ACS Infectious Diseases
·
2024
·
IF 4
Jing Wu,Yong Zhang,Wenqi Li,Hao Tang,Ying Zhou,Di You,Xiaohe Chu,Hanbing Li,Jinsai Shang,Nan Qi,Bang-Ce Ye
Cholesterol is a key carbon source for Mycobacterium tuberculosis (Mtb) survival and persistence within macrophages. However, little is known about the role of cholesterol metabolism by Mtb in host–Mtb interplay. Here, we report the immune suppression mediated by Mtb’s cholesterol metabolites. Conducting the cholesterol metabolic profiling and loss-of-function experiments, we show that the cholesterol oxidation products catalyzed by a thiolase FadA5 from Mtb H37Ra, 4-androstenedione (AD), and its derivant 1,4-androstenedione (ADD) inhibit the expression of pro-inflammatory cytokines and thus promote bacterial survival in bone marrow-derived macrophages (BMDMs). Our time-resolved fluorescence resonance energy transfer (TR-FRET)-based screening further identifies the nuclear receptor LXRα as the target of ADD. Activation of LXRα via ADD impedes the nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK) signaling and reduces cholesterol accumulation in lipid rafts upon TLR4 simulation, thereby compromising the inflammatory responses. Our findings provide the evidence that Mtb could suppress the host immunity through its cholesterol metabolic enzyme and products, which are potential targets for screening novel anti-tuberculosis (TB) agents.
研究领域:
微生物学
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免疫学
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传染病
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代谢
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PubMed
高影响因子
MICROCHEMICAL JOURNAL
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2021
·
IF 4.821
Mingying Ma,Tianhao Wu,Zhian Hu,Gongwei Sun,Sichun Zhang
High-throughput sample preparation is important in clinical mass spectrometry (MS) analysis. In this work, we developed a method by coupling Nano-ESI-MS with magnetic SPE using a MOF material, Fe 3 O 4 @UiO-66, as sorbent for simultaneous determination of testosterone (T) and androstenedione (4-AD) in serum samples. The proposed method presented good linearity in the range of 0.1–5 μg/L and the recoveries of serum samples were 90.7%-110.0% and 101.7%-108.7% for T and 4-AD, respectively. The results indicated that the Fe 3 O 4 @UiO-66 is an ideal sorbent for androgens extraction and the method is reliable for high-throughput quantitation of T and 4-AD in clinical applications.
研究领域:
分析化学
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临床医学
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DOI
高影响因子
Frontiers in Microbiology
·
2022
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IF 6.064
Huo Jinxi,Sun Yuqing,Pan Meiliang,Ma Huanyan,Lin Tianbao,Lv Zhiqiang,Li Yougui,Zhong Shi
Sanghuangprous vaninii is a wood-inhabiting fungus, and its mycelium and fruiting body show excellent medicinal values. Mulberry is one of the major hosts of S. vaninii, however, the mechanism of mulberry affecting the growth of S. vaninii has not been reported. In the present study, a mulberry-inhabiting strain of S. vaninii was selected to explore the effects of mulberry branch extracts (MBE) on the growth of the strain. Results showed that MBE could significantly promote the growth of S. vaninii mycelium at the concentration of 0.2 g/l. After 16 days of liquid culture, the dry weight of mycelium in 0.2 g/l MBE medium was higher by three times compared with that in the control. The non-targeted metabonomic analysis of the culture medium at different culture times and concentrations was conducted to find the key components in MBE that promoted the growth of S. vaninii mycelium. Under the different concentrations of MBE culture for 10 and 16 days, 22 shared differential metabolites were identified. Next, in accordance with the peak value trend of these metabolites, HPLC–MS and liquid culture validation, four components derived from MBE (i.e., scopoletin, kynurenic acid, 3,5-dihydroxybenzoic acid and 2,4-dihydroxybenzoic acid) could significantly increase the growth rate of mycelium at the concentration of 2 mg/l. Transcriptomic and qRT-PCR analyzes showed that MBE could upregulate hydrolase-related genes, such as serine–glycine–asparaginate–histidine (SGNH) hydrolase, alpha-amylase, poly-beta-hydroxybutyrate (PHB) depolymerase, glycosyl hydrolase family 61, cerato-platanin protein and Fet3, which might enhance the nutrient absorption ability of S. vaninii. Importantly, MBE could significantly increase the content of harmine, androstenedione and vesamicol, which have been reported to possess various medicinal effects. Results suggested that MBE could be an excellent additive for liquid culture of S. vaninii mycelium, and these hydrolase-related genes also provided candidate genes for improving the nutrient absorption capacity of S. vaninii.
研究领域:
真菌学
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代谢组学
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转录组学
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药理学
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DOI
PubMed
高影响因子
BIORESOURCE TECHNOLOGY
·
2023
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IF 11.4
Guojuan Yi,Hanlu Zou,Tao Long,Tolbert Osire,Lin Wang,Xiaoyun Wei,Mengfei Long,Zhiming Rao,Guojian Liao
Hydroxylated steroids are value-added products with diverse biological activities mediated by cytochrome P450 enzymes, however, few has been thoroughly characterized in fungi. This study introduces a rapid identification strategy for filamentous fungi P450 enzymes through transcriptome and bioinformatics analysis. Five novel enzymes (CYP68J5, CYP68L10, CYP68J3, CYP68N1 and CYP68N3) were identified and characterized in Saccharomyces cerevisiae or Aspergillus oryzae . Molecular docking and dynamics simulations were employed to elucidate hydroxylation preferences of CYP68J5 (11α, 7α bihydroxylase) and CYP68N1 (11α hydroxylase). Additionally, redox partners (cytochrome P450 reductase and cytochrome b5) and ABC transporter were co-expressed with CYP68N1 to enhance 11α-OH-androstenedione (11α-OH-4AD) production. The engineered cell factory, co-expressing CPR1 and CYP68N1, achieved a significant increase of 11α-OH-4AD production, reaching 0.845 g·L -1 , which increased by 14 times compared to the original strain. This study provides a comprehensive approach for identifying and implementing novel cytochrome P450 enzymes, paving the way for sustainable production of steroidal products.
研究领域:
分子生物学
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生物化学
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生物技术
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真菌遗传学
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PubMed
高影响因子
Microbial Biotechnology
·
2024
·Vol. 17
·期 2
·e14415
·
IF 4.8
Zhikuan Wang,Hailiang Qiu,Yulong Chen,Xuemin Chen,Chunhua Fu,Longjiang Yu
Green manufacture of steroid precursors from diosgenin by microbial replacing multistep chemical synthesis has been elusive. It is currently limited by the lack of strain and degradation mechanisms. Here, we demonstrated the feasibility of this process using a novel strain Mycolicibacterium sp. HK-90 with efficiency in diosgenin degradation. Diosgenin degradation by strain HK-90 involves the selective removal of 5,6-spiroketal structure, followed by the oxygenolytic cleavage of steroid nuclei. Bioinformatic analyses revealed the presence of two complete steroid catabolic gene clusters, SCG-1 and SCG-2, in the genome of strain HK-90. SCG-1 cluster was found to be involved in classic phytosterols or cholesterol catabolic pathway through the deletion of key kstD1 gene, which promoted the mutant m-∆kstD1 converting phytosterols to intermediate 9α-hydroxyandrostenedione (9-OHAD). Most impressively, global transcriptomics and characterization of key genes suggested SCG-2 as a potential gene cluster encoding diosgenin degradation. The gene inactivation of kstD2 in SCG-2 resulted in the conversion of diosgenin to 9-OHAD and 9,16-dihydroxy-pregn-4-ene-3,20-dione (9,16-(OH) 2 -PG) in mutant m-ΔkstD2 . Moreover, the engineered strain mHust-ΔkstD1,2,3 with a triple deletion of kstDs was constructed, which can stably accumulate 9-OHAD by metabolizing phytosterols, and accumulate 9-OHAD and 9,16-(OH) 2 -PG from diosgenin. Diosgenin catabolism in strain mHust-ΔkstD1,2,3 was revealed as a progression through diosgenone, 9,16-(OH) 2 -PG, and 9-OHAD to 9α-hydroxytestosterone (9-OHTS). So far, this work is the first report on genetically engineered strain metabolizing diosgenin to produce 21-carbon and 19-carbon steroids. This study presents a promising biosynthetic platform for the green production of steroid precursors, and provide insights into the complex biochemical mechanism of diosgenin catabolism.
研究领域:
生物技术
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微生物学
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生物化学
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药学
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DOI
PubMed
高影响因子
ACS Synthetic Biology
·
2023
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IF 4.7
Ruosi Zhang,Mingdong Yao,Haidi Ma,Wenhai Xiao,Ying Wang,Yingjin Yuan
Substrate competition within a metabolic network constitutes a common challenge in microbial biosynthesis system engineering, especially if indispensable enzymes can produce multiple intermediates from a single substrate. Androstenedione (4AD) is a central intermediate in the production of a series of steroidal pharmaceuticals; however, its yield via the coexpression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17α-hydroxylase/17,20-lyase (CYP17A1) in a microbial chassis affords a nonlinear pathway in which these enzymes compete for substrates and produce structurally similar unwanted intermediates, thereby reducing 4AD yields. To avoid substrate competition, we split the competing 3β-HSD and CYP17A1 pathway components into two separate Yarrowia lipolytica strains to linearize the pathway. This spatial segregation increased substrate availability for 3β-HSD in the upstream strain, consequently decreasing the accumulation of the unwanted intermediate 17-hydroxypregnenolone (17OHP5) from 94.8 ± 4.4% in single-chassis monocultures to 24.8 ± 12.6% in cocultures of strains expressing 3β-HSD and CYP17A1 separately. Orthologue screening to increase CYP17A1 catalytic efficiency and the preferential production of desired intermediates increased the biotransformation capacity in the downstream pathway, further decreasing 17OHP5 accumulation to 3.9%. Furthermore, nitrogen limitation induced early 4AD accumulation (final titer, 7.71 mg/L). This study provides a framework for reducing intrapathway competition between essential enzymes during natural product biosynthesis as well as a proof-of-concept platform for linear steroid production.
研究领域:
代谢工程
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生物技术
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药学
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微生物生物技术
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Xinqi Xu,Jinchang Zhong,Bingmei Su,Lian Xu,Xiaokun Hong,Juan Lin
The synthesis of steroids is challenging through multistep steroidal core modifications with high site-selectivity and productivity. In this work, a novel enzymatic cascade system was constructed for synthesis of testolactone by specific C17 lactonization/Δ 1 -dehydrogenation from inexpensive androstenedione using an engineered polycyclic ketone monooxygenase (PockeMO) and an appropriate 3-ketosteroid-Δ 1 -dehydrogenase ( Re KstD). The focused saturation mutagenesis in the substrate binding pocket was implemented for evolution of PockeMO to eliminate the bottleneck effect. A best mutant MU3 (I225L/L226V/L532Y) was obtained with 20-fold higher specific activity compared to PockeMO. The catalytic efficiency ( k cat / K m) of MU3 was 171-fold higher and the substrate scope shifted to polycyclic ketones. Molecular dynamic simulations suggested that the activity was improved by stabilization of the pre-lactonization state and generation of productive orientation of 4-AD mediated by distal L532Y mutation. Based on that, the three genes, MU3, Re KstD and a ketoreductase for NADPH regeneration, were rationally integrated in one cell via expression fine-tuning to form the efficient single cell catalyst E. coli S9. The single whole-cell biocatalytic process was scaled up and could generate 9.0 g/L testolactone with the high space time yield of 1 g/L/h without steroidal by-product, indicating the potential for site-specific and one-pot synthesis of steroid.
研究领域:
生物化学
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生物技术
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酶学
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合成生物学
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药物化学
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PubMed
高影响因子
Inorganic Chemistry Frontiers
·
2022
·Vol. 9
·期 14
·3470-3483
·
IF 7.779
Xiao-Meng Liu,Zhe Li,Xin-Rui He,Rui-Ping Liu,Zhong-Ying Ma,Xin Qiao,Shu-Qing Wang,Jing-Yuan Xu
The combination of endocrine therapy and chemotherapy is an attractive approach for treating breast cancers. Aromatase inhibitors (AIs) are the first-line drugs for postmenopausal ER-positive breast cancer and adjuvant therapy drugs for early stages of breast cancer. In this paper, we employed the FDA-approved aromatase inhibitor aminoglutethimide (AG) to design and prepare a new class of multi-targeting Pt(IV) prodrugs (aminoplatins 1–3) by utilizing the targeting, estrogen-manipulating, and chemo-sensitizing effects of the aromatase inhibitor. Aminoplatin 3, as a representative, exhibited an enhanced anticancer activity up to 190-fold greater than the parent Pt(II) drug cisplatin, and 3 had less toxicity to normal cells, displaying higher anticancer efficacy and a superior therapeutic index compared to those of cisplatin. Moreover, aminoplatin 3 could enhance intracellular accumulation, induce remarkable DNA damage, inhibit migration and metastasis, and result in S phase cell cycle arrest and apoptosis in ER-positive MCF-7 cells. Besides, molecular docking results showed that 3 simultaneously occupies the heme iron-binding domain and the ASD-binding site of aromatase by a highly efficient dual-binding pattern to aromatase, which further displayed a striking anti-estrogenic effect by significant downregulation of CPY19A1 and ERα in ER-positive MCF-7 cells. Most strikingly, our in vivo anticancer evaluation data also provided evidence that 3 effectively inhibited tumor growth and attenuated kidney toxicity compared to cisplatin in MCF-7 xenografted BALB/c nude mice. Our results suggest that the anticancer activity of aminoplatins is mechanistically distinct from that of the conventional platinum drugs, and modifying platinum drugs with aromatase inhibitors may represent an improved modality for the treatment of advanced postmenopausal breast cancer.
研究领域:
肿瘤学
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药理学
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分子生物学
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癌症治疗
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DOI
高影响因子
Frontiers in Pharmacology
·
2024
·
IF 4.4
Dou Peiyuan,Liu Linlin,Jin Mozhu,Huang Jing,Lekhooa Rose Makhotso,Ran Xiaoku,Yan Xiaohui
Introduction: Black ginseng (BG) was processed by “steaming and drying” (generally nine times) repeatedly to produce “rare saponins” and secondary ginsenosides. Both ginseng (GS) and red ginseng (RG) were commonly used in treating heart failure (HF), and the latter was confirmed to be more potent, implying the presence of rare ginsenosides that contribute positively to the treatment of heart failure. Previous research indicated that rare ginsenosides are more abundant in BG than in RG. Consequently, this study aims to investigate the effects of BG and its components on HF to elucidate the active substances and their underlying mechanisms in the treatment of HF.Methods: The effects of BG and its fractions (water-eluted fraction (WEF), total saponin fraction (TSF), and alcohol-eluted fraction (AEF)) on rats with isoproterenol (ISO)-induced HF were explored, and steroids belonging to the hypothalamic–pituitary–adrenal (HPA) and hypothalamic–pituitary–gonadal (HPG) axes were determined quantitatively using the ultra-performance liquid chromatography–triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) method. In addition, 16S rDNA sequencing was performed on the gut microbiota, followed by GC–MS analysis of short-chain fatty acids (SCFAs), and the biochemical indexes related to energy metabolism and the serum cyclic nucleotide system were also analyzed by ELISA.Results: Based on a thorough evaluation of energy metabolism and the endocrine system, it was observed that the effects of BG components on the hypothalamic–pituitary–thyroid (HPT) and HPA axes were more pronounced. Notably, the treatment efficacy of the low dose of the total saponin fraction (TSFL), water decoction (WD), and high dose of the polysaccharide fraction (PSFH) was superior based on pharmacodynamic indicators such as brain natriuretic peptide (BNP), creatine kinase (CK), and estradiol (E2)/T). Furthermore, the WD and BG components exhibited significant effects on androgens (T and androstenedione (A4)). The TSFL group exerts an anti-inflammatory effect by regulating Lactobacillus/Erysipelotrichales. The WD, PSFH, and TSFL may impact inflammatory cytokines through the gut microbiota (Lactobacillus/Erysipelotrichales) and their metabolites (acetate and butyrate), exerting an anti-inflammatory effect.Discussion: The BG and all its split components demonstrated varying levels of efficacy in alleviating HF, and TSF and PSF exhibited a significant protective effect on HF. The main active components in TSF were revealed to be ginsenosides Rk1, Rk3, 20-(S)-Rg3, and 20-(S)-Rh2 by the H9C2 cell experiment. The decoction of BG and its components exhibited a potent impact on androgen hormones, with an elevation trend. This phenomenon may be attributed to the activation of the eNOS-NO pathway through androgen regulation, thereby contributing to its anti-HF activities. The WD, PSFH, and TSFL may exert anti-inflammatory effects through the intestinal flora (Lactobacillaceae/Erysipelotrichaceae) and its metabolites (acetic acid and butyric acid), which affect the inflammatory factors. The different mechanisms of action of each component of HF also reflect the significance and necessity of the overall role of traditional Chinese medicine (TCM). Our research was the first to report that the E2/T is related to HF and can be used as an indicator to evaluate HF.
研究领域:
药理学
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心脏病学
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传统中药
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内分泌学
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微生物学
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代谢组学
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DOI
PubMed
高影响因子
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