1.SUN Yuanzheng's experience with yuan-primary and luo-connecting meridian-regulating acupuncture for diabetic retinopathy.
Hongju YOU ; Yuanzheng SUN ; Jiaohui LI ; Yuan ZHANG
Chinese Acupuncture & Moxibustion 2025;45(4):516-520
This paper summarizes Professor SUN Yuanzheng's academic thought and clinical experience in treating diabetic retinopathy (DR) with yuan-primary and luo-connecting meridian-regulating acupuncture. Professor SUN considers that the fundamental cause of DR lies in visceral dysfunction, with stagnation in the ocular collateral vessels and impairment of the mysterious orifices being the core pathogenesis. He proposes the treatment model of "regulating viscera, unblocking ocular collaterals, and opening mysterious orifices" based on yuan-primary and luo-connecting meridian-regulating acupuncture. Yuan-primary and luo-connecting points are used to balance qi, blood, yin, and yang of the viscera. Baihui (GV20) is stimulated with transcranial repetitive acupuncture to activate the yang qi of the governor vessel and invigorate qi in the twelve meridians, improving systemic microcirculation. Combining this with stimulation of ocular areas, Fengchi (GB20), and periocular points, the stasis in the ocular collaterals are cleared and the mysterious orifices are unblocked, addressing both the root and manifestations of DR.
Humans
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Diabetic Retinopathy/physiopathology*
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Acupuncture Therapy/history*
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Meridians
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Acupuncture Points
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Qi
2.Analysis of the correlation between blood lipid levels and cognitive dysfunction in elderly people aged 65 and above
Jinping HUANG ; Yuanzheng FU ; Yangjian PAN ; Yurong HU ; Jinquan ZHANG ; Xiaoyan DU
Chinese Journal of Preventive Medicine 2025;59(7):1084-1089
his cross-sectional study employed convenience sampling to enroll 1 994 community-dwelling older adults (aged ≥65 years) undergoing health examinations at a Guangzhou community hospital between January and December 2024, aiming to investigate associations between blood lipid profiles and cognitive impairment. Cognitive function was assessed using the AD8 scale, with demographic characteristics (age, sex, education, occupation), health status (hypertension, diabetes mellitus), and lifestyle factors (smoking/alcohol use) collected via questionnaires. Fasting blood samples quantified triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Propensity score matching (PSM) balanced baseline characteristics between lipid-level groups. Pre-PSM analyses revealed significant intergroup differences: TG groups differed in sex, BMI, alcohol use, hypertension, and self-rated health ( P<0.05); TC groups in sex, age, occupation, diabetes, and hypertension; LDL-C groups in sex, age, occupation, diabetes, hypertension, and daily living ability; HDL-C groups in sex, age, education, occupation, BMI, smoking, diabetes, and hypertension. Post-PSM adjustment eliminated baseline differences ( P>0.05). Multivariable logistic regression adjusted for demographic, clinical, and lifestyle factors demonstrated that elevated TG levels conferred a 48% reduced risk of cognitive dysfunction [ OR (95% CI): 0.52 (0.29-0.94)], whereas TC, LDL-C, and HDL-C showed no significant associations (all P>0.05). These findings suggest an inverse association between higher triglyceride levels and cognitive dysfunction risk in older adults, highlighting TG′s potential protective role in cognitive health.
3.Effects of electroheating needle on pathologic pain, endoplasmic reticulum stress and EGFR/JAK/STAT signaling pathway in rats with knee osteoarthritis
Jie ZHANG ; Mingjun XU ; Yuanzheng YANG
International Journal of Traditional Chinese Medicine 2025;47(3):342-347
Objective:To investigate the effects of electroheating on pathologic pain, endoplasmic reticulum stress and EGFR/JAK/STAT signaling pathway in rats with knee osteoarthritis.Methods:Totally 40 SPF SD male rats were divided into normal group (10 rats) and modeling group (40 rats) with random number table method. The model of knee osteoarthritis was prepared by injecting sodium iodoacetate into the joint cavity. The successfully modeled rats were divided into model group, sodium hyaluronate group, and electroheating needle group, with 10 rats in each group. After successful modeling, sodium hyaluronate group was injected with 0.1 ml sodium hyaluronate into joint cavity. The electrothermal acupuncture group was treated with electrothermal acupuncture, and the normal group and the model group were given the same volume of normal saline at the same time, once a day, for two weeks. The mechanical pain threshold (PWT) and thermal pain threshold (PWL) in rats were detected. The pathological morphology of cartilage was detected by HE staining. Immunohistochemical staining was used to detect the expressions of glucose regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP) in cartilage tissue. Western blot was used to detect the expressions of EGFR, JAK, and STAT proteins, while RT-PCR was used to detect the mRNA levels of GRP78, CHOP, EGFR, JAK, and STAT in cartilage tissue.Results:The normal group had normal cartilage tissue structure, orderly distribution of synovial cells, neat arrangement of chondrocytes, and no inflammatory cell infiltration. The model group had synovial hyperplasia and hypertrophy, irregular arrangement of synovial cells and a large number of hyperplasia, disordered arrangement of chondrocytes and increased number of inflammatory cell infiltration. Compared with the model group, the PWT and PWL in cartilage tissues of sodium hyaluronate group and electrothermal acupuncture group increased ( P<0.05), the expressions of GRP78 and CHOP in cartilage tissues decreased ( P<0.05), the protein expressions of EGFR, JAK1 and STAT1 in cartilage tissue decreased, and the mRNA expressions of GRP78, CHOP, EGFR, JAK1 and STAT1 in cartilage tissue decreased ( P<0.05). Conclusion:Electroheating needle can improve pathological pain in knee osteoarthritis model rats, and its mechanism may be related to inhibiting endoplasmic reticulum stress and regulating the EGFR/JAK/STAT signaling pathway.
4.Exploration of biomarkers for moyamoya disease and analysis of traditional Chinese medicine targets
Rulin ZHOU ; Yuanzheng HU ; Zongqing WANG ; Guoping ZHOU ; Baochao ZHANG ; Qian XU ; Fanghui BAI
Chinese Journal of Tissue Engineering Research 2025;29(32):6927-6938
BACKGROUND:The pathogenesis of moyamoya disease is unclear,and there is a lack of early diagnostic methods and effective medical treatments.OBJECTIVE:To identify biomarkers of moyamoya disease through machine learning,and analyze the therapeutic mechanism of Bu Huang Si Wu Tang in moyamoya disease.METHODS:Moyamoya disease gene dataset from gene expression omnibus database was retrieved.R language was used to screen differentially expressed genes and perform enrichment analysis on differentially expressed genes.Moyamoya disease biomarkers were screened using least absolute shrinkage and selection operator and support vector machine-recursive feature elimination machine learning algorithms.A diagnostic model was constructed by establishing a column chart,and its clinical utility and diagnostic value were evaluated and further validated through detecting clinical serum markers of patients with moyamoya disease.In vitro experiments were performed to observe the effect of Bu Huang Si Wu Tang on umbilical vein endothelial cells under oxygen glucose deprivation,and the main components of Bu Huang Si Wu Tang were selected for molecular docking and molecular dynamics simulation with the screened targets to explore its potential mechanism.RESULTS AND CONCLUSION:This study identified 76 differentially expressed genes,mainly enriched in immune and metabolic related pathways.Two potential biomarkers,complement factor D(CFD)and DKFZp434L192,were obtained through machine learning.The calibration curve,decision curve analysis,and clinical impact curve showed that the column chart had good predictive ability.qRT-PCR analysis showed that CFD and DKFZp434L192 expression levels were significantly upregulated in patients with moyamoya disease(P<0.001).In vitro experiments showed that Bu Huang Si Wu Tang significantly enhanced the activity and migration ability(P<0.01)of umbilical vein endothelial cells under oxygen glucose deprivation.Molecular docking results showed that all four active ingredients of Bu Huang Si Wu Tang could bind to the target site,and the dynamic simulation results further verified its binding stability.To conclude,CFD and DKFZp434L192 exhibit good diagnostic efficacy for moyamoya disease and can serve as potential diagnostic biomarkers and therapeutic targets for moyamoya disease.Bu Huang Si Wu Tang has a certain protective effect on vascular endothelial cells under ischemic conditions,which may be related to CFD.
5.Analysis of the correlation between blood lipid levels and cognitive dysfunction in elderly people aged 65 and above
Jinping HUANG ; Yuanzheng FU ; Yangjian PAN ; Yurong HU ; Jinquan ZHANG ; Xiaoyan DU
Chinese Journal of Preventive Medicine 2025;59(7):1084-1089
his cross-sectional study employed convenience sampling to enroll 1 994 community-dwelling older adults (aged ≥65 years) undergoing health examinations at a Guangzhou community hospital between January and December 2024, aiming to investigate associations between blood lipid profiles and cognitive impairment. Cognitive function was assessed using the AD8 scale, with demographic characteristics (age, sex, education, occupation), health status (hypertension, diabetes mellitus), and lifestyle factors (smoking/alcohol use) collected via questionnaires. Fasting blood samples quantified triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Propensity score matching (PSM) balanced baseline characteristics between lipid-level groups. Pre-PSM analyses revealed significant intergroup differences: TG groups differed in sex, BMI, alcohol use, hypertension, and self-rated health ( P<0.05); TC groups in sex, age, occupation, diabetes, and hypertension; LDL-C groups in sex, age, occupation, diabetes, hypertension, and daily living ability; HDL-C groups in sex, age, education, occupation, BMI, smoking, diabetes, and hypertension. Post-PSM adjustment eliminated baseline differences ( P>0.05). Multivariable logistic regression adjusted for demographic, clinical, and lifestyle factors demonstrated that elevated TG levels conferred a 48% reduced risk of cognitive dysfunction [ OR (95% CI): 0.52 (0.29-0.94)], whereas TC, LDL-C, and HDL-C showed no significant associations (all P>0.05). These findings suggest an inverse association between higher triglyceride levels and cognitive dysfunction risk in older adults, highlighting TG′s potential protective role in cognitive health.
6.Exploration of biomarkers for moyamoya disease and analysis of traditional Chinese medicine targets
Rulin ZHOU ; Yuanzheng HU ; Zongqing WANG ; Guoping ZHOU ; Baochao ZHANG ; Qian XU ; Fanghui BAI
Chinese Journal of Tissue Engineering Research 2025;29(32):6927-6938
BACKGROUND:The pathogenesis of moyamoya disease is unclear,and there is a lack of early diagnostic methods and effective medical treatments.OBJECTIVE:To identify biomarkers of moyamoya disease through machine learning,and analyze the therapeutic mechanism of Bu Huang Si Wu Tang in moyamoya disease.METHODS:Moyamoya disease gene dataset from gene expression omnibus database was retrieved.R language was used to screen differentially expressed genes and perform enrichment analysis on differentially expressed genes.Moyamoya disease biomarkers were screened using least absolute shrinkage and selection operator and support vector machine-recursive feature elimination machine learning algorithms.A diagnostic model was constructed by establishing a column chart,and its clinical utility and diagnostic value were evaluated and further validated through detecting clinical serum markers of patients with moyamoya disease.In vitro experiments were performed to observe the effect of Bu Huang Si Wu Tang on umbilical vein endothelial cells under oxygen glucose deprivation,and the main components of Bu Huang Si Wu Tang were selected for molecular docking and molecular dynamics simulation with the screened targets to explore its potential mechanism.RESULTS AND CONCLUSION:This study identified 76 differentially expressed genes,mainly enriched in immune and metabolic related pathways.Two potential biomarkers,complement factor D(CFD)and DKFZp434L192,were obtained through machine learning.The calibration curve,decision curve analysis,and clinical impact curve showed that the column chart had good predictive ability.qRT-PCR analysis showed that CFD and DKFZp434L192 expression levels were significantly upregulated in patients with moyamoya disease(P<0.001).In vitro experiments showed that Bu Huang Si Wu Tang significantly enhanced the activity and migration ability(P<0.01)of umbilical vein endothelial cells under oxygen glucose deprivation.Molecular docking results showed that all four active ingredients of Bu Huang Si Wu Tang could bind to the target site,and the dynamic simulation results further verified its binding stability.To conclude,CFD and DKFZp434L192 exhibit good diagnostic efficacy for moyamoya disease and can serve as potential diagnostic biomarkers and therapeutic targets for moyamoya disease.Bu Huang Si Wu Tang has a certain protective effect on vascular endothelial cells under ischemic conditions,which may be related to CFD.
7.Impact of ultra-early enteral nutrition support on the prognosis of young and middle-aged postoperative patients with cerebral hemorrhage
Lili BAI ; Qingguo FENG ; Hongyun TENG ; Liyan GUO ; Dangqing WANG ; Yuanzheng ZHANG
Chinese Critical Care Medicine 2024;36(9):985-988
Objective:To investigate the effect of ultra-early enteral nutrition (UEEN) support on the prognosis of young and middle-aged postoperative patients with cerebral hemorrhage.Methods:The clinical data of young and middle-aged patients (aged 18-59 years) admitted to Tianjin Fifth Central Hospital from January 2020 to June 2023 after surgery for intracerebral hemorrhage were retrospectively analyzed, and the general data, nutritional indexes, gastrointestinal complications, neurological function recovery and long-term prognosis of the patients were recorded. According to the time of initiation of enteral nutrition (EN) support, patients were divided into UEEN group (EN implementation within 12 hour after surgery) and early enteral nutrition (EEN) group (EN implementation within 24 to 48 hour after surgery). The differences of the above indexes between the two groups were analyzed and compared.Results:A total of 64 young and middle-aged postoperative patients with cerebral hemorrhage were enrolled, including 32 cases in the UEEN group and 32 cases in the EEN group. There were no significant differences in gender, age, proportion of hypertension and diabetes, Glasgow coma score (GCS) on admission and surgical methods between the two groups. In terms of nutritional indexes, serum total protein, albumin and hemoglobin levels of patients in both groups on day 7 after admission were lower than those on day 1, and higher than those on day 3, and the above indexes levels in UEEN group were significantly higher than those in EEN group on day 7 [total protein (g/L): 63.05±5.79 vs. 59.02±6.63, albumin (g/L): 40.40±5.26 vs. 37.66±4.63, hemoglobin (g/L): 133.33±12.58 vs. 123.80±22.12, all P < 0.05]. In terms of gastrointestinal complications, the incidence of stress ulcer in the UEEN group within 14 days after admission was significantly lower than that in the EEN group [12.5% (4/32) vs. 31.3% (10/32), P < 0.05], but there was no statistically significant difference in feeding intolerance symptoms between the two groups. In terms of neurological recovery and long-term prognosis, GCS scores and Barthel index scores of 14 days after admission were higher than those of 1 day after admission, but there was no statistical significance between the two groups. Six months after surgery, Glasgow outcome scale (GOS) and Barthel index score of the UEEN group were significantly higher than those of the EEN group (GOS score: 3.81±1.06 vs. 3.18±1.07, Barthel index score: 60.78±7.24 vs. 54.52±5.13, both P < 0.05). Conclusion:UEEN support can improve the nutritional level of young and middle-aged postoperative patients with cerebral hemorrhage, reduce the occurrence of postoperative gastrointestinal complications, promote the recovery of neurological function, and improve the long-term prognosis.
8.Effects and mechanisms of ATP-sensitive potassium channel openers on microglial inflammatory response induced by oxygen-glucose deprivation/reoxygenation
Shanshan YIN ; Chenming ZHAO ; Qiujun ZHANG ; Yuanzheng ZHAO
Chinese Journal of Behavioral Medicine and Brain Science 2024;33(9):776-782
Objective:To investigate the effects and mechanisms of Nicorandil, an ATP-sensitive potassium channel (KATP) opener, on pyroptosis and inflammatory responses in microglia(BV2) induced by oxygen-glucose deprivation/reoxygenation (OGD/R).Methods:BV2 cells were divided into control group, OGD/R group, and OGD/R+ Nicorandil group.And the cells were subjected to oxygen-glucose deprivation for 3 hours and then reoxygenated for 24 hours to establish an OGD/R cell model. OGD/R+ Nicorandil group cells were incubated with 5 μg/mL Nicorandil culture medium for 24 hours after oxygen-glucose deprivation for 3 hours.The cell proliferation activity was detected by CCK8 assay.Calcein/propidium iodide (calcein/PI) assay kit was used to detect the membrane porosity rupture rate of cell in each group.Western blot analysis was performed to detect the protein expression levels of nuclear factor-κB (NF-κB), phosphorylated NF-κB (p-NF-κB), inhibitor of nuclear factor-κB α(IκB-α), phosphorylated IκB-α (p-IκB-α), absent in melanoma 2 (AIM2), cleaved-caspase-1, gasdermin D-N (GSDMD-N), interleukin-18 (IL-18), and interleukin-1β (IL-1β). Immunofluorescence was used to detect the protein expression levels of AIM2 and GSDMD-N in each group. Statistical analysis was performed by SPSS 26.0 software. One-way ANOVA was used for multiple group comparisons, and LSD test was used for pairwise comparisons.Results:(1) There were statistically significant differences in the membrane porosity rupture rates among the three groups ( F=615.882, P<0.05). The membrane porosity rupture rate in the Nicorandil group was lower than that in the OGD/R group ((41.50±3.04)%, (59.44±3.66)%, P<0.05). (2) Western blot results showed that the protein expression levels of p-NF-κB, NF-κB, p-IκB-α, and IκB-α were significantly different among the three groups ( F=10.000, 62.652, 67.121, 101.023, all P<0.05). The levels of p-NF-κB, NF-κB and p-IκB-α in the OGD/R+ Nicorandil group ((0.60±0.13), (0.87±0.06), (0.55±0.06), respectively) were lower than those in the OGD/R group ((1.02±0.09), (1.03±0.09), (0.86±0.04), respectively) (all P<0.05). The level of IκB-α in the OGD/R+ Nicorandil group ((0.63±0.05), (0.46±0.06)) was higher than that in the OGD/R group( P<0.05). (3) The protein expression levels of AIM2, cleaved-caspase-1, GSDMD-N, IL-18, and IL-1β were significantly different among the three groups ( F=65.926, 12.428, 66.447, 44.831, 52.960, all P<0.05). The levels of AIM2, cleaved-caspase-1, GSDMD-N, IL-18 and IL-1β in the OGD/R+ Nicorandil group ((0.78±0.04), (0.71±0.09), (0.54±0.04), (0.72±0.07), (0.50±0.08), respectively) were lower than those in the OGD/R group ((0.94±0.09), (0.89±0.09), (0.85±0.04), (0.90±0.07), (0.99±0.03), respectively) (all P<0.05). (4) Immunofluorescence results showed statistically significant differences in the fluorescence intensity of pyroptosis marker proteins AIM2 and GSDMD-N among the three groups ( F=36.353, 46.817, both P<0.05). The fluorescence intensities of AIM2 ((124.36±7.91), (140.19±5.63)) and GSDMD-N ((134.16±5.18), (147.45±5.63))in the OGD/R+ Nicorandil group were lower than those in the OGD/R group (both P<0.05). Conclusion:Nicorandil can mitigate BV2 cell damage following oxygen-glucose deprivation, inhibiting the release of pro-inflammatory factors. The mechanism may be related to the downregulation of the expression of NF-κB related proteins and inhibition of AIM2 inflammasome-mediated pyroptosis after OGD/R.
9.Multicenter evaluation of the diagnostic efficacy of jaundice color card for neonatal hyperbilirubinemia
Guochang XUE ; Huali ZHANG ; Xuexing DING ; Fu XIONG ; Yanhong LIU ; Hui PENG ; Changlin WANG ; Yi ZHAO ; Huili YAN ; Mingxing REN ; Chaoying MA ; Hanming LU ; Yanli LI ; Ruifeng MENG ; Lingjun XIE ; Na CHEN ; Xiufang CHENG ; Jiaojiao WANG ; Xiaohong XIN ; Ruifen WANG ; Qi JIANG ; Yong ZHANG ; Guijuan LIANG ; Yuanzheng LI ; Jianing KANG ; Huimin ZHANG ; Yinying ZHANG ; Yuan YUAN ; Yawen LI ; Yinglin SU ; Junping LIU ; Shengjie DUAN ; Qingsheng LIU ; Jing WEI
Chinese Journal of Pediatrics 2024;62(6):535-541
Objective:To evaluate the diagnostic efficacy and practicality of the Jaundice color card (JCard) as a screening tool for neonatal jaundice.Methods:Following the standards for reporting of diagnostic accuracy studies (STARD) statement, a multicenter prospective study was conducted in 9 hospitals in China from October 2019 to September 2021. A total of 845 newborns who were admitted to the hospital or outpatient department for liver function testing due to their own diseases. The inclusion criteria were a gestational age of ≥35 weeks, a birth weight of ≥2 000 g, and an age of ≤28 days. The neonate′s parents used the JCard to measure jaundice at the neonate′s cheek. Within 2 hours of the JCard measurement, transcutaneous bilirubin (TcB) was measured with a JH20-1B device and total serum bilirubin (TSB) was detected. The Pearson′s correlation analysis, Bland-Altman plots and the receiver operating characteristic (ROC) curve were used for statistic analysis.Results:Out of the 854 newborns, 445 were male and 409 were female; 46 were born at 35-36 weeks of gestational age and 808 were born at ≥37 weeks of gestational age. Additionally, 432 cases were aged 0-3 days, 236 cases were aged 4-7 days, and 186 cases were aged 8-28 days. The TSB level was (227.4±89.6) μmol/L, with a range of 23.7-717.0 μmol/L. The JCard level was (221.4±77.0) μmol/L and the TcB level was (252.5±76.0) μmol/L. Both the JCard and TcB values showed good correlation ( r=0.77 and 0.80, respectively) and agreements (96.0% (820/854) and 95.2% (813/854) of samples fell within the 95% limits of agreement, respectively) with TSB. The JCard value of 12 had a sensitivity of 0.93 and specificity of 0.75 for identifying a TSB ≥205.2?μmol/L, and a sensitivity of 1.00 and specificity of 0.35 for identifying a TSB ≥342.0?μmol/L. The TcB value of 205.2?μmol/L had a sensitivity of 0.97 and specificity of 0.60 for identifying TSB levels of 205.2 μmol/L, and a sensitivity of 1.00 and specificity of 0.26 for identifying TSB levels of 342.0 μmol/L. The areas under the ROC curve (AUC) of JCard for identifying TSB levels of 153.9, 205.2, 256.5, and 342.0 μmol/L were 0.96, 0.92, 0.83, and 0.83, respectively. The AUC of TcB were 0.94, 0.91, 0.86, and 0.87, respectively. There were both no significant differences between the AUC of JCard and TcB in identifying TSB levels of 153.9 and 205.2 μmol/L (both P>0.05). However, the AUC of JCard were both lower than those of TcB in identifying TSB levels of 256.5 and 342.0 μmol/L (both P<0.05). Conclusions:JCard can be used to classify different levels of bilirubin, but its diagnostic efficacy decreases with increasing bilirubin levels. When TSB level are ≤205.2 μmol/L, its diagnostic efficacy is equivalent to that of the JH20-1B. To prevent the misdiagnosis of severe jaundice, it is recommended that parents use a low JCard score, such as 12, to identify severe hyperbilirubinemia (TSB ≥342.0 μmol/L).
10.Two types of coumarins-specific enzymes complete the last missing steps in pyran- and furanocoumarins biosynthesis.
Yucheng ZHAO ; Yuedong HE ; Liangliang HAN ; Libo ZHANG ; Yuanzheng XIA ; Fucheng YIN ; Xiaobing WANG ; Deqing ZHAO ; Sheng XU ; Fei QIAO ; Yibei XIAO ; Lingyi KONG
Acta Pharmaceutica Sinica B 2024;14(2):869-880
Pyran- and furanocoumarins are key representatives of tetrahydropyrans and tetrahydrofurans, respectively, exhibiting diverse physiological and medical bioactivities. However, the biosynthetic mechanisms for their core structures remain poorly understood. Here we combined multiomics analyses of biosynthetic enzymes in Peucedanum praeruptorum and in vitro functional verification and identified two types of key enzymes critical for pyran and furan ring biosynthesis in plants. These included three distinct P. praeruptorum prenyltransferases (PpPT1-3) responsible for the prenylation of the simple coumarin skeleton 7 into linear or angular precursors, and two novel CYP450 cyclases (PpDC and PpOC) crucial for the cyclization of the linear/angular precursors into either tetrahydropyran or tetrahydrofuran scaffolds. Biochemical analyses of cyclases indicated that acid/base-assisted epoxide ring opening contributed to the enzyme-catalyzed tetrahydropyran and tetrahydrofuran ring refactoring. The possible acid/base-assisted catalytic mechanisms of the identified cyclases were theoretically investigated and assessed using site-specific mutagenesis. We identified two possible acidic amino acids Glu303 in PpDC and Asp301 in PpOC as vital in the catalytic process. This study provides new enzymatic tools in the epoxide formation/epoxide-opening mediated cascade reaction and exemplifies how plants become chemically diverse in terms of enzyme function and catalytic process.

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