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.

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.

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).

Objective:To observe the clinical effect of acupuncture treatment based on Tiaoshen theory for the patients with functional dyspepsia (FD) and depression with liver-stomach disharmony syndrome. Methods:A total of 76 FD patients from May 2018 to August 2019 were randomly divided into 2 groups with 38 patients in each group. In the routine group, acupoints were selected routinely, and in Tiaoshen group, acupoints were selected by Tiaoshen theory. Both groups were treated for 28 days. The results were evaluated by Nepean Dyspepsia Index (NDI), TCM Syndrome Score and Hamilton Depression Scale-24 (HAMD-24). Results:The total effective rate of both groups was 94.6% (35/37) in Tiaoshen group and 75.0% (27/36) in routine group. There was significant difference between the two groups ( χ2=6.125, P=0.011). The NDI in Tiaoshen group was significantly lower than that of routine group ( t=3.038, P=0.003). The scores of interference domain, control domain, food and beverage domain and sleep disturbance domain in Tiaoshen group were significantly higher than those in routine group ( t=3.096, 2.460, 2.225, 2.732, P<0.05); the TCM Syndrome Score in Tiaoshen group was significantly lower than that of routine group ( t=3.241, P=0.002), and that of HAMD-24 was significantly lower than that of routine group ( t=2.767, P=0.007). Conclusion:Treatment based on Tiaoshen theory can improve the quality of life of FD patients in the fields of interference, control, food and beverage and sleep disturbance, and reduce the patients’depression.

Objective To fabricate an antibacterial controlled drug delivery system with PEG-hydrogel and gentamicin-loaded-CSt on titanium surface,and to investigate its surface characteristics,swelling behavior,drug release behavior in vitro,antiinfection performance in vivo,and tissue biocompatibility.Methods Cross-linked starch (CSt) was synthesized first and then CSt was loaded with gentamicin (GEN) as a carrier (GEN@CSt),then 4-arm-polyethylene glycol (PEG) was added to it which was mixed by ultrasound.The surface of titanium (Ti) was covered with a layer of poly dopamine (PDA).The drug-loaded hydrogel was fixed to the titanium surface,subsequently capped by poly lactic-co-glycolic acid (PLGA) membranes,and then the Ti-PDA-PEG (GEN@CSt)-PLGA composite coating was fabricated finally.Surface morphology of the system was observed,while the swelling behavior was characterized;release behavior of the composite coating was detected;the bacteriostatic experiments were carried out with staphylococcus aureus (SAU),staphylococcus epidermidis (SEP) and escherichia coli (ECO) in vitro.The animal models of infected bone defect was established in 36 New Zealand white rabbits.These animals were randomly divided into three groups.Group 1 animals were implanted with drug-loaded composite coatings.Group 2 animals were implanted with drug-free composite coatings.Group 3 animals were implanted with bare titanium rods.The infection data were collected periodically to carry out antiinfection experiments in vivo.Another 12 rabbits were divided into the experimental group and the control group randomly.Biocompatibility of the materials was observed by histopathology after implantation of the corresponding materials into the femoral condyle.Results The composite coating adhered to the titanium surface firmly,presenting a smooth and translucent shape.The ratio of CSt/PEG affects swelling behavior varied,starch-free gels maintained an equilibrium swelling of 7.4,after the ratio reached 1 ∶ 1,the equilibrium swelling ratio remained at 3.0.In-vitro the release rate of the first 8 h was fast,and the cumulative release amount accounted for 83％ of the total in the first 7 days,lasting more than 13 d.In vitro antibacterial test,the average diameter of the inhibition ring was 3.6±0.13 cm (SAU),3.4±0.11 cm (SEP),3.7±0.10 cm (ECO).In-vivo anti-infection experiment,the infection situation of the group 1 was better than the control groups 2 and 3.The pathological results indicated that inflammatory reaction in the experimental group was basically the same as the control group.Conclusion The study successfully fabricated the antibacterial controlled drug delivery system with PEG-hydrogel and gentamicin-loaded-CSt on titanium surface.The system has a reasonable drug release behavior,and effectively inhibited the growth of bacteria in vivo and in vitro.It also has good biocompatibility to stand a promising strategy to improve the orthopedics anti-infection.

Objective To explore the neuroprotection and mechanisms of bone marrow mononuclear cells (BMMNCs),and evaluate whether ERK1/2 signaling pathway was involved in it.Methods384 healthy male SD rats,which were 6-8 week old,weighting 250-280 g,were selected.The middle cerebral artery occlusion (MCAO) model was established in SD rats using the suture method.The rats were randomly divided into sham operation group,model group,BMMNCs group and ERK1/2 inhibitor group,with 96 rats in each group.At the time of 24 h after the successful modeling,200 μl PBS solution was injected into the caudal vein of the rats in the model group,200 μl PBS solution containing 5×106 BMMNCs was injected into the rats in the BMMNCs group and the ERK1/2 inhibitor group.meanwhile,5 μl PD98059 was injected into the lateral ventricle of the brain of rats in the ERK1/2 inhibitor group.At the time points of 3 d,7 d and 14 d,the modified neurological severity scores (mNSS) was used to evaluate the neurological function,the volume of cerebral infarction was assessed by TTC staining,the pERK1/2,Bax,Bcl-2 and caspase-3 levels were detected by Western blot,and the effect of BMMNCs on activation of microglia was detected by immunofluorescence assay.Results(1)At each time point,the mNSS and the volume of cerebral infarction of the model group were significantly higher than those of the sham operation group (P<0.05),while the mNSS and the volume of cerebral infarction of the BMMNCs group were lower than those of the model group,higher than those of the sham operation group,and it was gradually decreased with the treatment time extension (P<0.05).There was no difference in comparison between the ERK1/2 inhibitor group and the model group (P>0.05).(2)At each time point,the pERK1/2,Bcl-2,Bax and caspase-3 protein levels of the model group were significantly higher than those of the sham operation group (P<0.05).The pERK1/2 and Bcl-2 protein levels of the BMMNCs group(pERK1/2:(0.38±0.16),(0.39±0.15),(0.40±0.20),Bcl-2:(0.38±0.14),(0.39±0.15),(0.37±0.13)) were higher than those of the model group(pERK1/2:(0.17±0.05),(0.14±0.04),(0.13±0.03),Bcl-2:(0.23±0.11),(0.24±0.12),(0.27±0.14),Bax:(0.39±0.13),(0.40±0.14),(0.45±0.23),caspase-3:(0.52±0.26),(0.56±0.27),(0.58±0.28)),while Bax and caspase-3 protein levels(Bax:(0.25±0.13),(0.19±0.06),(0.21±0.08),caspase-3:(0.35±0.13),(0.34±0.16),(0.29±0.09)) were decreased (P<0.05).The pERK1/2 protein level of ERK1/2 inhibitor group was lower than other groups,There was no difference in the level of Bcl-2,Bax and caspase-3 between the ERK1/2 group and the model group.(P>0.05).(3) At each time point,microglia (Iba1 positive) in ischemic penumbra of the BMMNCs group was significantly more than those of the model group,and it was increased with the time extension (P<0.05).There was no difference in comparison between the ERK1/2 inhibitor group and the model group (P>0.05).ConclusionBMMNCs can reduce the apoptosis through ERK1/2 signaling pathway,thus improving the neurological function and reducing the infarct scope.

Objective To investigate the neuroprotective effect of thyroid hormones T3 on cerebral ischemia-reperfusion injury in rats and its mechanism.Methods SD rats were divided into four groups:sham+saline group,sham+T3 group,MCAO+saline group,MCAO+T3 group.The cerebral ischemia-reperfusion injury rat models were established by right middle cerebral artery occlusion.Thyroid hormones(10 μg/100 g)or normal saline were given respectively by intraperitoneal injection twice at 1 h after the onset of ischemia and 6 h after reperfusion.Neurobehavioral score was evaluated at 24 h after reperfusion;TTC staining was used to label infarction area;RT-PCR was used to detect the mRNA level of nerve growth factor(NGF)and brain derived neurotrophic factor(BDNF)in brain tissue;Western blot was employed to determine alterations in protein levels of NGF and BDNF.Results Compared with MCAO+saline group,the neurological deficit and the volume of cerebral infarction of MCAO+T3 group was decreased,and the mRNA and protein expression of NGF and BDNF of MCAO+T3 group were increased(P<0.05).Conclusion Thyroid Hormones could promote the nerve repair,stimulate the nerve regeneration and improve the nervous behavioral function by up-regulating the expression of NGF and BDNF.

Objective To investigate the clinical,imaging,pathological and molecular biological features of mitochondrial encephalomyopathy with lactic acidosis and stroke -like episodes(MELAS)in children.Methods The clinical,imaging,pathological and molecular biological features of 1 2 children with MELAS diagnosed through muscle biopsy or gene sequencing in the Fifth Affiliated Hospital of Zhengzhou University from January 201 1 to December 201 5 were retrospectively analyzed.Results (1 )Clinical features:the main manifestations included headache and vomiting in 1 1 cases,epileptic seizures in 9 cases,short stature in 8 cases,hairy in 7 cases,intolerance fatigue in 7 cases,cogni-tive decline in 7 cases,visual disturbance in 6 cases,hearing disturbance in 6 cases,and 5 cases had positive family history.In addition,7 cases had the serum lactic acid level increase in a rest for 1 0 min after exercise.(2)Imaging fea-tures:4 cases showed bilateral basal ganglia calcification symmetry in 8 patients who underwent head CT scan.The most frequently involved parts of the lesion were occipital in 1 0 cases,temporal in 9 cases and parietal lobe in 7 cases in stroke -like episodes.The lesions were lamellar necrosis.The abnormal areas by MRI showed low signal intensity on T1 weighted imaging,high signal intensity on T2 weighted imaging and fluid attenuated inversion recovery,high or equal signal intensity on diffusion weighted imaging,high or low signal intensity on apparent diffusion coefficient;the lactate peak significantly increased on magnetic resonance spectroscopy.The distribution was not in accordance with the control region of the cerebral vessels.Dynamic observation revealed that the lesions were reversible and migratory.(3)Myo-pathological features:muscle biopsy was performed in all children,and ragged -red fibers were found in 1 0 cases by im-proved Gomori staining,strongly succinate dehydrogenase -reactive were found in 9 cases,and the lipid droplets slight-ly increased in 8 cases by oil red O staining.Besides,the crystalline inclusion bodies in mitochondria were arranged in a parking lotpattern in 9 cases by electromicroscope.(4)Molecular biological characteristics:the mitochondrial gene mutations were analyzed in peripheral blood of 9 children and their parents,including 8 cases with A3243G muta-tion and 1 case with G13513A mutation.Five mothers had the same A3243G mutation site in 8 cases.Conclusions Children with MELAS have complex and varied clinical manifestations and certain characteristic of neuroimaging.More-over,muscle pathology and gene sequencing have important diagnostic value.Fully understanding the clinical,muscle pathology,imaging and molecular biological characteristics of children with MELAS can be helpful to the early diagnosis and treatment,also reduce misdiagnosis.

Objective To fabricate an anti?tuberculosis controlled drug release coating with Ti?PDA?PEG?PLGA?INH and to investigate its surface characteristics, in vivo and in vitro drug release behavior, and tissue biocompatibility. Methods 4?arm?polyethylene glycol (PEG) was synthesized first. Then cover the surface of titanium (Ti) with a layer of poly dopamine (PDA) by Michael addition reaction. Use porous starch and 4?arm?PEG as a carrier, load with isoniazid (INH), then attach to the surface of titanium by casting or sol?gel dip coating methods, and then cover with a layer of poly lactic?co?glycolic acid (PLGA) by the same method, to fabricate the Ti?PDA?PEG?PLGA?INH composite coating finally. The functional group of 4?arm?PEG was charac?terized by proton nuclear resonance spectroscopy (HNMR). The surface characteristics of Ti?PDA?PEG?PLGA?INH were evaluated by scanning electron microscope (SEM), while drug release behaviors were detected by high performance liquid chromatography (HPLC) and the cumulative release rate was calculated, and carry out the antibacterial performance in vitro. The animal model of femoral condyle bone defect was established in 25 New Zealand white rabbits. Titanium rods covered with PDA?PEG?PLGA?INH coating were implanted into defect area. INH concentrations were detected by HPLC in venous blood, muscle and bone tissue at each time point postoperatively. Another 12 rabbits were randomly divided into experimental group and control group, the experi?mental group was implanted with titanium tablets and titanium rods coated with PDA?PEG?PLGA?INH in the paraspinous muscle and left femoral condyles respectively, while the control group was implanted with a blank sheet of titanium tablets and titanium rods in the same place. Hematoxylin and Eosin Staining were used to observe the biocompatibility of the composite system in vivo at 28 and 56 days postoperatively. Results Ti?PDA?PEG?PLGA?INH controlled drug release coating uniformly distributed on the surface of plates and rods, with translucent form and smooth surface. In vitro INH release kinetics exhibited a short?burst release during the first 8h, and the cumulative release of the INH was about 65%. On the 9th day, the cumulative release of the INH was about 90%, and then the release tended to be flat, and the drug release behavior in vitro continued more than 20d. In vivo release test showed that the concentration of INH in vein blood, muscle and bone tissue around the composite system was increased steadi?ly postoperatively. On about the 28th day, the concentration reached the max. However, the INH concentrations in muscle and bone tissue around the composite system were still higher than the minimum inhibitory concentration (MIC) on the 56th day. The antibacterial test in vitro showed that the titanium tablets coated with PDA?PEG?PLGA?INH formed obvious bacterial inhibition zones. The pathological results indicated that mild inflammatory reaction was seen in the 4th week postoperatively, and the reac?tive capsule formed with loose connective tissue. In the 8th week postoperatively, there's no obvious inflammation occurred, and the reactive capsule became more dense and thicker. Conclusion The study successfully fabricated the Ti?PDA?PEG?PLGA?INH anti?tuberculosis controlled drug release coating, with reasonable release behavior both in vivo and in vitro, effective antibac?terial effect of Mycobacterium tuberculosis in vitro and good tissue biocompatibility, which is a potentially effective drug delivery system for spinal tuberculosis.

Objective To detect the effects of 17 β-estradiol(E2)on the expression of Calbindin-D9k (CaBP-9k) in pituitary GH3 cells,and to investigate the antagonistic effect of a selective estrogen receptor antagonist,ⅡCI 182780 on CaBP-9k expression.Methods A rat pituitary prolactinoma cell line,GH3 cell was used as the in vitro model.The localization of CaBP-9k in GH3 cells was observed by immunofluorescence.GH3 cells were cultured with exogenous E2-added medium for 24 hours,and the concentrations of E2 were 10-8,10-9,10-10M,respectively.mRNA and protein expression levels of CaBP-9k in different groups were analyzed by RT-PCR and Western blot analysis.The estrogen receptor antagonist,and ⅡCI 182780 was added to GH3 cells before E2 administration (10-8M)with the concentration of 10-6M,in order to investigate the regulation of ER-mediated pathway on the expression of CaBP-9k.Immunoprecipitation was used to detect the interaction between CaBP-9k and ERα.Results E2 had significant stimulatory effect on the CaBP-9k expression of GH3 cells in a dose dependent manner,and the expression level of CaBP-9k was higher when treated with a higher concentration of E2.ⅡCI 182780 could suppress the stimulatory effect of E2 on the CaBP-9k expression of GH3 cells.The expression level of CaBP-9k was significantly reduced by coadministration of E2 with ⅡCI 182780 in GH3 cells,which meant the CaBP-9k expression was mediated through ERα pathway.The immunoprecipitation results further illustrated the fact that CaBP-9k could directly interact with ERα,and E2 could increase the interaction between CaBP-9k and ERα.Conclusion Estrogen might induce CaBP-9k expression via ERα mediated pathway and CaBP-9k could directly combine with ERα,suggesting that CaBP-9k might be involved in the biological effects mediated by ER pathway in GH3 cells.