosterol biosynthesis causing loss of ergosterol and cell killing without harm to the host organism. Here, we examine recent advances in our understanding of sterol biosynthesis and the reaction differences in catalysis for sterol methylation-demethylation enzymes across kingdoms. In addition, the novelties and nuances of structure-guided or mechanism-based approaches based on crystallographic mappings and substrate specificities of the relevant enzyme are contrasted to conventional phenotypic screening of small molecules as an approach to develop new and more effective pharmacological leads.