Objective To establish an in vitro model of primnary osteoblasts fluorosis and to detect the influences of different doses of fluorosis on promoter methylation,transcription and expression of p16,then study the epigenetic effects of p16 gene on skeletal fluorosis.Methods Osteoblasts were isolated from Sprague-Dawley neonatal rats by enzyme digestion,and identified by morphology,alkaline phosphatasc staining and alizarin red staining.Osteoblast were treated with 0 (control group),200,400,800 and 1 600 μmol/L NaF for 72 h.The pl6 gene promoter region was amplified in the transcription initiation site-88-+ 143 region by bisulfatesequencing polymerase chain reaction (BSP).The mRNA transcription and the protein expression of p16 were detected by real-time quantitative PCR and Western blotting.Results Among the groups of osteohlasts treated with 200,400,800,1 600 μmol/L NaF,the positive rates of DNA methylation of promoter region in p16 gene of osteoblasts were 5.88％ (10/170),12.94％ (22/170),17.65％ (30/170) and 33.53％ (57/170),respectively.No DNA methylation was observed in the control group.There were significant differences between the control group and the NaF-treated osteoblasts groups (x2 =92.87,P ＜ 0.05).Average levels of p16 mRNA were 1.050 ± 0.073,0.869 ±0.037,1.065 ± 0.118 and 0.786 ± 0.148 in 200,400,800 and 1 600 μmol/L NaF-treated osteoblasts groups,compared with the control group (1.110 ± 0.315),there were significant differences among groups (all P ＜ 0.05) and 1 600 μmol/L NaF-treated osteoblasts group was much lower than other groups (P ＜ 0.05).Average levels of p16protein were 1.190 ± 0.050,1.214 ± 0.058,1.122 ± 0.123 and 0.320 ± 0.074 in 200,400,800 and 1 600 μmol/LNaF-treated osteoblasts groups,compared with the control group (1.115 ± 0.057),there were significant differences among groups (all P ＜ 0.05) and 1 600 μmol/L group was much lower than other groups.Conclusion NaF can cause hypermethylation in the promoter region of p16 gene,then suppress the expression of mRNA and protein,which might be one of the important mechanisms of cell proliferation change and cell cycle disorder in skeletal fluorosis.

0.05),but the average levels of DNMT1 mRNA of mild and moderate groups were significantly lower than that of the control group(P

Objective To investigate the insulin and glucagon levels in the patients with different islet cell antibody (ICA) subtypes and to explore the pathogenesis of latent autoimmune diabetes of adult (LADA). Methods Subjects were classified by immunohistology and 29 ICA-peripheral-positive DM patients, 28 ICA-diffused-positive DM patients and 17 controls (ICA-negative) were included. Serum glucose, insulin and plasma glucagon were measured at 0, 30, 60, 120 min after standard meal. Results (1) As compared with controls, glucagon in ICA positive groups were higher (both P

This paper discussed that how to cultivate high-quality medical talents by the use of basic education,medical moral education and psychologic education etc.

Pancreatic cancer is a highly malignant tumor with a high mortality.Surgery is the only potential hope of cure for patients with pancreatic cancer.Imaging examination plays an essential role in both the early diagnosis and preoperative assessment.Accurate tumor staging has guiding significance for clinical practice.Appropriate therapeutic schedule will be selected according to the tumor staging,and then patients' prognosis can be evaluated.Recently,the TNM staging systems for pancreatic cancer have been updated by American Joint Committee on Cancer (AJCC).Major changes were made in the T and N staging.This article will review the updates of the 8th edition of AJCC cancer staging for pancreatic cancer from radiography aspect.

Objective:To establish a protein-protein interaction (PPI) network of 5 microRNA (miRNA) target genes differentially expressed in the plasma of coal-burning fluoride exposed population, and to screen genes and gene modules with important roles.Methods:Five miRNA (hsa-miRNA-3131, hsa-miRNA-4516, hsa-miRNA-6501-5p, hsa-miRNA-10b-5p, hsa-miRNA-4683) target genes differentially expressed in the plasma of coal-burning fluoride exposed population screened by our previous study were mapped to the STRING online database (https://string-db.org), and the PPI network was screened. The Cytoscape v3.6.0 software was used to visualize the PPI network, the topological attribute values degree and betweenness centrality were obtained by the NetworkAnalyzer plug-in, and the central node was filtered in the network (the node with the highest degree and the highest betweenness centrality). At the same time, the maximal clique centrality (MCC) analysis method in the CytoHubba plug-in was used to determine the important nodes in the PPI network. The cluster analysis was conducted by the MCODE plug-in, and the gene modules were screened in the PPI network. The protein names contained in the gene modules were submitted online to the KOBAS v3.0 database (http://kobas.cbi.pku.edu.cn/), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed on the gene modules selected by the MCODE plug-in.Results:The PPI network of target genes was consisted of 1 035 nodes and 4 346 edges. The degree (101) and betweenness centrality (0.010 723 89) of ubiquitin A-52 residue ribosomal protein fusion product 1 (UBA52) were the highest, which was the central node of the PPI network. According to MCC analysis, UBA52 was an important node in the PPI network. The top 5 gene modules were selected from the PPI network, and the highly enriched gene pathways in the KEGG pathway enrichment analysis of the 5 gene modules included ubiquitin mediated proteolysis, spliceosome, endocytosis, neuroactive ligand-receptor interaction and vesicular transport.Conclusion:The PPI network of 5 miRNA target genes differentially expressed in the plasma of people exposed to coal-burning pollution of fluoride is successfully established, and the UBA52 gene and the 5 main pathways of gene modules are selected.

Objective To detect the early diversification of the bone mineral density in skeletal fluorosis of rabbits by quantitative computed tomography (QCT),and analyze the possible relationship between bone mineral density and bone injury in rabbits with fluorosis.Methods A total of 16 rabbits,half male and half female,were randomly divided into control and experimental groups according to body weight.The two groups of rabbits were given drinking water containing NaF 0 or 300 mg/L,respectively,for 90 days.After the experiment,their bone fluoride content was determined via the fluorine ion-selective electrode method.The alkaline phosphatase (ALP) and osteocalcin (BGP) in serum were measured using enzyme-linked immunosorbent assay (ELISA).Femurl bone mineral density was detected with QCT in vivo.Histopathological changes of femur were observed under light microscope and trabecular acreage was calculated.The results were analyzed with independent-samples t test(t') and partial correlations.Results The bone fluoride content [(3 232.16 ± 927.85) mg/kg],ALP [(42.69 ± 3.28) U/L],BGP concentration [(2 504.19 ± 276.79) μg/L],bone density [(653.49 ± 167.81) g/cm3] and trabecular number [(39.02 ± 3.33)Tb.Ar] of the experimental group were higher than those of control group [(554.01 ± 376.51)mg/kg,(20.50 ± 4.90)U/L,(1 294.60 ± 191.86)μg/L,(540.40 ± 41.99)g/cm3,(8.15 ± 2.34)Tb.Ar],and the differences were statistically significant (t'=7.565,10.641,10.158,2.615,14.494,all P ＜ 0.05).The tissue sclerosis,bone sclerosis and bone texture coarsening were observed through bone mineral density imaging taken by QCT in experimental group.The number of trabeculae increased and the arrangement of tra bec ulae was disorganized.Bone mineral density was positively correlated with bone fluoride,trabeculae,BGP and ALP (r =0.702,0.627,0.614,0.567,all P ＜ 0.05).Conclusions QCT bone density measurement in skeletal fluorosis of rabbits can be used to compute the threedimensional bone density.And it has a good correlation with bone fluoride content,bone histopathological changes and index of bone metabolism in skeletal fluorosis,which suggests that QCT may provide a useful reference for application in patients with skeletal fluorosis.

OBJECTIVETo screen DENV-2 binding proteins from Aedes albopictus and Culex. quinquefasciatus.

METHODSThe total proteins of Aedes albopictus and Culex. quinquefasciatus in different developmental stages were prepared and analyzed with SDS-12% polyacrylamide gel. After electrophoresis the proteins were transferred using Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad ) to a nitrocellulose membrane. Virus overlay protein-binding assay (VOPBA) was carried out using anti-dengue virus 1-4 monoclonal antibody.

RESULTSIn Aedes albopictus, VOPBA detected DEN-2 binding molecules of 25 000, 35 000, and 50 000 in larvae samples, molecules of 35 000 and 50 000 in pupae samples, a 50 000 molecule in male mosquito samples, and molecules of 35 000 and 50 000 in female mosquito samples. DENV-2 binding protein of 35 000 was found in the larvae, pupae, and female mosquitoes, but not in male mosquitoes. In Culex. Quinquefasciatus, VOPBA detected a molecule of 100 000 in larvae samples, molecules of 40 000, 100 000, and around 50 000 (48 000 and 60 000) in pupae samples, and molecules of 40 000 and 100 000 in male mosquitoes and female mosquito samples.

CONCLUSIONSeveral proteins capable of binding DENV are found in Aedes albopictus and Culex. quinquefasciatus in different development stages. The 35 000 molecule expressed in Aedes albopictus as a putative receptor protein may be related to virus tropism in mosquito tissues.

Aedes ; virology ; Animals ; Culex ; virology ; Dengue Virus ; Female ; Insect Proteins ; isolation & purification ; Larva ; Male ; Pupa ; Receptors, Virus ; isolation & purification

OBJECTIVETo evaluate the anti-viral effects of a plasmid expressing an inverted-repeat RNA targeting dengue virus type-2 (DENV-2) pre-membrane (prM) gene.

METHODSuckling mice were inoculated with live DENV-2 in the brain. The total RNA was extracted from the brain tissue of the infected mice, and the prM gene fragments were amplified by RT-PCR and then subcloned into XhoI/EcoR I of the pcDNA3.1(+) plasmid in antisense orientation to construct the plasmid pcDNA-asprM. DENV-2 prM sequences were also subcloned into pMD18-T-vector in sense orientation to construct the plasmid pMD18-T- prM. pcDNA-irRNA was constructed by inserting in sense orientation the prM fragment isolated from pMD18-T-prM into the NheI/Kpn I of pcDNA-asprM. The plasmid pcDNA-irRNA was transfected into BHK-21 cells and the anti-viral effects were analyzed by semi-quantitative PCR and real-time PCR.

RESULTSTransfection with the plasmid pcDNA-irRNA caused a reduction of NS3 mRNA expression level by 28% in BHK-21 cells following a 96-h challenge with DENV-2 as compared to the cells without plasmid transfection (positive control). The viral copies in pcDNA-irRNA-transfected cells was 1.44-fold lower than those in the positive control cells following a 72-h virus challenge, and the mRNA expression levels of NS1 were also significantly lower in the transfected cells at 96 h after viral challenge (P<0.05) as shown by real-time quantitative PCR.

CONCLUSIONThe inverted-repeat RNA for DENV-2 prM gene silencing can suppress DENV-2 replication in BHK-21 cells, which provides a basis for developing dengue virus gene vaccine.

Animals ; Base Sequence ; Cells, Cultured ; Cricetinae ; Dengue Virus ; physiology ; Gene Silencing ; Mice ; Mice, Inbred Strains ; RNA, Viral ; genetics ; Terminal Repeat Sequences ; Viral Envelope Proteins ; genetics ; Virus Replication ; genetics

BACKGROUND: Mild cognitive impairment (MCI) in elderly people is becoming increasingly a new hot spot for the clinical study of Alzheimer disease at present, and it is still unknown how many cases of MCI will convert into Alzheimer disease. OBJECTIVE: To analyze the incidence rate of mild cognitive impairment and the conversion rates into dementia and Alzheimer disease among elderly people.DESIGN: A prospective cohort study based on diagnosis. SETTING: Second Department of Cadre's Ward, Bethune International Peace Hospital of Chinese PLA.PARTICIPANTS: The subjects were 216 MCI patients and 2 302 subjects with normal cognition selected in the baseline investigation to the retired cadres aged 60 years and above, in 26 military cadres' sanatoriums of Shijiazhuang city between August and September 2001.METHODS: The MCI diagnosis was according to the MCI diagnostic standard in Diagnostic and Statistical Manual of Mental Disorders - Fourth Edition (DSM-Ⅳ), and the diagnosis of dementia was according to the standard in DSM-Ⅳ revised by American Psychiatric Association. Alzheimer disease was diagnosed according to the standards of National Institute of Neurological and Communicative Disorders and Stroke/the Alzheimer Disease and Related Disorders Association (NINCDS-ADRDA). All the 216 MCIpatients and 2 302 subjects with normal cognition were enrolled in the 3-year cohort study. MAIN OUTCOME MEASURES: The incidences of MCI among subjects with normal cognition; the annual conversion rates into Alzheimer disease in MCI patients and subjects with normal cognition; the relative risks (RR) and 95% confidence interval (95% CI) of MCI and normal cognition developing into Alzheimer disease. RESULTS: The MCI group and normal cognition group were followed up once every year for 3 years. Of the 216 MCI patients, 7 died, and 209 were followed up actually, and the follow-up rate was 96.8%. Of the 2 302 subjects with normal cognition, 36 died, and 2 266 were followed up actually,and the follow-up rate was 98.4%. ① In the elderly subjects with normal cognition, the annual incidence of MCI was 4.8%, and those of dementia and Alzheimer disease were 1.3% and 0.8 respectively. ② The annual in cidence rates of dementia and Alzheimer disease in MCI patients were 8.1% and 5.6% respectively, which were insignificantly different between males and females (P ＞ 0.05). ③ The incidence rates of dementia and Alzheimer disease showed a descending trend with the elevation of educational level in both the MCI patients (trend x2 =5.57, P=0.02; trend x2 =4.92, P=0.03) and the subjects with normal cognition (trend x2 =23.1, P＜ 0.001; trend x2 =18.0, P ＜ 0.001). ④ The annual incidence rates of dementia and Alzheimer disease showed an ascending trend with aging in both the MCI patients (trend x2 =14.6, P ＜ 0.01; trend x2 =13.9, P ＜ 0.01) and the subjects with normal cognition (trend x2 =32.3, P ＜ 0.01; trend x2=23.8, P ＜ 0.01). ⑤ The relative risks of the conversion into dementia and Alz heimer disease in MCI patients were 6.4 and 7.4 times as many as those in the subjects with normal cognition.CONCLUSION: The risks of conversion into dementia and Alzheimer disease in elderly MCI patients are far higher than those in elderly people with normal cognition. It should be reinforced to monitor elderly MCI patients, who are the of high risk group of dementia.