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Over 800 known carotenoids are synthesized from phytoene or 4,4'-diapophytoene (dehydrosqualene) characterized by three conjugated double bonds. In this paper, we report that carotenoid desaturase CrtN from Staphylococcus aureus and Methylomonas can accept oxidosqualene, which is the precursor for plant- or animal-type triterpenoids, yielding the yellow carotenoid pigments with 8, 9, or 10 conjugated double bonds. The resulting pathway is the second nonnatural route for carotenoid pigments and the first pathway for carotenoid pigments not biosynthesized via (diapo)phytoene.

Background Ginseng, officially known as Panax ginseng Meyer, has been traditionally used as a medicinal herb, particularly in Asia. Ginseng is propagated from seeds; however, seed germination is challenging, especially in its natural environment on farms. The seeds typically exhibit morphophysiological dormancy and require release from both morphological and physiological dormancy before germination. Although some studies have proposed methods for increasing seed germination rates, the underlying mechanisms of its dormancy release process remain unclear. Here, we investigated metabolic alterations during dehiscence in P. ginseng to determine their potential roles in dormancy release. Methods We compared the ginseng seed metabolome before and after dehiscence and the ginsenoside and phytosterol compositions of the seeds in both periods in the presence of related enzymes. Results After seed dehiscence, the sugar, amino acid, and squalene concentrations were significantly altered, phytosterols associated with the stigmasterol biosynthesis pathway were increased, while ginsenoside and brassinosteroid levels were not significantly altered. In addition, squalene epoxidase, cycloartenol synthase, 24-methylenesterol C-methyltransferase, and the stigmasterol biosynthesis pathway were activated. Conclusion Overall, our findings suggest that morphological activities that facilitate ginseng seed growth are the primary phenomena occurring during the dehiscence process. This study improves the understanding of P. ginseng germination processes and promotes further research of its germination and cultivation.

Cholesterol is vital for membrane function, yet toxic in excess and associated with cardiovascular disease and cancer. Squalene monooxygenase (SM) catalyses the rate-limiting and first oxygen-dependent step of the committed cholesterol synthesis pathway, and past research has shown that cholesterol modulates its protein levels to ensure an appropriate amount of pathway flux according to supply and demand. This is mediated by the N‑terminal regulatory domain of SM (SM‑N100), which senses high cholesterol levels and accelerates entry into the endoplasmic reticulum-associated degradation (ERAD) pathway. A mechanistic understanding of how SM is regulated by such stimuli is critical, as aberrant SM activity is oncogenic in a broad range of cancers. Thus, this thesis sought to identify additional metabolic factors controlling SM degradation. Using a chemical genetics screen and SM‑N100 reporter constructs, we found that SM is protected from degradation by the accumulation of its substrate, squalene. This feedforward regulation involves allosteric binding of squalene to the SM‑N100 domain, preventing its recognition by ERAD effectors. We next studied a lower molecular-weight form of SM routinely detected by immunoblotting, and through SM mutagenesis and targeting of the ERAD pathway found that it arises through the rare phenomenon of partial proteasomal degradation. This disrupts the cholesterol-sensing SM‑N100 domain but not the catalytic domain, rendering truncated SM constitutively active. Truncated SM, but not full-length SM, is also capable of localising to lipid droplets. Finally, we identified hypoxia as a physiological trigger for truncation through a combination of accelerated entry into ERAD and the accumulation of squalene, which prevents complete degradation of SM at the proteasome. Analysis of endometrial cancer tissues revealed a marked upregulation of SM truncation that was well-correlated with a hypoxic marker protein, suggesting that hypoxia-induced truncation occurs in vivo and plays a role in the oncogenic functions of SM. In summary, this thesis identifies dual mechanisms of substrate-induced SM regulation that impair its proteasomal degradation and preserve catalytic capacity. Beyond these fundamental insights into the control of cholesterol synthesis, our data highlight the significance of SM feedforward regulation under both physiological and pathophysiological conditions.

Background: The resistance to treatment of onychomycosis is increasingly reported. The present study aimed to assess the antifungal activity of itraconazole, terbinafine, luliconazole, and efinacon...

BACKGROUND The T indotineae population shows a high amount of terbinafine resistant isolates based on different point mutations of squalene epoxidase erg1 (ergosterol) gene. A significant proportion of these isolates also show azole resistance. OBJECTIVES Elucidation of the molecular mechanism for azole resistance, especially the identification of mutations in the sterol 14-α demethylase Erg11 genes, which encode for enzymes interacting with azoles. METHODS Sequencing of putative Erg11 genes and analysis of phenotypic resistance pattern using a microplate-laser-nephelometry-based growth assay. RESULTS Four different types of Erg11B mutants were detected; two double mutants with Ala230Thr/Asp441Gly, respectively, Ala230/Tyr444His and single mutants with Gly443Glu, Tyr444Cys and Tyr444His. All isolates featured the wild type genotype of Erg11A. All strains demonstrated different combinations of Erg1 and Erg11 genotypes. CONCLUSION Resistance against terbinafine and azoles developed several times independently within the T indotineae population. The challenge for fungal treatment is, therefore, that species identification is not enough for prediction of therapeutic efficacy of antifungals. In the future, it will also become important to analyse genes involved in resistance mechanisms.

A narrow genetic base in cultivated pigeonpea (Cajanus cajan [L] Millsp.) limits the host plant resistance (HPR) to the devastating pest Helicoverpa armigera (Hubner). Earlier research on pod borer resistance in wild Cajanus relatives has mainly explored the tertiary genepool, making breeding for resistance difficult due to linkage drag. Previous investigations of a wide range of Australian native germplasm from the secondary and tertiary gene pools revealed resistance in the secondary gene pool, associated with total phenolic content (TPC). This study reports the application of the extreme phenotype genome-wide association (XP-GWAS) to associate SNPs with resistance traits. A total of 16,489 and 16,227 non-synonymous SNPs were identified for the resistance and TPC traits, respectively, by mapping reads from 19 accessions, including 12 wild accessions, to a reference pigeonpea genome sequence. These SNPs were subjected to functional analysis, and KEGG pathway analysis revealed the presence of candidate genes coding for the crucial enzymes in five defence related pathways that could be contributing to resistance. These pathways involve “Jasmonic acid synthesis” (Palmitoyl-CoA hydrolase, phospholipase A1 and phospholipase A2), “insect hormone biosynthesis” (aldehyde dehydrogenase) “Steroid biosynthesis” (squalene monooxygenase, farnesyl transferase, and lanosterol synthase), “Cutin, suberine and wax synthesis” (O-acyltransferase), and “Brassinosteroid biosynthesis” (Det2). Analysis of SNPs associated with the TPC trait led to the identification of a divergent pathway of “secondary metabolite synthesis (“flavonoid 3′ hydroxylase, L- rhamnosyltransferase) in wild Cajanus species consistent with a high concentration of isoorientin and the possible presence of its derivative maysin; a strong insecticidal compound in leaves.

SQLE (squalene epoxidase) is a cell membrane-bound enzyme. It is a target of fungicides and may become a new target for cancer therapy. Therefore, monitoring the content and distribution of the key enzyme in living cells is very challenging. To achieve this goal, tetraphenyl ethylene-Ter (TPE-Ter) was first designed as a new fluorescent probe to SQLE based on its active cavity. Spectral experiments discovered that SQLE/TPE-Ter shows stronger emission with fast response time and low interference from other analytes. Molecular dynamics simulation clearly confirmed the complex structure of SQLE/TPE-Ter, and the key residues contribute to restriction of TPE-Ter single-molecular motion in the cavity. TPE-Ter-specific response to SQLE is successfully demonstrated in living cells such as LO2, HepG2, and fungi. Imaging of TPE-Ter-treated fungi indicates that it can be used to rapidly assess antifungal drug susceptibility (30 min at least). The present work provides a powerful tool to detect content and distribution of SQLE in living cells.

Vietnamese ginseng (Panax vietnamensis Ha et Grushv.), also known as Ngoc Linh ginseng, is a high-value herb in Vietnam. Vietnamese ginseng has been proven to be effective in enhancing the immune system, human memory, anti-stress, anti-inflammatory, anti-cancer, and prevent aging. The present study reports the first draft whole-genome of Vietnamese ginseng and the identification of potential genes involved in the triterpenoid metabolic pathway. De novo whole-genome assembly was performed successfully from a data of approximately 139 Gbps of 394,802,120 high quality reads to generate 9815 scaffolds with an N50 value of 572,722 bp from the leaf of Vietnamese ginseng. The assembled genome of Vietnamese ginseng is 3,001,967,204 bp long containing 79,374 gene models. Among them, there are 55,012 genes (69.30%) were annotated by various public molecular biology databases. The potential genes involved in triterpenoid saponin biosynthesis in Vietnamese ginseng and their metabolic pathway were also predicted.“ Three genes encoding squalene monooxygenase isozymes in Vietnamese ginseng were cloned, sequenced and characterized. Moreover, expression levels of several key genes involved in terpenoid biosynthesis in different parts of Vietnamese ginseng were also analyzed. The SSR markers were detected by various programs from both of assembly full dataset of Vietnamese ginseng genome and predicted genes. The present work provided important data of the draft whole-genome of Vietnamese ginseng for further studies to understand the role of genes involved in ginsenoside biosynthesis and their metabolic pathway at the molecular level of this rare medicinal species.

Background Ferroptosis, a new form of programmed cell death, has great potential for cancer treatment. However, the roles of ferroptosis-related (FR) genes in breast cancer (BC) remain elusive. Materials and methods Using TCGA database, a novel FR risk signature was constructed through the Lasso regression analysis. Meanwhile, its prognostic value was assessed by a series of survival analyses. Besides, a nomogram was constructed to predict the overall survival rate (OSR) of individual at 1,3,5 year. Four validation cohorts (n = 2248), including METABRIC, GSE58812, GSE20685 and ICGC-KR datasets, were employed to test the prognostic value of FR risk signature. The effects of FR risk signature on BC immune microenvironment were explored by CIBERSORT algorithm and ssGSEA method. The histological expressions of FR risk genes were presented by HPA database. The biofunctions of SQLE were determined by qPCR, MTT, wound-healing and Transwell assays. Results We constructed a novel FR risk signature consisting of eight genes. High FR risk led a poor prognosis and was identified as an independent prognostic factor. Besides, A higher proportion of patients with luminal A type was observed in low-risk group (53%), while a higher proportion of patients with basal type in high-risk group (24%). FR risk score could discriminate the prognostic difference of most clinical subgroups, except for M1 stage, HER2 and basal types. Moreover, its prognostic value was successfully validated in other four cohorts. Through immune analyses, we found that the reduced infiltration levels of CD8+ and NK cells, whereas the enhanced activity of antigen presentation process appeared in high FR risk. Then, FR risk score was found to weakly correlate with the expressions of six immune checkpoints. Through the experiments in vitro, we confirmed that overexpression of SQLE could promote, whereas blocking SQLE could inhibit the proliferative, migrative and invasive abilities of BC cells. Conclusions FR risk signature was conducive to BC prognostic assessment. High FR risk level was closely associated with BC immunosuppression, but may not predict ICIs efficacy. Moreover, SQLE was identified as a crucial cancer-promoting gene in BC. Our findings provide new insights into prognostic assessment and molecular mechanism of BC.

Prostate cancer (PCa) shows strong dependence on the androgen receptor (AR) pathway. Here, we show that squalene epoxidase (SQLE), an enzyme of the cholesterol biosynthesis pathway, is overexpressed in advanced PCa and its expression correlates with poor survival. SQLE expression is controlled by micro-RNA 205 (miR-205), which is significantly downregulated in advanced PCa. Restoration of miR-205 expression or competitive inhibition of SQLE led to inhibition of de novo cholesterol biosynthesis. Furthermore, SQLE was essential for proliferation of AR-positive PCa cell lines, including abiraterone or enzalutamide resistant derivatives, and blocked transactivation of the AR pathway. Inhibition of SQLE with the FDA approved antifungal drug terbinafine also efficiently blocked orthotopic tumour growth in mice. Finally, terbinafine reduced levels of prostate specific antigen (PSA) in three out of four late-stage PCa patients. These results highlight SQLE as a therapeutic target for the treatment of advanced PCa., Cholesterol metabolism is involved in the progression of aggressive prostate cancer (PCa). Here the authors show that miR-205 downregulation promotes cholesterol synthesis and androgen receptor signalling in PCa through enhancing the expression of the rate-limiting enzyme of cholesterol synthesis, squalene epoxidase.

Background Atractylodes chinensis (DC.) Koidz is a well-known medicinal plant containing the major bioactive compound, atractylodin, a sesquiterpenoid. High-performance liquid chromatography (HPLC) analysis demonstrated that atractylodin was most abundant in 3-year old A. chinensis rhizome, compared with those from 1- and 2-year old rhizomes, however, the molecular mechanisms underlying accumulation of atractylodin in rhizomes are poorly understood. Results In this study, we characterized the transcriptomes from rhizomes of 1-, 2- and 3-year old (Y1, Y2 and Y3, respectively) A. chinensis, to identify differentially expressed genes (DEGs). We identified 240, 169 and 131 unigenes encoding the enzyme genes in the mevalonate (MVA), methylerythritol phosphate (MEP), sesquiterpenoid and triterpenoid biosynthetic pathways, respectively. To confirm the reliability of the RNA sequencing analysis, eleven key gene encoding factors involved in the sesquiterpenoid and triterpenoid biosynthetic pathway, as well as in pigment, amino acid, hormone and transcription factor functions, were selected for quantitative real time PCR (qRT-PCR) analysis. The results demonstrated similar expression patterns to those determined by RNA sequencing, with a Pearson’s correlation coefficient of 0.9 between qRT-PCR and RNA-seq data. Differential gene expression analysis of rhizomes from different ages revealed 52 genes related to sesquiterpenoid and triterpenoid biosynthesis. Among these, seven DEGs were identified in Y1 vs Y2, Y1 vs Y3 and Y2 vs Y3, of which five encoded four key enzymes, squalene/phytoene synthase (SS), squalene-hopene cyclase (SHC), squalene epoxidase (SE) and dammarenediol II synthase (DS). These four enzymes directly related to squalene biosynthesis and subsequent catalytic action. To validate the result of these seven DEGs, qRT-PCR was performed and indicated most of them displayed lower relative expression in 3-year old rhizome, similar to transcriptomic analysis. Conclusion The enzymes SS, SHC, SE and DS down-regulated expression in 3-year old rhizome. This data corresponded to the higher content of sesquiterpenoid in 3-year old rhizome, and confirmed by qRT-PCR. The results of comparative transcriptome analysis and identified key enzyme genes laid a solid foundation for investigation of production sesquiterpenoid in A. chinensis.

Background Colorectal cancer (CRC) is one of the most malignant tumors with high incidence, yet its molecular mechanism is not fully understood, hindering the development of targeted therapy. Metabolic abnormalities are a hallmark of cancer. Targeting dysregulated metabolic features has become an important direction for modern anticancer therapy. In this study, we aimed to identify a new metabolic enzyme that promotes proliferation of CRC and to examine the related molecular mechanisms. Methods We performed RNA sequencing and tissue microarray analyses of human CRC samples to identify new genes involved in CRC. Squalene epoxidase (SQLE) was identified to be highly upregulated in CRC patients. The regulatory function of SQLE in CRC progression and the therapeutic effect of SQLE inhibitors were determined by measuring CRC cell viability, colony and organoid formation, intracellular cholesterol concentration and xenograft tumor growth. The molecular mechanism of SQLE function was explored by combining transcriptome and untargeted metabolomics analysis. Western blotting and real‐time PCR were used to assess MAPK signaling activation by SQLE. Results SQLE‐related control of cholesterol biosynthesis was highly upregulated in CRC patients and associated with poor prognosis. SQLE promoted CRC growth in vitro and in vivo. Inhibition of SQLE reduced the levels of calcitriol (active form of vitamin D3) and CYP24A1, followed by an increase in intracellular Ca2+ concentration. Subsequently, MAPK signaling was suppressed, resulting in the inhibition of CRC cell growth. Consistently, terbinafine, an SQLE inhibitor, suppressed CRC cell proliferation and organoid and xenograft tumor growth. Conclusions Our findings demonstrate that SQLE promotes CRC through the accumulation of calcitriol and stimulation of CYP24A1‐mediated MAPK signaling, highlighting SQLE as a potential therapeutic target for CRC treatment., SQLE increases the levels of calcitriol and CYP24A1. Then MAPK signaling is induced to accelerate CRC tumor growth.

Appropriate light intensity is favorable for the photosynthesis, biomass accumulation, key enzyme activity, and secondary metabolite synthesis of medicinal plants. This study aims to explore the influence of light intensity on growth and quality of Panax quinquefolius. To be specific, sand culture experiment was carried out in a greenhouse under the light intensity of 40, 80, 120, and 160 μmol·m~(-2)·s~(-1), respectively. The growth indexes, photosynthetic characteristics, content of 6 ginsenosides of the 3-year-old P. quinquefolius were determined, and the expression of ginsenoside synthesis-related enzyme genes in leaves, main roots, and fibrous roots was determined. The results showed that the P. quinquefolius growing at 80 μmol·m~(-2)·s~(-1) light intensity had the most biomass and the highest net photosynthetic rate. The total biomass of P. quinquefolius treated with 120 μmol·m~(-2)·s~(-1) light intensity was slightly lower than that with 80 μmol·m~(-2)·s~(-1). The root-to-shoot ratio in the treatment with 120 μmol·m~(-2)·s~(-1) light intensity was up to 6.86, higher than those in other treatments(Plt;0.05),and the ginsenoside content in both aboveground and underground parts of P. quinquefolius in this treatment was the highest, which was possibly associated with the high expression of farnesylpyrophosphate synthase(FPS), squalene synthase(SQS), squalene epoxidase(SQE), oxidosqualene cyclase(OSC), dammarenediol-Ⅱ synthase(DS), and P450 genes in leaves and SQE and DS genes in main roots. In addition, light intensities of 120 and 160 μmol·m~(-2)·s~(-1) could promote PPD-type ginsenoside synthesis in leaves by triggering up-regulation of the expression of upstream ginsenoside synthesis genes. The decrease in underground biomass accumulation of the P. quinquefolius grown under weak light(40 μmol·m~(-2)·s~(-1)) and strong light(160 μmol·m~(-2)·s~(-1)) was possibly attributed to the low net photosynthetic rate, stomatal conductance, and transpiration rate in leaves. In the meantime, the low expression of SQS, SQE, OSC, and DS genes in the main roots might led to the decrease in ginsenoside content. However, there was no significant correlation between the ginsenoside content and the expression of synthesis-related genes in the fibrous roots of P. quinquefolius. Therefore, the light intensity of 80 and 120 μmol·m~(-2)·s~(-1) is beneficial to improving yield and quality of P. quinquefolius. The above findings contributed to a theoretical basis for reasonable shading in P. quinquefolius cultivation, which is of great significance for improving the yield and quality of P. quinquefolius through light regulation.

Chemoresistance in patients with glioblastoma multiforme (GBM) is a common reason hindering the success of treatment. Recently, ferroptosis has been reported to be associated with chemoresistance in different types of cancer, while the role of ferroptosis-related genes in GBM have not been fully elucidated. This study aimed to demonstrate the roles and mechanism of ferroptosis-related genes in chemoresistance and metastasis of GBM. First, two candidate genes, squalene epoxidase (SQLE) and FANCD2, were identified to be associated with ferroptosis-related chemoresistance in GBM from three temozolomide (TMZ) therapeutic datasets and one ferroptosis-related gene dataset. Then, comprehensive bio-informatics data from different databases testified that SQLE was significantly downregulated both in GBM tissue and cells and displayed a better prognosis in GBM. Clinical data identified lower expression of SQLE was significantly associated with WHO grade and 1p/19q codeletion. Moreover, through in vitro experiments, SQLE was confirmed to suppress ERK-mediated TMZ chemoresistance and metastasis of GBM cells. The KEGG analysis of SQLE-associated co-expressed genes indicated SQLE was potentially involved in the cell cycle. Furthermore, SQLE was found to have the most significant correlations with tumor-infiltrating lymphocytes and immunomodulators. These findings highlighted that SQLE could be a potential target and a biomarker for therapy and prognosis of patients with GBM.

Lanosterol, an oxysterol molecule, has been proposed to help maintain lens transparency by inhibiting the formation of protein aggregates. This sterol is produced by the enzyme lanosterol synthase and is part of a metabolic pathway that forms cholesterol as a final step. Abnormalities in lanosterol synthase are responsible for congenital cataracts. The αA-crystallin protein, which acts as a molecular chaperone to lanosterol synthase, has been reported to have anti-protein aggregation, anti-inflammatory and anti-apoptotic properties. In this work, we evaluated the correlation of lanosterol synthase and αA-crystallin in human cataractous lenses with the grade of opacity, as well as the expression of lanosterol synthase, farnesyl DPP, geranyl synthase and squalene epoxidase genes. Lanosterol synthase and αA-crystallin were overexpressed in cataractous lenses as well as farnesyl-DP synthase, squalene epoxidase, lanosterol synthase and geranyl synthase genes in cataratous lenses in comparison with normal lenses. Our data confirm that lanosterol synthase and the sterol pathway are upregulated in cataractous lenses. This argues for a functional role of the oxysterol pathway and its products as an important mediator in the pathogenesis of human cataracts.

Panax quinquefolius L., a medicinal plant of Araliaceae family, is used in traditional Chinese medicine. Arbuscular mycorrhizal fungi (AMF) has been shown to promote plant growth and the accumulation of secondary metabolites in many plants. Our group has found a significant positive correlation between AMF infection rate and the accumulation of ginsenoside, while the effects and underlying mechanism of AMF on the growth and accumulation of ginsenoside in P. quinquefolius have been poorly studied. In this study, the effects on the biomass, photosynthesis and expressions of genes involved in biosynthesis of ginsenoside: hydroxymethylglutaryl-CoA reductase (HMGR), geranylgeranyl pyrophosphate synthase (GPS), farnesyl diphosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), dammarendiol synthase (DS) and cytochrome P450 enzyme (CYP716A47, CYP716A53v2) in P. quinquefolius during AMF inoculation were investigated. Plant biomass and photosynthesis of P. quinquefolius were stimulated by AMF treatment. Chlorophyll concentration in P. quinquefolius leaves treated by R. intraradices was higher than those in non-inoculated leaves. The concentration of Rb1 and Re was markedly increased by 26.15% and 22.38% (P

Background: Dermatophytosis is commonly encountered in the dermatological clinics. The main aetiological agents in dermatophytosis of skin and nails in humans are Trichophyton (T.) rubrum, T. mentagrophytes and T. interdigitale (former T. mentagrophytes-complex). Terbinafine therapy is usually effective in eradicating infections due to these species by inhibiting their squalene epoxidase (SQLE) enzyme, but increasing numbers of clinically resistant cases and mutations in the SQLE gene have been documented recently. Resistance to antimycotics is phenotypically determined by antifungal susceptibility testing (AFST). However, AFST is not routinely performed for dermatophytes and no breakpoints classifying isolates as susceptible or resistant are available, making it difficult to interpret the clinical impact of a minimal inhibitory concentration (MIC). Summary: PubMed was systematically searched for terbinafine susceptibility testing of dermatophytes on October 20, 2020, by two individual researchers. The inclusion criteria were in vitro terbinafine susceptibility testing of Trichophyton (T.) rubrum, T. mentagrophytes and T. interdigitale with the broth microdilution technique. The exclusion criteria were non-English written papers. Outcomes were reported as MIC range, geometric mean, modal MIC and MIC50 and MIC90 in which 50 or 90% of isolates were inhibited, respectively. The reported MICs ranged from 64 mg/L. The huge variation in MIC is partly explained by the heterogeneity of the Trichophyton isolates, where some originated from routine specimens (wild types) whereas others came from non-responding patients with a known SQLE gene mutation. Another reason for the great variation in MIC is the use of different AFST methods where MIC values are not directly comparable. High MICs were reported particularly in isolates with SQLE gene mutation. The following SQLE alterations were reported: F397L, L393F, L393S, H440Y, F393I, F393V, F415I, F415S, F415V, S443P, A448T, L335F/A448T, S395P/A448T, L393S/A448T, Q408L/A448T, F397L/A448T, I121M/V237I and H440Y/F484Y in terbinafine-resistant isolates.

Background Treatment-resistant dermatophytosis caused by Trichophyton mentagrophytes/interdigitale complex has emerged as a global public health threat, particularly in endemic countries like India and has spread to many other countries. This veritable spread is alarming due to increase in resistance to terbinafine, which targets the ergosterol biosynthetic pathway by inhibiting the enzyme squalene epoxidase (SQLE). About two third of studies worldwide have reported amino acid substitutions Phe397Leu and Leu393Phe in the SQLE protein to be responsible for high terbinafine MICs. Objectives We evaluated the efficacy of the newly developed DermaGenius® Resistance real-time PCR assay to rapidly identify Trichophyton isolates harbouring most common SQLE mutant (Phe397Leu and Leu393Phe) conferring high terbinafine resistance from wild-type susceptible isolates. Methods A total of 97 Trichophyton isolates confirmed by ITS sequencing as T. mentagrophytes/interdigitale (recently named T. indotineae n = 90), T. rubrum/T. soudanense (n = 3), T mentagrophytes (n = 2) and T tonsurans (n = 2) were analysed to evaluate DermaGenius® Resistance real-time PCR assay. All 40 T. indotineae isolates exhibiting amino acid substitutions Phe397Leu or Leu393Phe identified by SQLE gene sequencing were evaluated for detection of non-wild-type strains by real-time PCR. Antifungal susceptibility testing for terbinafine was done by CLSI microbroth dilution method. Results All terbinafine-resistant isolates harbouring amino acid substitutions Phe397Leu or Leu393Phe in SQLE gene were correctly recorded as SQLE mutants by the DermaGenius® Resistance real-time PCR assay. Conclusions The DermaGenius® Resistance real-time PCR assay effectively identified Trichophyton species and distinguished wild-type from SQLE mutant genotype that harbour Phe397Leu and Leu393Phe amino acid substitutions.

The histone methyltransferase EZH2 silences gene expression via H3 lysine 27 trimethylation and has been recognized as an important antitumour therapeutic target. However, the clinical application of existing EZH2 inhibitors is not satisfactory for the treatment of solid tumours. To discover novel strategies against head and neck squamous cell carcinoma (HNSCC), we performed genomics, metabolomics and RNA omics studies in HNSCC cells treated with EZH2 inhibitors. It was found that EZH2 inhibitors strongly induced the expression of genes in cholesterol synthesis. Through extensive drug screening we found that inhibition of squalene epoxidase (a key enzyme of endogenous cholesterol synthesis) synergistically increased the squalene content and enhanced the sensitivity of HNSCC cells to EZH2 inhibitors. Our findings provide an experimental and theoretical basis for the development of new combinations of EZH2 inhibitors to treat HNSCC.

Background:Squalene epoxidase gene is an azole antifungal target enzyme in the ergosterol biosynthesis pathway in fungi—the inhibition of enzyme aggregate squalene, a type of ergosterol derivative depletion that leads to fungal growth inhibition. Squalene epoxidase widely diverges in three fungal groups of dermatophytes Trichophyton, Microsporum and Epidermophyton. Methodology:The study has demonstrated a variety of squalene epoxidase genes in the dermatophyte genome. Squalene epoxidase gene was also studied for possible single nucleotide polymorphism (SNPs) in the Trichophyton group's homologs and found multiple SNP positions that induce non-synonymous mutation and change the sequence of amino acid into protein structure that can change phenylalanine to leucine. Results:Squalene epoxidase is widely present in dermatophytes. Mutation in the squalene epoxidase gene can mislead normal sterol formation in the fungal cell membrane that induces fungal resistance against several drugs, including azoles and polyenes. Squalene epoxidase gene carries 2 - 3 transcripts and 2 - 3 number of exons. Squalene epoxidase gene has FAD-dependent oxidoreductases and NADP binding domain conserved domains in fungal groups of dermatophytes. Conclusion:This study analyzed the abundance of the squalene epoxidase gene, suggesting that squalene epoxidase gene-resistant mutants can occur naturally. Thus squalene epoxidase gene should be extensively studied in order to increase the potential of available antifungals. Link:http://aeirc-edu.com/ojs14/index.php/IJEHSR/article/view/574/651

Aralia elata (Miq.) Seem is widely used as a medicinal plant and functional food in China. In this study, A. elata plants were exposed to full sunlight (CK), 40% shading (LS), 60% shading (MS), and >80% shading (ES) condition to investigate the effects of shading treatments on growth, stress levels, antioxidant enzymes activity, araloside content and related gene expression. The greatest growth and leaf biomass were achieved in 40% shading, and leaf biomass per plant increased by 16.09% compared to the non-shading treatment. Furthermore, the lowest reactive oxide species (ROS) production and lipid peroxidation resulting from increasing antioxidant enzyme activity were also observed in LS treatment. Overall, shading percentage negatively regulated the expression of key enzymes (squalene synthase, SS; squalene epoxidase, SE and β-amyrin synthase, bAS) involved in the saponin biosynthesis, resulting in the greatest yields of total and four selected aralosides in A. elata leaves were achieved in sunlight group. However, the greatest yield of total saponin in the leaves was observed in the 40% shading group due to higher leaf biomass. The results suggest that optimizing the field growing conditions would be important for obtaining the greatest yield of bioactive components. Total saponin and selected aralosides also have a significant correlation with ROS production and antioxidant enzyme activity, these indicated the increased yield of these saponins may be part of a defense response. The study concludes that the production of saponin was the interaction of oxidative stress and photosynthesis.

Tinea capitis is local alopecia caused by a dermatophyte infection of the scalp. Trichophyton rubrum produces the squalene epoxidase enzyme, which has a crucial role in prolonged dermatophyte infection, as well as in synthesizing fatty acids in this dermatophyte group. This study analyzes Trichophyton cacao compounds as anti-alopecia by inhibiting the squalene epoxidase enzyme formation, in silico. The structure of T. cacao compounds was prepared using the MolView Web application. The compound docked to squalene epoxidase using AutoDock Vina in PyRx 0.8, followed by PyMOL for visualization, and the Proteins Plus program to analyze the complexity. The binding affinity value of catechin, epicatechin (−8.0 kcal/mol), and anthocyanin (−7.8 kcal/mol) compounds was higher than the positive control (terbinafine, −6.7 kcal/mol). Pre-ADMET demonstrated that catechin and epicatechin had moderate Human Intestinal Absorption (66.71%), but anthocyanin was very good (100%). Caco-2 parameters for catechin and epicatechin were relatively low (

To clarify the terbinafine (TRF) resistance mechanism in highly TRF-resistant [minimum inhibitory concentration (MIC) >32 μg/mL] strains of Trichophyton indotineae (NUBS19006T and NUBS19007), we investigated the expression of squalene epoxidase (SQLE), pleiotropic drug resistance 1 (PDR1), multidrug resistance 2 (MDR2), and MDR4 genes by real-time quantitative PCR analysis, given the known interaction of the corresponding proteins with antifungals and the efflux blocker tacrolimus (FK506). SQLE, PDR1, MDR2, and MDR4 transcript levels in TRF-resistant strains cultured in SDB were not significantly higher than those of the respective genes in TRF-susceptible strains (1 and 10). By contrast, PDR1, MDR2, and MDR4 transcript levels in TRF-resistant and TRF-susceptible strains cultured in SDB containing 10 μg/mL TRF were 5-100 times higher than those of the respective genes in strains grown in the absence of TRF. However, no differences in PDR1, MDR2, and MDR4 transcript levels were found between TRF-resistant (NUBS19006T and NUBS19007) and TRF-susceptible strains cultured in SDB containing 10 μg/mL TRF. The interaction between TRF and FK506 on antifungal activity was not detected in TRF-resistant strains. These results indicate that ATP-dependent efflux pumps do not confer TRF-resistance mechanisms in TRF-resistant strains.

Over 50% of all patients with cancer are treated with radiotherapy. However, radiotherapy is often insufficient as a monotherapy and requires a nontoxic radiosensitizer. Squalene epoxidase (SQLE) controls cholesterol biosynthesis by converting squalene to 2,3-oxidosqualene. Given that SQLE is frequently overexpressed in human cancer, this study investigated the importance of SQLE in breast cancer and non–small cell lung cancer (NSCLC), two cancers often treated with radiotherapy. SQLE-positive IHC staining was observed in 68% of breast cancer and 56% of NSCLC specimens versus 15% and 25% in normal breast and lung tissue, respectively. Importantly, SQLE expression was an independent predictor of poor prognosis, and pharmacologic inhibition of SQLE enhanced breast and lung cancer cell radiosensitivity. In addition, SQLE inhibition enhanced sensitivity to PARP inhibition. Inhibition of SQLE interrupted homologous recombination by suppressing ataxia-telangiectasia mutated (ATM) activity via the translational upregulation of wild-type p53-induced phosphatase (WIP1), regardless of the p53 status. SQLE inhibition and subsequent squalene accumulation promoted this upregulation by triggering the endoplasmic reticulum (ER) stress response. Collectively, these results identify a novel tumor-specific radiosensitizer by revealing unrecognized cross-talk between squalene metabolites, ER stress, and the DNA damage response. Although SQLE inhibitors have been used as antifungal agents in the clinic, they have not yet been used as antitumor agents. Repurposing existing SQLE-inhibiting drugs may provide new cancer treatments. Significance: Squalene epoxidase inhibitors are novel tumor-specific radiosensitizers that promote ER stress and suppress homologous recombination, providing a new potential therapeutic approach to enhance radiotherapy efficacy.

Biodiesel and natural products derived from microalgae require a smaller land area and have higher production rates compared to plants and animals and has recently attracted considerable interest. However, biodiesel production from microalgal triacylglycerol is still far from commercial realization due to its high production cost. One way to overcome this obstacle is to improve the triacylglycerol accumulation and couple its production with other high-value compounds. Of particular interest is the sterol biosynthetic pathway with squalene as an intermediate due to its close relationship with triacylglycerol and carotenoid biosynthetic pathways. Besides, both squalene and carotenoids are isoprenoid lipids that have health benefits. Perturbation of one pathway has been suggested to affect other pathways. Three terbinafine-sensitive mutants of the green microalga Chlamydomonas reinhardtii were isolated using terbinafine, a drug that inhibits squalene epoxidase, leading to squalene accumulation. One of the mutants, tfs2, accumulated twice the amount of wild-type triacylglycerol. As well as squalene accumulation, the presence of terbinafine further increased the triacylglycerol content. The level of prenyl lipid carotenoid and chlorophyll was also more significant than that of the wild type. Growth and photosynthesis were not compromised in this mutant. This is the first study that has demonstrated a mutant screening method to improve the co-production of TAG and isoprenoid lipids in a green microalgae.

Sterol synthesis is a highly complex and integrated pathway in mammals. In the present review, we briefly summarize the main steps of this pathway, especially concerning its main rate-limiting enzymes, HMG-CoA reductase (HMGCR) and squalene epoxidase (SQLE), in relation with cancer. We focus on studies reporting key findings linking cholesterol with cancer. The inhibition of HMGCR and SQLE to prevent and inhibit cancer are reviewed. Finally, a pan-cancer review of publicly available data on genomic aberrations in the main enzymes involved in sterol biosynthesis and their transcription factors is reported, providing hitherto unexplored findings that may be the subject of future research in cancer metabolomics and tumor targeted treatment.

Squalene epoxidase (also called squalene monooxygenase) catalyses the conversion of squalene into 2,3-oxidosqualene by epoxidation and is regarded as the rate-limiting enzyme for sterol and saponin biosynthesis. However, the role of the squalene epoxidase gene in saponin biosynthesis in plants is not yet well understood. Here, we investigated the effects of overexpression of a Panax ginseng squalene epoxidase gene (PgSE1) on the production of phytosterols and ginsenoside saponins in ginseng adventitious roots. For the functional complementation test, the two squalene epoxidase sequences (PgSE1 and PgSE2) of P. ginseng were expressed in a yeast erg1 mutant (ergosterol auxotroph). The yeast mutant expressing PgSE1 or PgSE2 can restore growth on medium lacking ergosterol. Transgenic ginseng roots overexpressing the PgSE1 gene were constructed by Agrobacterium-mediated genetic transformation. The transgenic ginseng roots resulted in the enhanced production of both ginsenosides (ginsenoside Rg1, Re, Rf, Rc, Rb1, Rb2, and Rd) and phytosterols (campesterol, stigmasterol, and β-sitosterol). qPCR analysis revealed that overexpression of PgSE1 in transgenic ginseng roots clearly enhanced the expression of dammarenediol-II synthase (PgDDS) and cycloartenol synthase (PgPNX), which are key enzymes for ginsenoside and phytosterol biosynthesis in P. ginseng. This result indicates that the P. ginseng squalene epoxidase gene (PgSE1) encodes an efficient enzyme responsible for not only phytosterol production but also ginsenoside production in P. ginseng.

Background The Indian ITS genotype VIII T. mentagrophytes population shows a high amount of different erg1 (ergosterol) mutants encoding for squalene epoxidase, which catalyzes the first step of ergosterol biosynthesis. Objectives Illumination of the implication of point mutations at position Ala448Thr in single and double erg1 T. mentagrophytes mutants because mutants of this type were abundantly found within the Indian fungal population. Methods Growth in fluconazole or terbinafine containing medium was analyzed using a microplate-laser-nephelometry (MLN)-based growth assay. Results Ala 448 Thr erg1 single mutants were terbinafine sensitive but about 50% of isolates showed an increased fluconazole resistance, whereas 95% of the double mutants (Phe 397 Leu, Ala 448Thr) demonstrated combined terbinafine and increased fluconazole resistance. Conclusion The new Indian T. mentagrophytes populations show several point mutations in erg1. Point mutations at position 397 were previously described and cause terbinafine resistance. A large part of the double mutants exhibit resistance to terbinafine and fluconazole, demonstrating a selective advantage of the combination of both mutations.

Background Dermatomycoses are the most common fungal infections in the world affecting a significant part of the human and animal population. The majority of zoophilic infections in humans are caused by Trichophyton mentagrophytes. Currently, the first-line drug for both oral and topical therapy is terbinafine. However, an increasing number of cases that are difficult to be cured with this drug have been noted in Europe and Asia. Resistance to terbinafine and other allylamines is very rare and usually correlated with point mutations in the squalene epoxidase gene resulting in single amino acid substitutions in the enzyme, which is crucial in the ergosterol synthesis pathway. Purpose Here, we report terbinafine-resistant T. mentagrophytes isolates among which one was an etiological factor of tinea capitis in a man and three were obtained from asymptomatic foxes in Poland. Methods We used the CLSI protocol to determine antifungal susceptibility profiles of naftifine, amphotericin B, griseofulvin, ketoconazole, miconazole, itraconazole, voriconazole, and ciclopirox. Moreover, the squalene epoxidase gene of the terbinafine-resistant strains was sequenced and analysed. Results In the genomes of all four resistant strains exhibiting elevated MICs to terbinafine (16 to 32 µg/ml), single-point mutations leading to Leu393Phe substitution in the squalene epoxidase enzyme were revealed. Among the other tested substances, a MIC50 value of 1 µg/ml was shown only for griseofulvin. Conclusion Finally, our study revealed that the terbinafine resistance phenomenon might not be acquired by exposure to the drug but can be intrinsic. This is evidenced by the description of the terbinafine-resistant strains isolated from the asymptomatic animals.

Fritillariae Bulbus are the most commonly used antitussive and edible herbs in China. Based on UPLC-QTOF-MS and UPLC-QQQ-MS, the validated MRM-based non-targeted quantitative method was applied to determinate the contents of 48 Fritillaria alkaloids (FAs) in three Fritillaria species (F. thunbergii Miq., F. unibracteata and F. ussuriensis). The RNA-Seq results showed that gene transcript levels have different expression patterns in three Fritillaria species. Based on transcriptome data, the full-length cDNA sequences of squalene epoxidase gene were cloned and characterized. Natural evolution of squalene epoxidase genes resulted in four mutations (C236R, M489L, G510A and K517R) in three Fritillaria species. Molecular docking analysis showed that the 236 residue is located inside the pocket and the binding center while other three residues are located on the surface of the protein. Functional verification indicated the mutations of SQE (C236R) could effectively increase the activity of SQE and obtain higher yield of 2,3-oxidosqualene in recombinant yeast. And the mutations of SQE (M489L and G510A), which increased the hydrophobicity of the protein surface, could also enhance the activity of SQE. This study provides major insights into the metabolites differentiation of FAs biosynthesis, and a firm foundation for the quality control and metabolic engineering of Fritillariae bulbus. Deeper insights into the differentiation of metabolomics and transcriptomics and natural mutations of squalene epoxidase genes in three Fritillaria Bulbus.

The study reports a protocol for the regeneration of Glycyrrhiza glabra plantlet from the leaf explant. Notably, the chemical analysis of various developmental stages revealed presence of glycyrrhizin in the underground (7.0–29.8 µg/g) & aerial (7.3–23.4 µg/g) tissues of the in vitro regenerated plants, which was otherwise not detected in the aerial tissues of the field plant. Glycyrrhizin accumulation was reduced or undetected in the regenerated plants transferred to the glasshouse and subsequently under field conditions. Further, spatio-temporal relative gene expression analysis of aerial tissues of in-vitro regenerated G. glabra showed expression of all the known genes committed to glycyrrhizin pathway. In the shoot system, maximum expression of squalene epoxidase (7.9 fold), β-amyrin synthase (21.8 folds), Licorice β-amyrin 11-oxidase (5.9 folds) and UDP-glucosyltransferase (1.7 folds) was observed in different months. However, no expression was detected in the aerial tissues of the field grown plant. Also, a correlation was found between the expression patterns of Licorice β-amyrin 11-oxidase with glycyrrhizin accumulation. This is the first study demonstrating the presence of glycyrrhizin in the aerial tissues of the in-vitro regenerated Glycyrrhiza plant. This study will help in understanding glycyrrhizin regulation, precursor accumulation and their transport. Also, it will unlock the possibilities of understanding and enhancing glycyrrhizin biosynthesis and accumulation in the plant, under in-vitro conditions.

In this report, we describe the first isolation of two highly terbinafine (TRF)-resistant Trichophyton interdigitale-like strains from a Nepali patient and an Indian patient with tinea corporis in Japan. These strains (designated NUBS19006 and NUBS19007) exhibited a TRF minimal inhibitory concentration (MIC) of > 32 mg/L and contained a missense mutation (Phe397Leu) in squalene epoxidase (SQLE) gene. The internal transcribed spacer (ITS) region sequences amplified from the isolates (NUBS19006 and NUBS19007) were 99.5% identical to Japanese isolates of T. interdigitale and T. interdigitale strain CBS 428.63. The homology of region sequences were also 97.6% identical to T. mentagrophytes strain CBS 318.56. Moreover, the ITS sequences amplified from the isolates were 100% identical to highly TRF-resistant strains of T. interdigitale, which were isolated in Delhi, India, and harbored mutations in SQLE. The urease test on Christensen’s urease agar was positive for T. mentagrophytes and T. interdigitale after 7 days of incubation. On the other hand, the type strain of T. rubrum CBS 100081 T and highly TRF-resistant strains (NUBS19006 and NUBS19007) were negative on Christensen urease agar after 7 and 14 days of incubation. Moreover, NUBS19006 and NUBS19007 were also negative reaction on the hair perforation test. To avoid confusion in the taxonomy of the T. mentagrophytes/T. interdigitale complex, we suggest that the highly TRF-resistant Indian strains be considered a new species independent of T. interdigitale, according to clinical and mycological features.