Objective To study the structures of the plasmids of Klebsiella pneumoniae KF3 at the genome metagenome level througth with whole plasmid DNA sequencing, to analyze the functional genes carried by plasmid and to identify the correlation of resistance and pathogenicity between the plasmids and the host strains. Methods The alkaline lysis method was used to extract plasmids. We constructed the small insert pUC18 library and the large insert Forsmid library, sequenced and used the Phred / Phrap / Consed package to assemble these sequences and gained a complete sequence. The open reading frame(ORFs) were predicted by the Glimmer software and annotated, analyzed the functions of these genes. Results We successfully constructed the pUC18 library and the Fosmid libraries for the plasmid DNA and obtained three circular double-stranded DNA plasmids: pKF3-70 (69 477 bp), pKF3-90 (91 327 bp) and pKF3-147 ( 147 416 bp). There were drug resistant genes, conjugative transfer genes and mobile DNA elements identified on three plasmids. Conclusion The three plasmids of KF3 could be transferred among different strains. It would lead to the dissemination of the resistant genes.

OBJECTIVETo prepare the trimeric subunits of recombinant human mannan-binding lectin (MBL) with biological activities.

METHODSA prokaryotic expression vector containing human MBL N-terminal deletant (rhMBLδN) gene we previously constructed was transformed into E. coli for efficient expression of rhMBLδN fusion protein. Based on the principle that the collagen polypeptides tend to self-assembly into the tertiary structure of proteins by forming a triple helix due to the characteristic properties of the collagen proteins, rhMBLδN fusion protein was limitedly hydrolyzed with thrombin. The obtained rhMBLδN polypeptide was repeatedly dialyzed in 50 mmol/L PBS (pH7.2) and ddH(2)O, and the final product was analyzed for its bioactivities using a ligand-binding assay and a C4d deposition assay.

RESULTSrhMBLδN polypeptide with a relative molecular mass of about 20 000 was obtained by limited proteolysis of rhMBLδN fusion protein with thrombin. Repeated dialyses of rhMBLδN polypeptides in 50 mmol/L PBS and ddH(2)O resulted in the isolation of the trimeric subunit trhMBLδN (with a relative molecular mass of about 50 000), which contained a collagen-like helix. The trhMBLδN protein had a higher ligand-binding activity than rhMBLδN polypeptide, and acquired the activity to initiate the lectin pathway of complement activation, but the activities were lower than those of natural MBL.

CONCLUSIONWe have successfully obtained the bioactive trimeric subunit of rhMBL, trhMBLδN, and this structural subunit is also the functional subunit of the MBL molecule.

Complement Activation ; Escherichia coli ; metabolism ; Genetic Vectors ; Humans ; Mannose-Binding Lectin ; biosynthesis ; genetics ; isolation & purification ; Recombinant Proteins ; biosynthesis

Objective To study the relationship between glutamine metabolism and radiotherapy resistance in nasopharyngeal carcinoma (NPC).Methods The glutaminase 1 (GLS1) knockout CNE1,6-10B cell lines and the control cell lines were established by lentivirus transfection technology.The CNE2 cell lines with GLS1 overexpression and control cell lines were established by liposome transfection technology.BPTES,one of Glutamine enzyme inhibitors,was used to inhibit the activity of GLS1.Glutamine/Glutamate(Gln/Glu) and Glu kits were used to detect glutamine metabolism in NPC cells;in vitro radiation clone formation experiment and flow cytometre mediated apoptosis assay were adopted to detect the effects of glutamine metabolism modulation on NPC radiosensitivity.Western blot and autophagic puncta monitor assay were carried out to detect the effects of glutamine metabolism modulation on autophagy status in NPC cells.Results Inhibiting the glutamine metabolism led to decreased clone formation,increased apoptotic rates,and decreased autophagic level in NPC cells;while activating the glutamine metabolism led to increased clone formation,decreased apoptotic rates and increased autophagic level in NPC cells.Conclusions The reduction of glutamine metabolism inhibits autophagy and increases radio sensitivity of NPC.

Objective To investigate the relationship between the severity of white matter lesions (WMLs) and the hematoma volume in patients with spontaneous intracerebral hemorrhage. Methods Patients with intracerebral hemorrhage (age ≥40 years) who were included in the Image Registration Center, Drum Tower Hospital, Nanjing University School of medicine from June 1, 2012 to May 31, 2017 were enrolled retrospectively. The head CT and baseline head MRI data were collected within 12 h after onset. The volume of hematoma on the baseline CT was calculated using ITK-SNAP software. The WMLs volume was semi-automatically segmented and calculated in the WMLs area by MRIcron software and ITK-SNAP software. According to the Fazekas score, the severity of WMLs was divided into mild ( 0-2), moderate (3-4) and severe (5-6). According to the median volume of hematoma, the patients were divided into smaller hematoma volume group and larger hematoma volume group. The baseline data in patients of both groups were compared. Multivariate logistic regression analysis was used to determine the independent related factors of hematoma volume. Results Age, National Institutes of Health Stroke Scale (NIHSS) score, and hematoma volume increased with the severity of WMLs, while the total cholesterol and low-density lipoprotein cholesterol levels decreased with the severity of WMLs. Hematoma volume was significantly associated with the NIHSS scores, apolipoprotein A1, D-dimer, WML volume, and intracerebral hemorrhage site. Multivariate logistic regression analysis showed that high WML score (odds ratio [OR] 1.001, 95% confidence interval [ CI] 1.002-1.008; P=0.049), intracerebral hemorrhage site ( OR 1.441, 95% CI 1.090-1.911; P=0.010), and NIHSS score (OR 1.081, 95% CI 1.011-1.152; P=0.031) were the independent risk factors for larger hematoma volume. Conclusion The severity of WMLs was significantly positively correlated with the baseline hematoma volume in patients with spontaneous intracerebral hemorrhage.

Objective:To analyze the value of the modified 5-factor frailty index in assessing postoperative complications and mortality in elderly hip fracture patients.Methods:In this retrospective study, clinical data were collected of hip fracture patients aged 60 years and above surgically treated at Beijing Luhe Hospital affiliated to Capital Medical University between January 2015 and December 2019.Patients' group assignment was based on whether the modified frailty index score was ≤1 or ≥2, and a post-surgery follow-up was conducted for survival at 30 days, 1 year, 2 years, and 4 years, which was analyzed by the Kaplan-Meier method.Multivariate Cox regression analysis was used to identify factors affecting death in elderly patients.Results:A total of 1 208 patients were included, with 890 in the group with the index score ≤1 and 318 in the group with the index score ≥2.There was no difference in mortality at 30 days(1.6% or 14/890 vs.1.9% or 6/318, P=0.707), 1-year(11.3% or 99/874 vs.11.6% or 36/310, P=0.917), 2-years(19.7% or 168/852 vs.24.3% or 73/300, P=0.099)and 4-years(44.0% or 238/541 vs.51.5% or 106/206, P=0.071). The incidence of postoperative complications in the group with the score ≥2 was higher(14.8% or 47/318 vs.9.7% or 86/890, P=0.012), including the incidence of stroke(6.3% or 20/318 vs.1.8% or 16/890, P<0.001)and the incidence of postoperative pneumonia(6.0% or 19/318 vs.3.1% or 28/890, P=0.029), and the differences were statistically significant.Multivariate Cox regression analysis showed that age, being female, the Charlson comorbidity index score and low hemoglobin at admission were risk factors for 1-year, 2-year and 4-year mortality post-surgery(all P<0.05), while the modified frailty index score had no correlation with postoperative mortality. Conclusions:A modified frailty index ≥2 is predictive of increased risk of postoperative pneumonia and stroke in patients with hip fractures, but is not correlated with the risk of postoperative mortality.

Objective:To investigate the expressions of miR-1254 and its target gene colony-stimulating factor-1 (CSF-1) in glioma tissues and glioma cells, and the effects of miR-1254 and CSF-1 on the proliferation and invasion of glioma cells.Methods:The clinicopathological specimens and paracancerous tissues of 30 patients with glioma who underwent surgical treatment in Shiyan Taihe Hospital of Hubei Province from April 2017 to May 2019 were collected. Quantitative real-time fluorescent PCR (qRT-PCR) was used to detect the expression levels of miR-1254 and CSF-1 mRNA in glioma tissues, paracancerous tissues, U87 cells and human brain normal glial cells HEB. The glioma U87 cells were divided into blank control group, mimic NC group, miR-1254 mimic group, and siRNA NC group, CSF-1 siRNA group. The expression levels of miR-1254 and CSF-1 mRNA were detected by qRT-PCR. The protein expression levels of CSF-1 in each group were detected by Western blotting. Dual luciferase reporter gene assay was used to verify the targeting relationship between miR-1254 and CSF-1 gene. CCK-8 method and Transwell invasion experiment were used to detect the proliferation and invasion ability of cells.Results:The relative expression of miR-1254 mRNA in glioma tissues was 0.44±0.16, that in adjacent tissues was 1.15±0.28, and there was a statistically significant difference ( t=12.914, P<0.001). The relative expression of CSF-1 mRNA in glioma tissues was 1.96±0.27, that in adjacent tissues was 0.98±0.22, and there was a statistically significant difference ( t=14.970, P<0.001). The relative expression of miR-1254 mRNA in glioma cells U87 was 0.39±0.11, that in human brain normal glial cells HEB was 1.03±0.02, and there was a statistically significant difference ( t=10.113, P=0.008). The relative expression of CSF-1 mRNA in glioma cell U87 was 1.02±0.03, that in human brain normal glial cell HEB was 0.32±0.13, and there was a statistically significant difference ( t=9.037, P=0.009). The expression levels of CSF-1 mRNA and protein decreased with the increase of miR-1254 after transfection of miR-1254. The results of dual luciferase reporter gene assay showed that compared with the mimic NC group (1.04±0.12), the fluorescence activity of CSF-1-WT cells in the miR-1254 mimic group (0.31±0.02) was significantly reduced ( t=10.430, P<0.001). The difference in cell proliferation ability among the blank control group (0.71±0.01), mimic NC group (0.68±0.04) and miR-1254 mimic group (0.35±0.01) was statistically significant 48 h after transfection ( F=252.651, P<0.001). The difference was statistically significant between miR-1254 mimic group and blank control group ( P<0.001), and the difference was also statistically significant between miR-1254 mimic group and mimic NC group( P<0.001). The difference in cell proliferation ability among the blank control group (0.71±0.01), siRNA NC group (0.68±0.04) and CSF-1 siRNA group (0.25±0.01) was statistically significant ( F=320.309, P<0.001). The difference was statistically significant between CSF-1 siRNA group and blank control group ( P<0.001), and the difference was also statistically significant between CSF-1 siRNA group and siRNA NC group ( P<0.001). Invasion experiments showed that the difference of transmembrane cells number among the blank control group (365±27), mimic NC group (388±24) and miR-1254 mimic group (83±15) was statistically significant ( F=173.915, P<0.001). The blank control group (365±27), siRNA NC group (404±32) and CSF-1 siRNA group (87±14) had statistically significant difference in the number of transmembrane cells ( F=141.294, P<0.001). Conclusion:The expressions of miR-1254 in glioma tissues and glioma cells U87 are significantly decreased, and the expressions of CSF-1 in glioma tissues and glioma cells U87 are significantly increased. Overexpression of miR-1254 may inhibit the proliferation and invasion of glioma U87 cells by reducing the expression of CSF-1 targetedly.

OBJECTIVE：To establish the method for the content determination of inorganic elements in Cyperus rotundus ，and to compare the contents of 14 kinds of inorganic elements in C. rotundus from different producing areas ，and to provide theoretical basis for its quality control and high quality resources development . METHODS ：The samples were processed by microwave digestion，and ICP-MS method was used to determine the contents of Na ，Mg，K，Ca，Mn，Fe，Ni，Cu，Zn，As，Se，Sr，Cd and Pb. SPSS 23.0 software were used for principal component analysis （PCA）and cluster analysis. RESULTS ：The average contents of above 14 kinds of inorganic elements in C. rotundus from different producing areas were 168.62，753.71， 6 938.33，24.31，14.69，197.77，0.60，2.43，26.89，0.21，0.06，5.64，0.05，0.32 mg/kg，respectively. The results of PCA showed that the cumulative variance contribution rate of the first four principal components was 86.203％，which could reflect most of the information of the original data. C. rotundus from Shandong ，Jiangxi，Shanxi，Hubei and Yunnan ranked the top five places in terms of comprehensive score of inorganic element contents. The results of cluster analysis showed that the samples from 9 producing areas were clustered into 5 categories，showing the characteristics of clustering by producing area. From the perspective of inorganic elements ，the quality of C. rotundus from East China ，Central China ，North China and Southwest China was better than that from South China. CONCLUSIONS ：Essential trace elements like Na ，Mg，K，Ca，Mn，Fe，Cu，Zn，Sr are rich in C. rotundus，and there are small amounts of Ni ，As，Se，Cd，Pb elements in it. The contents difference of inorganic elements in C. rotundus from different origins may related to the geographical area it belongs to.

On August 2-4,2023,the"Third Summit Forum on'Building a Community of Shared Future for Doctors and Patients'"was jointly organized by institutions such as the Chinese Medical Ethics,the Hospital Humanities Management and Talent Training Special Committee of the China Population and Culture Promotion Association,Center for Ethical Studies of Renmin University of China,the Newspaper for China's Physicians,the China Health Law Society,the China Anti-Cancer Association,and the China Association For Ethical Studies in Harbin.The conference arranged a sub-forum for the"Seminar on the Construction of Chinese Medical Humanities",with domestic medical humanities scholars attending the conference.After heated discussions at the seminar,the Scholars'Consensus on the Construction and Development of Chinese Medical Humanities was formed.It was proposed that in the new era,it is urgent to build the medical humanities discipline,as well as lead the academic integration and development of medical humanities under the core socialist values.At the same time,for the construction of the medical humanities discipline,it is necessary to optimize the organizational mechanism,prosper and develop the overall framework of the medical humanities discipline,accelerate the construction of a professional teaching team for the medical humanities discipline,promote the establishment of a new carrier medical humanities education and teaching in cultivating morality and nurturing talents,as well as focus on solving problems related to the cultivation of medical humanities graduate students.

OBJECTIVE： To estab lish the method for simultaneous determination of the contents of cyperotundone ， nootkatone，α-cyperone and aristolone in the volatile oil of Cyperus rotundus ，compare the content differences of 4 components in C. rotundus samples from different origins ，and to provide reference for germplasm screening ，development and utilization of the medicinal material. METHODS ：The volatile oil was extracted from 46 batches of C. rotundus from 12 origins. The contents of cypermenone，nootkatone，α-cyperone and aristolone in volatile oil were determined by HPLC. The determination was performed on Kromasil C 18 column with mobile phase consisted of methanol-water （68∶32，V/V）at the flow rate of 1.0 mL/min；the column temperature was 30 ℃；the detection wavelength was set at 242 nm；the sample size was 20 μL. Using the contents of above 4 components as evaluation indexes ，radar image analysis ，cluster thermal map analysis and principal component analysis were performed for comparing the quality of C. rotundus from different origins. RESULTS ：The results of content determination methodology investigation met relevant requirements ；the total contents of 4 components in volatile oil from C. rotundus from different origins ranged from 136.986 4 to 538.832 1 mg/g，of which the total content of samples from Yunnan was the highest （the average value was 476.059 2 mg/g）. Radar image analysis results showed that the whole contour in the 4 origins of Guangdong ， Jiangxi，Guangxi and Yunnan was large relatively and better balanced ，among which the samples from Yunnan had the largest overall contour and the best balance. The cluster thermal map analysis results showed that the samples from 12 origins could be grouped into 2 categories，the first category was from Hubei ，Jiangxi，Yunnan，Sichuan，Guangdong，Shandong，Henan and Shaanxi；the second category was from Guangxi ，Shanxi，Anhui and Hainan ；the quality of samples from the first category were better than that of samples from the second category. The principal component analysis results showed that the cumulative contribution rate of the first three principal components was 96.1％，and the samples from 12 origins were mainly clustered into two categories ，which was consistent with the results of cluster thermal map analysis. CONCLUSIONS ：Established HPLC method can be used for simultaneous determination of cypermenone ，nootkatone，α-cyperone and aristolone in volatile oil of C. rotundus from different origins. Among the samples from 12 origins，the quality of medicinal material from Yunnan is better.