(+)-Borneol, the main component of "Natural Borneol" in the Chinese Pharmacopoeia, is a high-end spice and precious medicine. Plant extraction cannot meet the increasing demand for (+)-borneol, while microbial biosynthesis offers a sustainable supply route. However, its production was extremely low compared with other monoterpenes, even with extensively optimizing the mevalonate pathway. We found that the key challenge is the complex and unusual dephosphorylation reaction of bornyl diphosphate (BPP), which suffers the side-reaction and the competition from the cellular dephosphorylation process, especially lipid metabolism, thus limiting (+)-borneol synthesis. Here, we systematically optimized the dephosphorylation process by identifying, characterizing phosphatases, and balancing cellular dephosphorylation metabolism. For the first time, we identified two endogenous phosphatases and seven heterologous phosphatases, which significantly increased (+)-borneol production by up to 152%. By engineering BPP dephosphorylation and optimizing the MVA pathway, the production of (+)-borneol was increased by 33.8-fold, which enabled the production of 753 mg/L under fed-batch fermentation in shake flasks, so far the highest reported in the literature. This study showed that rewiring dephosphorylation metabolism was essential for high-level production of (+)-borneol in Saccharomyces cerevisiae, and balancing cellular dephosphorylation is also helpful for efficient biosynthesis of other terpenoids since all whose biosynthesis involves the dephosphorylation procedure.

Background Exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with the development of metabolic syndrome (MS). However, the role of amino acids in PAH-induced MS remains unclear. Objective To explore the impact of PAHs exposure on the incidence of MS among coking workers, and to determine potential modifying effect of amino acid on this relationship. Methods Unmatched nested case-control design was adopted and the baseline surveys of coking workers were conducted in two plants in Taiyuan in 2017 and 2019, followed by a 4-year follow-up. The cohort comprised 667 coking workers. A total of 362 participants were included in the study, with 84 newly diagnosed cases of MS identified as the case group and 278 as the control group. Urinary levels of 11 PAH metabolites and plasma levels of 17 amino acids were measured by ultrasensitive performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Logistic regression was used to estimate the association between individual PAH metabolites and MS. Stratified by the median concentration of amino acids, Bayesian kernel machine regression (BKMR) model was employed to assess the mixed effects of PAHs on MS. Due to the skewed data distribution, all PAH metabolites and amino acids in the analysis were converted by natural logarithm ln (expressed as lnv). Results The median age of the 362 participants was 37 years, and 83.2% were male. Compared to the control group, the case group exhibited higher concentrations of urinary 2-hydroxyphenanthrene (2-OHPhe), 9-hydroxyphenanthrene (9-OHPhe), and hydroxyphenanthrene (OHPhe) (P=0.005, P=0.049, and P=0.004, respectively), as well as elevated levels of plasma branched chain amino acid (BCAA) and aromatic amino acid (AAA) (P<0.05). After being adjusted for confounding factors, for every unit increase in lnv_2-OHPhe in urine, the OR (95%CI) of MS was 1.57 (1.11, 2.26), and for every unit increase in lnv_OHPhe, the OR (95%CI) of MS was 1.82 (1.16, 2.90). Tyrosine, leucine, and AAA all presented a significant nonlinear correlation with MS. At low levels, tyrosine, leucine, and AAA did not significantly increase the risk of MS, but at high levels, they increased the risk of MS. In the low amino acid concentration group, as well as in the low BCAA and low AAA concentration groups, it was found that compared to the PAH metabolite levels at the 50th percentile (P₅₀), the log-odds of MS when the PAH metabolite levels was at the 75th percentile (P₇₅) were 0.158 (95%CI: 0.150, 0.166), 0.218 (95%CI: 0.209, 0.227), and 0.262 (95% CI: 0.241, 0.282), respectively, However, no correlation between PAHs and MS was found in the high amino acid concentration group. Conclusion Amino acids modify the effect of PAHs exposure on the incidence of MS. In individuals with low plasma amino acid levels, the risk of developing MS increases with higher concentrations of mixed PAH exposure. This effect is partly due to the low concentrations of BCAA and AAA.

Objective:To investigate the influence of contrast-enhanced computed tomography(CECT)on dose calculation in magnetic resonance imaging(MRI)-guided online adaptive radiotherapy(oART)based on the electron density(ED)assignment method for rectal cancer.Methods:A retrospective analysis was conducted on the medical data of 15 patients with locally advanced rectal cancer at middle-low segments,who admitted to the Chinese PLA General Hospital between December 2023 and April 2025.All patients underwent both plain computed tomography(PCT)and CECT scans during location.The average HU and ED value of all organs that were extracted from PCT and CECT images in the treatment plan system were obtained,and the influences of contrast agent of intake on image characteristics of the structure of each organ(small intestine,femoral head,bladder)were analyzed.PCT was used as referred image to design reference plan(Pref).The synthetic CT(sCT)was simulated and generated on the basis of PCT and CECT,respectively.The beam flow field that was same with Pref was used to recalculate dose on sCT,and then,the online plan(PPCT)based on PCT,and the online plan(PCECT)based on CECT were obtained,respectively,which can simulate the online dose calculation of MRI-guided online adaptive radiotherapy(oART).The Pref was used as reference to compare dosimetric parameters for target region and organ at risk(OAR)through dose volume histogram(DVH)and planed evaluation indicators.Additionally,three dimension(3D)slicer software was used to perform γ analysis for the results of dose distribution,and explore the differences among PPCT,PCECT and Pref on dose distribution.Results:In terms of image characteristics,the HU values of soft-tissue organs(intestine,bladder,spinal cord,soft tissue)and planning target volume(PTV)in CECT were higher than those in PCT,and the differences of them were statistically significant(Zintestines=-2.188,Zbladder=-3.196,tspinal cord=-3.767,tsoft tissue=-10.083,tPTV=-4.693,P<0.05),while its influence was less on bone tissue.The statistical results of ED were consistent with those of HU.Regarding to dosimetric parameters,there was no statistically significant difference in target coverage rate between PPCT and Pref(P>0.05),and the D50%of the PPCT[(2724.25±19.91)cGy]was higher than that of the Pref[(2718.99±21.13)cGy],and the difference was statistically significant(t=-3.679,P=0.002).However,the target coverage rate of PCECT was 94.65(94.04,95.27)%,and the difference of that between PCECT and Pref was statistically significant(Z=-2.158,P=0.031).For OAR,the differences of Dmax value of the small intestine,and the V20 of the left femoral head between PPCT and Pref were significant(Z=-2.556,-2.529,P<0.05).The differences of the Dmax of small intestine,and the D50%of bladder between PCECT and Pref were significant(t=-4.821,2.171,P<0.05).The comparative γ passed rates of PPCT,PCECT and Pref under the standards of 3 mm/3%and 2 mm/2%were all above 95%,and the differences were not significant(P>0.05).Conclusions:The influence of CECT on dose calculation in MRI-guided oART based on ED assignment method for rectal cancer is relatively small,which can be directly used in the design of reference plan,but the maximum dose of radiation-sensitive organs such as the small intestine should be paid attention.

Objective:To investigate the influence of contrast-enhanced computed tomography(CECT)on dose calculation in magnetic resonance imaging(MRI)-guided online adaptive radiotherapy(oART)based on the electron density(ED)assignment method for rectal cancer.Methods:A retrospective analysis was conducted on the medical data of 15 patients with locally advanced rectal cancer at middle-low segments,who admitted to the Chinese PLA General Hospital between December 2023 and April 2025.All patients underwent both plain computed tomography(PCT)and CECT scans during location.The average HU and ED value of all organs that were extracted from PCT and CECT images in the treatment plan system were obtained,and the influences of contrast agent of intake on image characteristics of the structure of each organ(small intestine,femoral head,bladder)were analyzed.PCT was used as referred image to design reference plan(Pref).The synthetic CT(sCT)was simulated and generated on the basis of PCT and CECT,respectively.The beam flow field that was same with Pref was used to recalculate dose on sCT,and then,the online plan(PPCT)based on PCT,and the online plan(PCECT)based on CECT were obtained,respectively,which can simulate the online dose calculation of MRI-guided online adaptive radiotherapy(oART).The Pref was used as reference to compare dosimetric parameters for target region and organ at risk(OAR)through dose volume histogram(DVH)and planed evaluation indicators.Additionally,three dimension(3D)slicer software was used to perform γ analysis for the results of dose distribution,and explore the differences among PPCT,PCECT and Pref on dose distribution.Results:In terms of image characteristics,the HU values of soft-tissue organs(intestine,bladder,spinal cord,soft tissue)and planning target volume(PTV)in CECT were higher than those in PCT,and the differences of them were statistically significant(Zintestines=-2.188,Zbladder=-3.196,tspinal cord=-3.767,tsoft tissue=-10.083,tPTV=-4.693,P<0.05),while its influence was less on bone tissue.The statistical results of ED were consistent with those of HU.Regarding to dosimetric parameters,there was no statistically significant difference in target coverage rate between PPCT and Pref(P>0.05),and the D50%of the PPCT[(2724.25±19.91)cGy]was higher than that of the Pref[(2718.99±21.13)cGy],and the difference was statistically significant(t=-3.679,P=0.002).However,the target coverage rate of PCECT was 94.65(94.04,95.27)%,and the difference of that between PCECT and Pref was statistically significant(Z=-2.158,P=0.031).For OAR,the differences of Dmax value of the small intestine,and the V20 of the left femoral head between PPCT and Pref were significant(Z=-2.556,-2.529,P<0.05).The differences of the Dmax of small intestine,and the D50%of bladder between PCECT and Pref were significant(t=-4.821,2.171,P<0.05).The comparative γ passed rates of PPCT,PCECT and Pref under the standards of 3 mm/3%and 2 mm/2%were all above 95%,and the differences were not significant(P>0.05).Conclusions:The influence of CECT on dose calculation in MRI-guided oART based on ED assignment method for rectal cancer is relatively small,which can be directly used in the design of reference plan,but the maximum dose of radiation-sensitive organs such as the small intestine should be paid attention.

Background: Ophiopogon japonicus (L.f) Ker-Gawl. growing in Zhejiang is recognized as the Dao-di medicinal herb for the production of Ophiopogonis Radix. Borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside, a prominent pharmacologically active compound, serves as a marker distinguishing O. japonicus in Zhejiang from those in other geographical areas. It is synthesized from borneol through glycosylation, with terpene synthase (TPS) being the critical enzyme catalyzing the conversion of terpene precursors into borneol. Objective: The aim of the study was to define key genes involved in biosynthesis of borneol in O. japonicus. Methods: The candidate terpene synthase genes were identified from the root and leaf transcriptome data of O. japonicus in Zhejiang and the functions of these enzymes were identified using engineered Escherichia coli. Results: This study developed a rapid expression system for monoterpene and sesquiterpene synthases based on engineered E. coli. Seven terpene synthase genes (OjTPS1 to OjTPS7) were identified in different terpene synthase subfamilies, including 2 from TPS-a, 4 from TPS-b, and 1 from TPS-g. Biochemical analysis using an engineered system E. coli demonstrated that all the 7 terpene synthases produced monoterpenes, and OjTPS3, OjTPS5, and OjTPS6 also yielded sesquiterpenes. Conclusions: These 7 terpene synthases produced 13 monoterpenes and 8 sesquiterpenes. Notably, OjTPS1 produced borneol establishing the groundwork for elucidating the biosynthetic pathways of borneol-7-O-[β-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside and other volatile oil components.

Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.
Humans ; Salvia miltiorrhiza/metabolism* ; Biosynthetic Pathways ; Quinones/metabolism* ; Plant Roots/metabolism* ; Gene Expression Regulation, Plant

OBJECTIVE: To investigate the relationship of polycyclic aromatic hydrocarbons( PAH) metabolites,DNA oxidative damage and ring finger protein 2( RING2) expression in coke oven workers. METHODS: A judgment sampling method was used to select 497 coke oven workers in a steel plant as exposure group and 175 water treatment workers in the same plant as control group. The levels of urinary 1-hydroxypyrene, 2-hydroxynathalene, 2-hydroxyfluorene,9-hydroxyphenanthrene and 8-hydroxy deoxyguanosine(8-OHd G) were detected by high performance liquid chromatography.The RING2 expression in whole blood was measured by reverse transcription-polymerase chain reaction. RESULTS: The relative expression of urinary 1-hydroxypyrene,2-hydroxynathalene,2-hydroxyfluorene,9-hydroxyphenanthrene and RING2 in exposure group were higher than that in control group( P < 0. 01). The logistic regression analysis indicated that the higher the level of 1-hydroxypyrene,the higher the risk of high-RING2 expression( P < 0. 05) after adjusting for factors such as sex,age,smoking status,alcohol drinking,2-hydroxynathalene,2-hydroxyfluorene and 9-hydroxyphenanthrene.In 1-hydroxypyrene middle and high level groups,the 8-OHd G concentration of high-RING2 expression workers was significantly higher than those of low-RING2 expression workers( P < 0. 05). CONCLUSION: With the increase of urinary1-hydroxypyrene,the risk of high-RING2 expression was elevated,the degree of DNA oxidative damage was gradually increased.

Objective To investigate the relationship between urinary polycyclic aromatic hydrocarbon metabolite and cell cycle of lymphocyte in coke oven workers.Methods 437 coke oven workers and 163 workers in water treatment department were recruited in this study.Flow cytometry was used to detect the cell cycle of lymphocyte.For the measurement of urinary metabolites,urine samples were treated with β-glucuronidase and analyzed using HPLC with a fluorescence detector.Results The concentrations of urinary 2-naphthol,2-hydroxyfluorene,9-phenanthrol and 1-hydroxypyrene 1 in coke oven workers were significantly higher than those in control group (P＜0.01).The distributions of cell cycle were analyzed in high exposure group (the content of urinary metabolites high than P75) and low exposure group (the content of urinary metabolites low than P25).According to the content of 1-hydroxypyrene,the proportions of S phase in high exposure group were significantly higher than those of low exposure group (Z=-2.496,P=0.013),but the proportions of G0/G1 phase were significantly lower than low exposure group (Z=-2.074,P=0.038).The similar results were not been found in other hydroxylated metabolites as internal exposure group.Conclusion Increasing levels of urinary 1-hydroxypyrene might resulting in cell cycle of lymphocyte disorders,mainly for G0/G1 phase shorten and S phase arrest.

Objective To investigate the relationship between urinary polycyclic aromatic hydrocarbon metabolite and cell cycle of lymphocyte in coke oven workers.Methods 437 coke oven workers and 163 workers in water treatment department were recruited in this study.Flow cytometry was used to detect the cell cycle of lymphocyte.For the measurement of urinary metabolites,urine samples were treated with β-glucuronidase and analyzed using HPLC with a fluorescence detector.Results The concentrations of urinary 2-naphthol,2-hydroxyfluorene,9-phenanthrol and 1-hydroxypyrene 1 in coke oven workers were significantly higher than those in control group (P＜0.01).The distributions of cell cycle were analyzed in high exposure group (the content of urinary metabolites high than P75) and low exposure group (the content of urinary metabolites low than P25).According to the content of 1-hydroxypyrene,the proportions of S phase in high exposure group were significantly higher than those of low exposure group (Z=-2.496,P=0.013),but the proportions of G0/G1 phase were significantly lower than low exposure group (Z=-2.074,P=0.038).The similar results were not been found in other hydroxylated metabolites as internal exposure group.Conclusion Increasing levels of urinary 1-hydroxypyrene might resulting in cell cycle of lymphocyte disorders,mainly for G0/G1 phase shorten and S phase arrest.