Objective:To investigate the effect and mechanism of esophageal squamous cell carcinoma exosomal miR-181b-5p to the polarization of M2 macrophages.Methods:Extracted and identified exosomes from esophageal squamous cell carcinoma,and detected the expression of miR-181b-5p in esophageal squamous cell carcinoma cells and their exosomes by qRT-PCR.M0 type macro-phages were divided into PBS group,HEEC exo group,Eca-109 exo group,miR-NC exo group,miR-181b-5p exo group,miR-NC group,miR-181b-5p mimic group,si-NC group,si-PTEN group,miR-181b-5p exo+PTEN group.qRT-PCR was used to detect the expressions of CD163,CD206,iNOS and TNF-α in each group.The targeting relationship between miR-181b-5p and PTEN were veri-fied by double luciferase reporter gene experiment.Results:miR-181b-5p was significantly overexpressed in esophageal squamous cell carcinoma cells TE-13,TE-12,TE-10,Eca-109,KYSE30 and their exosomes(P<0.001).Compared with miR-NC group,the expression of CD163 and CD206 in cells were significantly upregulated in the miR-181b-5p mimic group,as well as the expressions of iNOS and TNF-α were significantly downregulated(P<0.001).The results of double luciferase reporter genes showed that PTEN was the target gene of miR-181b-5p.Compared with si-NC group,the expressions of CD163 and CD206 in cells were significantly upregu-lated in the si-PTEN group,as well as the expressions of iNOS and TNF-α were significantly downregulated(P<0.001).Compared with PBS group,the expressions of CD163 and CD206 in cells were significantly upregulated in the Eca-109 exo group,as well as the expressions of iNOS and TNF-α were significantly downregulated(P<0.001).Compared with miR-NC exo group,the expressions of CD163 and CD206 in cells were significantly upregulated in the miR-181b-5p exo group,as well as the expressions of iNOS and TNF-α were significantly downregulated(P<0.001).Compared with miR-181b-5p exo group,the expressions of CD163 and CD206 in cells were significantly downregulated in the miR-181b-5p exo+PTEN group,as well as the expressions of iNOS and TNF-α were significantly upregulated(P<0.001).Conclusion:Exosomal miR-181b-5p inhibits PTEN expressions to promote M2 macrophage polarization.

L-theanine has been shown to have a therapeutic effect on depression.However,whether L-theanine has an excellent preventive effect on depression in children and adolescents and what its mechanism is have not been well explained.Given the complexity of the pathogenesis of depression,this study investigated the preventive effect and mechanism of L-theanine on depression in juvenile rats by combining serum and hippocampal metabolomic strategies.Behavioral tests,hippocampal tissue sections,and serum and hippocampal biochemical indexes were studied,and the results confirmed the preventive effect of L-theanine.Untargeted reversed-phase liquid chromatography-quadrupole-time-of-flight mass spec-trometry and targeted hydrophilic interaction liquid chromatography-triple quadrupole mass spec-trometry were developed to analyze the metabolism changes in the serum and hippocampus to screen for potential biomarkers related to L-theanine treatment.The results suggested that 28 abnormal me-tabolites in the serum and hippocampus that were considered as potential biomarkers returned to near-normal levels after L-theanine administration.These biomarkers were involved in various metabolic pathways,mainly including amino acid metabolism and lipid metabolism.The levels of amino acids and neurotransmitters in the phenylalanine,tryptophan,and glutamic acid pathways were significantly reduced after L-theanine administration compared with chronic unpredictable mild stress-induced rats.In summary,L-theanine had a significant preventive effect on depression and achieved its preventive results on depression by regulating various aspects of the body,such as amino acids,lipids,and inflammation.This research systematically analyzed the mechanism of L-theanine in preventing depression and laid the foundation for applying L-theanine to prevent depression in children and adolescents.

Objective:To analyze the mechanism of Jindan Tablets, Xiaoyan Lidan Tablets and ursodeoxycholic acid in the treatment of gallstone and cholecystitis based on network pharmacology; To conduct a comparative analysis.Methods:The chemical components of Jindan Tablets, Xiaoyan Lidan Tablets and ursodeoxycholic acid and their drug targets were collected from Traditional Chinese Medicine Database and Analysis Platform (TCMSP). DAVID 6.8 database was used to search for the associated diseases of the drug targets. The disease targets of gallstone and cholecystitis were collected from GeneCards and other databases. The protein-protein interactions network was established based on the intersecting targets of three drugs and two diseases. KEGG enrichment analysis was performed based on the DAVID 6.8 database. Cytoscape 3.7.1 software was used to construct a complex network and topology analysis of component- target- disease between three drugs and diseases.Results:222 chemical components and 3 133 drug targets were collected for Jindan Tablets. 104 chemical components and 1 425 action targets were collected for Xiaoyan Lidan Tablets. 1 chemical component and 119 action targets were collected for ursodeoxycholic acid. The three drugs were associated with 31 diseases. 1 382 disease targets for gallstones and cholecystitis were collected. There were 237, 163 and 33 targets for gallstones and cholecystitis in the three drugs, of which 17 were shared by the three drugs and 20 were shared by Jindan Tablets and Xiaoyan Lidan Tablets. Based on the DAVID database, 113, 74 and 10 significant KEGG enrichment pathways were obtained for the three drugs respectively.Conclusions:The three drugs shared many targets and pathways in the treatment of gallstones and cholecystitis, which all had the function of regulating metabolism and inhibiting inflammatory response, while participating in apoptosis, oxidative stress and cancer pathology process. However, they had their own special effects, with Jindan Tablets favoring involving in the cancer process and inhibition of inflammation, and promoting angiogenesis. Xiaoyan Lidan Tablets and ursodeoxycholic acid focused on regulating cholesterol metabolism, and Xiaoyan Lidan Tablets also regulated steroid metabolism and inhibit inflammation, while ursodeoxycholic acid regulated bile acid metabolism.

Objective:To investigate the effect of activation of microglia in prefrontal cortex on long-term spatial memory in post-stroke depression mice.Methods:Forty-eight male C57BL/6 mice were divided into sham operation group, stroke group, post-stroke depression group and depression group according to the random number table method with 12 in each group, and 36 mice were divided into solvent group, enrofloxacin group and minocycline group according to the random number table method with 12 in each group.Middle cerebral artery occlusion (MCAO) was use to establish the stroke model, and forced swimming was used to establish the depression model.The post-stroke depression model mice were received MCAO first and then received forced swimming on the 4th day after stroke to establish the model.Mice in enrofloxacin group and minocycline group were treated with enrofloxacin and minocycline injection once a clay for 14 days from the 5th day after stroke, respectively.Forced swimming test and sugar water preference test were used to evaluate the depression of mice in each group, Morris water maze test was used to detect the spatial memory function of mice in each group, and Nissl staining and immunofluorescence staining were used to detect the neuronal function and the number and type of microglia activation.The expression of inflammatory cytokines IL-6 and IL-1β were detected by Western blot.GraphPad Prism 8.0.1 statistical software was used for statistical analysis.The single factor variance analysis was used to compare the difference among multiple groups, and pairwise comparison was performed with SNK- q test. Results:(1) There were statistically significant differences in depression, learning and memory, neuron damage, activation of microglia, inflammatory factors and other indicators in sham operation group, stroke group, post-stroke depression group and depression group ( F=43.58-255.70, all P<0.05). Compared with stroke group, post-stroke depression group had longer floating immobility time ((222.70±29.12) s, (79.25±46.78) s, P<0.05), the preference rate of sugar water was significantly lower ( (49.44±6.19) %, (84.49±4.73) %, P<0.05), and the average value of platform approach after correction was higher((125.00±9.95) mm, (96.79±12.57) mm, P<0.05), Nissl bodies expression was lower ((53.50±15.78) cells /mm 2, (85.67±17.52) cells /mm 2, P<0.05), NeuN positive expression rate was lower ((29.78±3.70) %, (45.73±4.51) %, P<0.05), the percent of M1 microglia expression was significantly higher ((75.55±8.84) %, (58.19±5.69) %, P<0.05), the percent of M2 microglia expression was lower ((43.46±5.11)%, (57.14±5.40)%, P<0.05), and the expression levels of IL-6 ((1.14±0.03), (0.94±0.05), P<0.05) and IL-1β((1.17±0.03), (0.56±0.04), P<0.05) were significantly higher.(2) Depression, learning and memory, neuron injury, activation of microglia, inflammatory factors and other indicators of mice in solvent group, enrofloxacin group and minocycline group were significantly different ( F=7.13-94.35, all P<0.05). Compared with enrofloxacin group, mice in minocycline group had shorter floating immobility time ((169.30±13.04) s, (224.30±22.60) s, P<0.05) and higher sugar water preference rate ((62.81±7.75) %, (47.71±8.11) %, P<0.05), the mean value of platform approach estimation after water maze correction was lower ((97.66±14.56) mm, (120.20±12.08) mm, P<0.05), and the expression level of Nissl bodies was higher ((80.17±10.55) cells /mm 2, (52.00±8.94) cells /mm 2, P<0.05), NeuN expression rate was high ((45.04±3.62) %, (28.88±4.50) %, P<0.05), Iba-1 expression was lower ((97.33±10.67) cells/mm 2, (112.50±6.54)cells/mm 2, P<0.05), the percent of M1 microglia expression was lower ((54.43±5.22) %, (73.82±6.88) %, P<0.05), and the percent of M2 microglia expression was significantly higher ((51.86±6.22) %, (36.30±5.72) %, P<0.05). The expression levels of IL-6 ((0.75±0.06), (1.21±0.07), P<0.05) and IL-1β ((0.61±0.06) (1.09±0.09), P<0.05) were lower. Conclusion:The long-term spatial memory impairment of post-stroke depression mice is aggravated, which is related to the neuron damage caused by increased activation of M1 microglia in PFC area.Inhibition of M1 microglia by minocycline can effectively improve the spatial memory ability of mice.

【Objective】 To study the yielding rate and distribution of unexpected antibodies in blood transfusion children with thalassemia in Yunnan province, and to explore the blood transfusion strategies. 【Methods】 From January 2016 to December 2021, 298 children with thalassemia, who received blood transfusion treatment in Kunming, Xishuangbanna, Wenshan, Dehong, Yuxi and Baoshan hospitals across Yunnan Province, were selected. The unexpected antibodies of blood plasma were screened by microcolumn gel card. The samples with positive antibodies were identified for alloantibody specificity. 【Results】 Unexpected antibodies were yielded in 67 out of 298(22.48%) transfused children with thalassemia. The positive rates of unexpected antibodies in boys and girls were 16.55%(24/145) and 28.10%(43/153), respectively. The positive rates of unexpected antibodies in Han, Dai, Zhuang, Yi, Bulang, Jinuo and Miao people were 14.06%(18/128), 30.80%(32/104), 35.71%(10/28), 36.36%(8/22), 50.00%(4/8), 60.00%(3/5)and 66.67%(2/3), respectively, with statistically significant differences between each other. The positive rate of unexpected antibodies in ethnic minorities was higher than that in Han. The positive rates of unexpected antibodies in children who received the first transfusion at birth-one year old, 1~3 years old, 3~6 years old and above 6 years old were 12.50%(3/24), 10.14%(7/69), 24.54%(40/163)and 40.48%(17/42), respectively. The positive rates of unexpected antibodies in children with first transfusion after 3 years old were significantly higher than those before 3 years old. The positive rates of unexpected antibodies in children with one transfusion, 1~3, 3~10, 10~20 and more than 20 transfusions were 4.76%(1/21), 12.07%(7/58), 23.71%(23/97), 28.16%(29/103)and 36.84%(7/19), respectively, with statistically significant differences between each other. The number of blood transfusions was positively correlated with the unexpected antibody yielding. The yielding rate of unexpected antibodies in children with α thalassemia, βthalassemia, δ+ βthalassemia and untyped thalassemia was 7.50%(3/40), 17.62%(34/193), 53.70%(29/54)and 9.09%(1/11), respectively(P<0.05). The yielding rate of unexpected antibodies in transfused children with δ+ βthalassemia was the highest. And 57 unexpected antibodies of Rh blood group system were yielded, 6 anti-M antibodies, 2 anti-N antibodies and 2 undetermined. 【Conclusion】 The positive rate of unexpected antibodies in transfused children with thalassemia in Yunnan province is high. Routine antibody screening should be carried out for transfusion children with thalassemia, and blood units, compatible with ABO, Rh and MNS typing results, should be selected to ensure the safety and effectiveness of clinical blood use.

Objective: To analyze the epidemiological characteristics of human brucellosis and trace back source of infection of human brucellosis in Hunan province during 2010-2018, and provide evidence for the prevention and control of human brucellosis. Methods: The surveillance data of human brucellosis in Hunan during 2010-2018 were analyzed with software Excel 2016 and ArcGIS 10.5, the epidemic characteristics were described using cases number, constituent ratio and rate. The conventional biotype methods were used for the identification of Brucella species, UTS-PCR was applied to further confirm the results from conventional biotype detections, then six virulence genes of two clinical Brucella strains were detected by PCR assay. Cluster analysis of two Brucella strains were performed with Multiple locus variable-number tandem repeat analysis (MLVA) for the investigation of the infection source of human brucellosis. Results: From 2010 to 2018, a total of 728 human brucellosis cases were reported in Hunan with the annual incidence rate of 0.12/100 000. The incidence rate was 2.50/100 000 in Chenzhou and 1.90/100 000 in Yongzhou, higher than those in other areas. The number of counties reporting cases increased from 5 in 2010 to 69 in 2018. Most cases were reported in age group 45-54 years, accounting for 38.32% (279/728). The cases in farmers accounted for 59.07% (430/728) of the total. The male to female ratio of the cases was 2.75 ∶ 1. The reported case number was highest during May-July, accounting for 45.33% (330/728). The incidence was high in summer and autumn, and the peak was in May. The conventional identification showed that two strains were all Brucella melitensis biovar 1, consistent with UTS-PCR amplification results. Six virulence genes were found in two isolated strains, suggesting that the Brucella melitensis strains in this study had strong virulence. MLVA results confirmed that two strains detected in Hunan had complete identical MLVA-16 genotype with strains isolated from goat and camel in Inner Mongolia Autonomous Region, indicating that there was molecular epidemiology relationship between these strains and the source of infection were originated from Inner Mongolia. Conclusions The epidemic of human brucellosis in Hunan is becoming serious, and disease has spread to general population and non-epidemic areas. Two Brucella melitensis strains detected in Hunan were originated from Inner Mongolia. The quarantine and inspection in animal transportation should be strengthened to prevent human outbreaks of brucellosis.

The discovery of hydroxylases in the anticancer drug taxol biosynthesis pathway is a hotspot and difficulty in current research. In this study, a new hydroxylase gene TcCYP725A22 (GenBank accession number: MF448646.1) was used to construct a sub-cellular localization vector pCAMIBA1303-TcCYP725A22-EGFP to get the transient expression in onion epidermal cells. Laser confocal microscopy revealed that the protein encoded by this gene was localized in the cell membrane. Furthermore, the recombinant plant expression plasmid pBI121-TcCYP725A22 was constructed. After transient transformation to the Taxus chinensis mediated by Agrobacterium tumefaciens LBA4404, qRT-PCR and LC-MS were utilized to analyze the effects of TcCYP725A22 overexpression on the synthesis of taxol. The results showed that, in the TcCYP725A22 overexpressed cell line, expression levels of most defined hydroxylase genes for taxol biosynthesis were increased, and the yield of taxanes were also increased. It was concluded that the hydroxylase gene TcCYP725A22 is likely involved in the biosynthetic pathway of taxol.
Biosynthetic Pathways ; Mixed Function Oxygenases ; Paclitaxel ; Taxoids ; Taxus

Objective To investigate the clinical therapeutic effect of sigmoid neobladder after laparoscopic radical cystectomy in the treatment of invasive bladder cancer .Methods 21 patients with invasive bladder cancer accepted sigmoid neobladder after laparoscopic radical cystectomy and pelvic lymphadenectomy .The postoperative complications and clinical therapeutic effect were analyzed .Results All surgeries were completed successfully .The operation time was (245 ±125) min,blood lost was (98 ±45) mL.All the 21 patients were followed up for 3-24 months,mean of 12 months.Both side mild hydronephrosis in 6 cases (28.6%) and unilateral hydronephrosis in 1 case(0.5%).Average bladder reservoir was (548.8 ±221.6)mL,the maximum bladder pressure during the new bladder urine storage period was (2.26 ±0.24) kPa,the maximum bladder pressure during voiding period was (6.18 ±0.29) kPa,volume of residual urine was (87 ±52) mL.The mean maximum uroflow rate was (83.2 ± 5.6)mL/s.The continence of micturition was 90.4% in day-time and 76.1% in night-time.1 case had urethral stricture and got better after the urethral dilatation .Conclusion The sigmoid neobladder after laparoscopic radical cystectomy has more reliable results,without serious postoperative complications and patients with high quality of life after operation.This operation may be applied in clinical practice .

Objective To establish genotyping methods for rapid identification of Brucella melitensis (B.melitensis) biovar 1,2 and 3 and to verify these method.Methods Single nucleotide polymorphism of RpoB gene and tandem repeat sequence (TRS) Bru42 of standard reference strain 16M were used to design primers,then the RpoB-PCR and TRS-PCR method were established for identification of B.melitensis standard reference strains,these two methods were used to identify clinical isolates of B.melitensis and compared with the conventional methods.Results The results of B.melitensis standard reference strains (biotype 1,2,3) identified by RpoB-PCR and TRS-PCR were consistent with those of the conventional identification methods.Totally 50 clinical isolates [including B.melitensis biovar 1 (17),2 (3) and 3 (30)] were identified as RpoB-2 genotype,only one B.melitensis biovar 1 strain was identified as RpoB-3 genotype.Genotype identification results of standard reference strains and clinical isolates with the same biotype were not exactly the same.Fothermore,TRS-PCR experiment displayed that 51 clinical isolates were all genotype 2 of B.melitensis (genotype TRS-2).Conclusions There is no clear relationship between biovars and genotypes within B.melitensis,and significant difference exists between B.melitensis standard reference strains and clinical isolates within RpoB gene.Bru42 can not be used for genotyping clinical isolates of B.melitensis.

Objective To evaluate whether second-look endoscopy could prevent endoscopic submucosal dissection complicated with delayed bleeding and to clarify what kind of lesions that need second-look endoscopy. Methods 98 patients with early gastric cancer by histological diagnosis from Oct. 2014 to Sep. 2016 were included in this study. Mucosal damage related bleeding within 24 h after ESD was considered delayed bleeding. Clinical data of patients, lesions, surgical factors, and the incidences of bleeding before and after the second endoscopy examination were retrospectively studied. Results The en bloc resection rate of 98 patients was 100.0%, and all incisal margins were negative. No gastrointestinal perforation, death or severe complications occurred. 5.1%of the patients (5/98) occurred delayed bleeding after ESD and had been successfully stopped in a second-look endoscopy, and none received operations. No delayed bleeding occurred in follow-ups of patients with negative delayed bleeding. 40.0 % of the patients (2/5) with delayed bleeding received blood transfusion. The median time of second-look endoscopy after ESD was the second day after operation (1 ~ 3 d). The median bleeding time of 5 patients with delayed bleeding after ESD was the first day after operation (1 ~ 10 d). The median duration of surgery was 75 min (60~150 min), and the prediction success rate was 94.9 %. The results of univariate analysis showed that age [(69.6 ± 7.9) vs (60.9 ± 10.1) years old, P = 0.003] and operation time [(90.0 ± 41.0) vs (66.0 ± 42.0) min, P = 0.000] were both the risk factors in delayed bleeding and non-bleeding group. Binary Logistic regression analysis showed that operation time (OR^ =1.07; 95%CI:0.73 ~ 14.63, P = 0.010) was the only predictor of delayed bleeding after ESD. Conclusions Second-look endoscopy might be effective in preventing delayed bleeding after gastric ESD, especially within 48 hours after ESD. The operation time was an independent risk factor for delayed bleeding after gastric ESD.