Objective:To investigate the methylation status of N-myc downstream regulated gene-1(NDRG-1) gene in breast cancer and the effects of methylation enzyme inhibitor 5-Aza-2'-deoxycytidine (5-Aza-CdR) on growth and expression of NDRG-1 mRNA in human breast cancer cell line T47D.Methods:Sensitive methylation-specific (MSP)-PCR was used to detect the methylation status in the promoter regions of NDRG-1 gene in 47 samples of breast cancer and tumor adjacent tissues and 15 cases of benign breast disease. The change in expression of the tumor suppressor gene NDRG-1 mRNA in cultured T47D cells was detected by RT-PCR before and after 5-Aza-CdR treatment. Cell proliferation was observed by MTT assay.Results:Hypermethylation frequencies of NDRG-1 gene promoter were 46.8% in breast cancer tissues and 21.3% in tumor adjacent tissues. No hypermethylation of NDRG-1 gene was observed in the tissues of breast benign disease. The growth of T47D cells was suppressed obviously after 5-Aza-CdR treatment compared with the control group. RT-PCR showed that compared with the control group, NDRG-1 mRNA expression was increased at different concentrations in 5-Aza-CdR treatment group. Conclusion:The promoter methylation status of NDRG-1 gene was significantly related with the occurrence of breast carcinoma. 5-Aza-CdR could effectively reverse the methylation of NDRG-1 gene and recover its expression, thereby inhibiting the growth of tumor cells.

Background and purpose:CpG methylation in promoter region is an essential mechanism for the dysfunction of the tumor suppression gene.Folate metabolism provides active methyl for organism methylation.Methionine synthase(MS) plays a vital role in the process of folate metabolism.This study aimed to explore the genetic polymorphism of MS, CHD5 methylation and their association with breast cancer morbidity.Methods:Fortyseven patients with primary breast cancer, 52 healthy subjects and 15 breast hyperplasia patients were enrolled in this experiment.The mRNA expression of CHD5 was determined by reverse transcription PCR(RT-PCR).Methylationspecific PCR(MSP) was used to detect the methylation status of CHD5.Polymorphisms in the MS gene were analyzed through the polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP).Results:The mRNA expression of CHD5 in breast cancer tissues(0.27?0.19) and adjacent non-cancerous tissues(0.33?0.17) were both significantly lower than that in the breast hyperplasia tissues(0.67?0.14)(P

Objective:To analyse the evaluation and experience of clinical effect of minimally invasive drainage and craniotomy in the treatment of patient with epidural hematoa.Methods:100 cases of patients who were diagnosed as epidural hematoma from January 2015 to January 2016 were selected and randomly divided into two groups,where the control group were given craniotomy,and the observation group were given minimal invasive drainage.The effect of the surgery and the life qualities before and after treatment of the two groups were compared.Results:The effect of observation group is better than that of control group (P ＜ 0.05);craniocerebral defect and the re-hemorrhage incidence occurrence in the observation group were obviously lower than those in the control group,(0 ％,6 ％ vs 12 ％,20 ％) (P＜0.05).Conclusion:Compared with craniotomy,minimal invasive drainage in clinical treatment of epidural hematoma showed advantage in at smaller trauma,faster recovery,and better effect,thus deserving further promotion of clinic application.

Aim Toinvestigatetheinducementeffect of isatin on apoptosis of breast cancer cell line MCF-7 , andexploreitsdetailedmechanism.Methods MCF-7 cell lines were exposed to isatin at different concentra-tions(0,50,100,200 μmol·L-1 )for 48 h.Apop-totic features were demonstrated by nuclei staining with Hoechst 33258.Bcl-2,Bax and p53 mRNA were ana-lyzed by RT-PCR.Caspase-9 activation and mitochon-drial depolarization were assayed by flow cytometry. Bcl-2,Bax,p53 and cytochrome c proteins were ana-lyzedbyWesternblot.Results Isatininducesapopto-sis of MCF-7 cells.Furthermore,Bcl-2 expression was decreased and the ratio of Bcl-2 to Bax was significant-ly decreased by isatin.While,p53 expression relative-ly decreased.The mitochondrial transmembrane poten-tial was markedly reduced and the release of cyto-chrome c into the cytosol was increased after treatment with isatin.Simultaneously,caspase-9 was activated. Conclusions Isatinsignificantlyinducedtheapopto-sis of MCF-7 cells in vitro.These results strongly sug-gest that the p53 dependent mitochondrial pathway is involved in apoptosis.

Objective:To investigate the mRNA expression of the WIF-1 gene and the methylation of its promoter in breast can-cer, and to determine the correlation between the epigenetic aberrant WIF-1 DNA methylation and the clinicopathological significance of WIF-1 in breast cancer . Methods:RT-PCR and sensitive methylation-specific-PCR (MSP) were used to detect WIF-1 mRNA ex-pression and the methylation of the WIF-1 promoter in 30 breast cancer samples as well as in tumor-adjacent tissue samples and 9 be-nign breast tissues. Results:The WIF-1 mRNA expression in 30 breast cancer samples significantly decreased compared with those of the other two groups. In addition, WIF-1 methylation was more frequent in breast-tumor tissues compared with those in tumor-free tis-sues. Meanwhile, WIF-1 mRNA expression in breast cancer tissues involved the abnormal methylation of its promoter. Clinicopatholog-ical correlation analysis showed that the abnormal methylation of the WIF-1 gene promoter was not associated with age, TNM stage, histotype, or lymph node metastasis. Conclusion:WIF-1 mRNA expression loss due to abnormal methylation may be a crucial factor in breast cancer development and can thus be used in the prognosis and progression of the disease.

Objective To investigate the effects of simulated microgravity by RCCS on proliferation and cell cytoskeleton of human HaCaT keratinocyte. Methods The rotary cell culture system (RCCS) was used to simulate the microgravity environment, and human HaCaT keratinocytes were divided randomly(random number) into the simulated microgravity group (SMG) and normal gravity group (NG). HaCaT cells in the two groups were harvested respectively after 32, 36 and 42 h culture. The HaCaT cells proliferation and cycles were detected by flow cytometry, the concentration of hb-EGF in supernatant was detected by ELISA, and the cell cytoskeleton was observed after 42 hours' culture under laser confocal microscope with FITC-labeled technique. SPSS 23.0 statistical software was used for statistical analysis, and P <0.05 was considered statistically significant. Results The flow cytometry showed that the proportions of human HaCaT keratinocytes in G1 and G2/M phases were increased while the proportion of HaCaT cells in S stage was decreased significantly after 32, 36 and 42 h RCCSculture compared with those in the normal gravity group. The HaCaT cells in G1 stage were declined along with incubation time. ELISA results showed that the hb-EGF concentration in HaCaT supernatant under simulated microgravity culture for 24 and 36 h was lower than that in the normal control group (P<0.01). The laser confocal microscope revealed that the HaCaT fluorescence intensity was decreased,and there were disordered microfilaments, structural ambiguity, pseudopodia reduction and irregularshape among FITC-labeled HaCaT cells cultured 42 h in RSSC compared with the normal gravity group.Conclusions RCCS simulated microgravity environment could inhibit the cell cycle transformation and proliferation of human HaCaT keratinocyte, affect the keratinocyte-secreting function, and induce alterations of the cell cytoskeleton.

The rotary cell culture system(RCCS)was used to simulate the microgravity environment, and FRTL-5 cells were divided into simulated microgravity group(SMG)and normal gravity group(NG). FRTL-5 cells were harvested after treatment for 6, 12, 24, and 36 h, the cell viability was measured by MTT assay, and the cells cycles were detected by flow cytometry. The ultrastructure of FRTL-5 cells was observed under laser confocal microscope with FITC-labeled technique. The MTT assay showed that the proliferation of FRTL-5 cells was significantly inhibited after RCCS treatment for 6, 12, 24, and 36h compared with NG(P<0.05), in which the most obvious effect was observed at 24h. The flow cytometry showed that the proportion of FRTL-5 cells at G1 stage in RCCS group was increased significantly after 6, 12, 24, and 36h compared with NG(P<0.05), while the proportion of FRTL-5 cells at S stage was decreased significantly(P<0.05)except that cultured with RCCS for 6 h. The proportion of FRTL-5 cells at G2/M stage was decreased in early phase(6-12 hours)of RCCS culture, with the lowest at 12h and transient increase at 24h of RCCS culture. The laser confocal microscope revealed that there were local microfilament depolymerization, tension fibers decrease, structure disorder, cellular pseudopodia reduction, and irregular shape among FITC-labeled FRTL-5 cells cultured with RCCS for 36h. (Chin J Endocrinol Metab, 2018, 34: 598-601)

Objective:To analyze food intolerance status in children in Qingdao by detecting the serum levels of food-specific IgG (sIgG).Methods:In this cross-sectional study, a total of 4 249 children aged 0 to 14 years (all were permanent residents of Qingdao City) admitted to Women and Children′s Hospital Affiliated to Qingdao University from May 2017 to December 2020 for suspected food intolerance were selected as the study objects with the whole sampling method. According to the age, the objects were divided into 4 groups: 0-<1 year group (440 cases), 1-<3 years group (1 761 cases), 3-<6 years group (1 193 cases), and ≥6 years group (855 cases). Positive condition of serum sIgG antibodies of 14 kinds of food in the children were detected by enzyme-linked immunosorbent assay. Chi-square test was used to compare the positive rate of the antibodies among different foods, gender and age groups.Results:The total positive rate of food sIgG antibody in 4 249 children was 95.32% (4 050/4 249), the highest positive rate was found in eggs (81.50%) and the lowest positive rate was found in pork (1.15%). The positive rates of sIgG antibody in milk (54.98% vs 49.69%, χ2=11.627), crab (5.59% vs 3.71%, χ2=8.049) and shrimp (4.62% vs 2.75%, χ2=9.784) in boys were significantly higher than those in girls, and the positive rates of sIgG antibody in tomato (49.19% vs 45.54%, χ2=5.510), cod (8.53% vs 5.96%, χ2=10.512) and beef (2.58% vs 1.70%, χ2=3.959) in girls were significantly higher than those in boys (all P<0.05). The total positive rate of sIgG antibody in 14 foods was the lowest in 0-<1 year group (89.09%), and it was the highest in 3-<6 years group (96.98%) ( χ2=63.950, P<0.001). The highest positive rate in 0-<1 year group was found in tomato (56.36%), and it was eggs (85.29%, 88.94%, 85.50%) in all the other 3 groups. The positive rates of corn and beef decreased with age ( χ2=44.098, 20.106, P<0.001), while those of cod and mushroom increased with age ( χ2=32.315, 40.338, P<0.001). The positive rate of wheat (57.13%, χ2=42.273), tomato (57.01%, χ2=209.862), soybean (24.99%, χ2=92.580), crab (6.81%, χ2=33.201), shrimp (6.25%, χ2=47.863) were all the highest in 1-<3 years group among the 4 groups (all P<0.001), and the positive rate of chicken was the highest in 3-<6 years group (7.88%, χ2=29.875; P<0.001). Conclusions:Children in Qingdao have a high level of food intolerance, and the highest positive rate is for eggs. Milk, crab and shrimp should be focused on for boys, while tomatoes, cod and beef shoud be paid more attention to for girls. Children of different ages have different kinds of food intolerance, and their diets should be adjusted reasonably according to the characteristics.

The 4-hydroxyphenylacetate 3-hydroxylase (4HPA3H), originated from Escherichia coli, converts p-coumaric acid to caffeic acid. In order to improve the efficiency of caffeic acid biosynthesis, we engineered E. coli for overexpression of 4HPA3H. The high-density fermentation of the engineered E. coli was conducted in a 5 L bioreactor. Subsequently, the conditions for whole-cell biocatalysis were optimized. The dry cell weight of the 4HPA3H-expressed strain reached 34.80 g/L. After incubated in the bioreactor for 6 h, 18.74 g/L (0.85 g/(L·OD₆₀₀)) of caffeic acid was obtained, with a conversion rate of 78.81% achieved. To the best of our knowledge, the titer of caffeic acid is the highest reported to date. The high-density fermentation of E. coli for overexpression of 4HPA3H and the efficient biosynthesis of caffeic acid may facilitate future large-scale production of caffeic acid.
Caffeic Acids ; Escherichia coli/metabolism* ; Fermentation ; Metabolic Engineering ; Mixed Function Oxygenases/metabolism* ; Phenylacetates

OBJECTIVE To investigate the mechanism of Mayuan tongbian zhitong decoction on improving slow-transmission constipation（STC）in rats by regulating AMP activated protein kinase （AMPK）/endothelial nitric oxide synthase （eNOS）signaling pathway. METHODS The rats were randomly divided into blank group ，model group ，Mayuan tongbian zhitong decoction low-dose，medium-dose and high-dose groups （6，12，18 mg/kg），with 10 rats in each group. Except for blank group ，other groups were given Compound diphenoxylate suspension to induce STC model. After modeling ，blank group and model group were given normal saline intragastrically ，and Mayuan tongbian zhitong decoction groups were given relevant medicine intragastrically ， once a day ，for consecutive 2 weeks. The number of feces and water content of feces in each group were observed before and after treatment；the carbon powder propulsion rate of rats in each group was calculated ；the pathological structure of colon in each group was observed ；the levels of nitric oxide （NO）and nitric oxide synthase （NOS）in colon tissues of rats in each group were detected；the expressions of AMPK ，eNOS，mammalian target of rapamycin （mTOR），tuberous sclerosis complex 1（Tsc-1）， Tsc-2 and eukaryotic promoter 4E binding protein （4ebp）were also detected. The active ingredients of Cannabis sativa ，Citrus aurantium and Rehmannia glutinosa were screened from Mayuan tongbian zhitong decoction. The active ingredients with high Degree value were docked with AMPK and eNOS ，to verify the interaction. RESULTS Compared with before treatment ，the number and water content of feces were increased significantly in Mayuan tongbian zhitong decoction groups （P＜0.05）. Compared with blank group ，carbon powder propulsion rate of model group was decreased significantly （P＜0.05）； colonic structure was disordered ，and a large number of inflammatory cells were seen in submucosa ；the levels of NO and NOS in colon tissue as well as the protein expressions of AMPK ，eNOS，mTOR，Tsc-1，Tsc-2 and 4ebp were increased significantly （P＜0.05）. Compared with model group ，above indexes of Mayuan tongbian zhitong decoction groups （except for NOS in low-dose group ）were reserved significantly （P＜0.05）. In the molecular docking experiment ，the active components with the highest Degree values in C. sativa ，C. aurantium and R. glutinosa were（Z）-3-（4-hydroxy-3-methoxy-phenyl）-N-[2-（4-hydroxyphenyl）ethyl] acrylamide，nobiletin and stigmasterol. The binding energies of AMPK with these three components were －5.15，－4.61 and －4.83 kJ/mol，the binding energies of eNOS with these three components were －6.11，－5.40 and －5.91 kJ/mol. The conformations of these three compounds with AMPK and eNOS were stable and their binding activities were high. CONCLUSIONS Mayuan tongbian zhitong decoction can improve the constipation symptoms and intestinal function in STC model rats ，and its specific mechanism may be related to the inhibition of AMPK/eNOS signaling pathway.