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.

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 [

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.

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.

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.

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.

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.

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.

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.

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.

Metabolite profiling and histochemical localization of alkaloids in Hippeastrum papilio (Ravena) van Scheepen.

Hippeastrum papilio (Amaryllidaceae) is a promising new source of galanthamine - an alkaloid used for the cognitive treatment of Alzheimer's disease. The biosynthesis and accumulation of alkaloids are tissue - and organ-specific. In the present study, histochemical localization of alkaloids in H. papilio's plant organs with Dragendorff's reagent, revealed their presence in all studied samples. Alkaloids were observed in vascular bundles, vacuoles, and intracellular spaces, while in other plant tissues and structures depended on the plant organ. The leaf parenchyma and the vascular bundles were indicated as alkaloid-rich structures which together with the high proportion of alkaloids in the phloem sap (49.3% of the Total Ion Current - TIC, measured by GC-MS) indicates the green tissues as a possible site of galanthamine biosynthesis. The bulbs and roots showed higher alkaloid content compared to the leaf parts. The highest alkaloid content was found in the inner bulb part. GC-MS metabolite profiling of H. papilio's root, bulb, and leaves revealed about 82 metabolites (>0.01% of TIC) in the apolar, polar, and phenolic acid fractions, including organic acids, fatty acids, sterols, sugars, amino acids, free phenolic acids, and conjugated phenolic acids. The most of organic and fatty acids were in the peak part of the root, while the outermost leaf was enriched with sterols. The outer and middle parts of the bulb had the highest amount of saccharides, while the peak part of the middle leaf had most of the amino acids, free and conjugated phenolic acids.

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.

Fluorescent probes and degraders of the sterol transport protein Aster-A.

Our understanding of sterol transport proteins (STPs) has increased exponentially in the last decades with advances in the cellular and structural biology of these important proteins. However, small molecule probes have only recently been developed for a few selected STPs. Here we describe the synthesis and evaluation of potential proteolysis-targeting chimeras (PROTACs) based on inhibitors of the STP Aster-A. Based on the reported Aster-A inhibitor autogramin-2, ten PROTACs were synthesized. Pomalidomide-based PROTACs functioned as fluorescent probes due to the intrinsic fluorescent properties of the aminophthalimide core, which in some cases was significantly enhanced upon Aster-A binding. Most PROTACs maintained excellent binary affinity to Aster-A, and one compound, NGF3, showed promising Aster-A degradation in cells. The tools developed here lay the foundation for optimizing Aster-A fluorescent probes and degraders and studying its activity and function in vitro and in cells.

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.

Taraxasterol enhanced bladder cancer cells radiosensitivity via inhibiting the COX-2/PGE2/JAK2/STAT3/MMP pathway.

PURPOSE: Radiotherapy with bladder preservation is highly acceptable among patients bearing bladder cancer (BCa), but the occurrence of secondary tolerance (ARR) during treatment is one of the important reasons for the failure of clinical radiotherapy. COX-2 has been frequently reported to be highly expressed and associated with radio-resistance in various cancers. In this study, the feasibility of Taraxasterol (Tara) as a radiosensitizer was investigated, and the target effect of Tara on COX-2 and its underlying mechanism were explored. METHODS AND MATERIALS: The toxicity of Tara toward BCa cells was detected with the MTT method and cells in response to IR or Tara + IR were compared by clone formation assay. Next, a small RNA interference system (siRNA) was employed to decrease endogenous COX-2 expression in BCa cells, and the stem cell-like features and motion abilities of BCa cells under different treatments were investigated using microsphere formation and transwell chamber assay, respectively. Meanwhile, the expression of a series of inflammation-related molecules and stem cell characteristic molecules was determined by qRT-PCR, western blot and ELISA method. In vivo studies, BCa cells were subcutaneously injected into the right flank of each male mouse. Those mice were then grouped and exposed to different treatment: Tara, IR, IR + Tara and untreated control. The volumes of each tumor were measured every two days and target proteins were detected with immunohistochemical (IHC) staining. RESULTS: The results show that COX-2 decline, due to COX-2 knocking-down or Tara treatment, could greatly enhance BCa cells' radiosensitivity and significantly decrease their migration, invasion and microsphere formation abilities, companied with the reduce of JAK2, phos-STAT3, MMP2 and MMP9 expression. However, Tara could not further reduce the expression of an above molecule of cells in COX-2-deficient BCa cells. Correspondingly, Tara treatment could not further enhance those siCOX-2 BCa cells response to IR. CONCLUSIONS: Our data support that Tara can improve the radiosensitivity of BCa cells by targeting COX-2/PGE2. The mechanism may involve regulating STAT3 phosphorylation, DNA damage response protein activation, and expression of MMP2/MMP9.

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.