ObjectiveTo investigate the methylation status of multiple genes in plasma and tumor tissues and its application in molecular diagnosis of esophageal squamous cell carcinoma (ESCC).Methods Methylation specific polymerase chain reaction (MSP) was used to detect methylation status of 5 tumor suppressor genes,such as adenomatous polyposis coli ( APC ),retinoic acid receptor-beta2 ( RARβ2 ),E-cadherin (CDH1),cyclin-dependent kinase inhibitor4A (p16INK4α) and ras association domain family member 1 A (RASSF1A) in tumour tissues,adjacent normal tissues and plasma which obtained 1 d preoperative and 7 d postoperative in 76 cases with ESCC.60 healthy volunteers were randomly selected as a control which were age-matched and sex-matched.Chi square test was used to analyze DNA methylation rates of 5 genes in various groups of tissue and plasma samples; Kappa test was used to compare the consistency of DNA methylation in the plasma samples and tissue samples,and their correlation was analyzed by Spearman correlation test; Receiver operating characteristic curve (ROC) was used to evaluate the sensitivity and specificity for single gene detection and 5 genes joint detection for diagnosis of esophageal squamous cell carcinoma.ResultsThe methylation rates of APC,RARβ2,CDH1,p16INK4α and RASSF1A in tumour tissues of patients with ESCC were 44.7％ ( 34/76),72.4％ ( 55/76 ),72.4％ (55/76),86.8％ ( 66/76 ),55.3％ (42/76),respectively,which were significantly higher than that in the corresponding adjacent normal tissues [ 6.6％ ( 5/76 ),3.9％ ( 3/76 ),3.9％ ( 3/76 ),3.9％ ( 3/76 ),2.6％ ( 2/76 ),x2 =29.01,75.39,75.39,105.34,57.18,all P ＜ 0.001 ].The methylation rates of above 5 genes in patients' plasma were 42.1％ ( 32/76 ),63.2％ ( 48/76 ),63.2％ ( 48/76 ),71.1％ ( 54/76 ),50.0％ ( 38/76 ),respectively,which were significantly higher than that of control group [3.3％ (2/60),3.3％ (2/60),1.7％ ( 1/60),3.3％ (2/60),1.7％ (1/60),x2 =26.88,51.62,55.01,63.48,38.30,all P ＜ 0.001 ].The methylation consistency was favorable or well between plasma and tumour tissues in patients with ESCC ( Kappa value was 0.679,0.791,0.791,0.542 and 0.895,respectively.all P ＜0.001 ).In single-gene detection for patients' plasma,methylation,the sensitivity of 5 genes was 42.1％ ( 32/76 ),63.2％ ( 48/76 ),63.2％ ( 48/76 ),71.1％ ( 54/76 ),50.0％ ( 38/76 ),respectively.The specificity was 96.7％ ( 58/60 ),96.7％ ( 58/60 ),98.3％ (59/60),96.7％ (58/60),98.3％ (59/60),respectively.The area under curve (AUC) of ROC was 0.694 [95％ confidence interval( CI)0.606 - 0.782 ) ],0.799 ( 95％ CI 0.723 - 0.875 ),0.807 ( 95％CI 0.733 - 0.882),0.839 ( 95 ％ CI 0.769 - 0.908 ) and 0.742 ( 95 ％ CI 0.659 - 0.824 ),respectively.In united testing of 5 genes,the sensitivity was 80.3％ and the specificity was 88.3％,AUC was 0.843 (95％CI 0.773 -0.913 ).The sensitivity of united testing was significantly higher than that of single-gene detection of APC and RASSF1A(x2 =23.30,15.33 ; P ＜ 0.001 ),except RARβ2,CDH1 and p16INK4α (x2 =5.48,5.48,1.75; P =0.019,0.019,0.186);There was no significant differences in specificity between united testing and single-gene detection (x2 =1.922,1.922,3.348,1.922,3.348,all P ＞ 0.05 ).Conclusions The methylation consistency is favorable or well between tumour tissues and plasma in patients with ESCC.There is no significant superior in diagnosing ESCC with united testing of multiple tumor suppressor genes methylation in plasma than with single-gene detection.But the sensitivity of the former is better than the latter.

Objective: To study the change of the blood level of CORT and expression of CRHmRNA in asthma-tic rats and the effects of Bushendingchuan Decoction(Cornu Cervi, Herba Cistanches, Radix Morindae Officinalis, Radix Rehmanniae Preparata, etc.) on them. Methods: The change of level in CORT was measured by radioimmunoassay, the change of level in hypothalamic CRHmRNA was measured by RT-PCR(Reverse transcription and polymerase chain reaction by the semi-quantitative determination). Results: The blood level of CORT decreased(P

ObjectiveTo investigate the mechanism of action and main active components of Xiaoyaosan in the treatment of diabetic mellitus-induced erectile dysfunction （DMED）. MethodStreptozotocin （STZ） was used to induce a diabetic rat model. The therapeutic efficacy of Xiaoyaosan was evaluated by measuring intracavernous pressure/mean arterial pressure （ICP/MAP） and using Masson's trichrome staining. The main active components， key targets， and potential signaling pathways of Xiaoyaosan for the treatment of DMED were predicted by network pharmacology and molecular docking. The predicted results were then validated by in vitro and in vivo experiments. ResultThe ICP/MAP measurements and Masson's staining results showed that compared with the results in the control group， the erectile function of rats in the model group was significantly reduced （P<0.01）， and the ratio of smooth muscle/collagen fibers was significantly reduced （P<0.01）. After treatment with Xiaoyaosan， compared with the results in the model group， the ICP/MAP value of the diabetic rats was significantly elevated （P<0.01）， and the ratio of smooth muscle/collagen fibers was significantly higher （P<0.01）. The results of network pharmacology showed that Xiaoyaosan acted on key targets such as albumin （ALB）， protein kinase B1 （Akt1）， interleukin-6 （IL-6）， and tumor necrosis factor （TNF） through its main active components， including quercetin， kaempferol， β-sitosterol， and stigmasterol. These components were involved in the regulation of the advanced glycation end-products/receptor for advanced glycation end-products （AGE/RAGE） signaling pathway and the phosphoinositide 3-kinases（PI3K）/Akt signaling pathway in diabetic complications. The results of molecular docking showed that the key components of Xiaoyaosan had good binding capabilities with core targets， with β-sitosterol showing the strongest binding affinity with ALB. In vivo experiments demonstrated that Xiaoyaosan could significantly increase the protein and mRNA expression of ALB and Akt1 in serum， and inhibit the expression of IL-6 and TNF-α. It also significantly upregulated the expression of protein and mRNA of phosphorylation（p）-PI3K and p-Akt， and inhibited the RAGE expression. The results of cellular thermal shift assay （CETSA） showed that β-sitosterol could significantly inhibit the degradation of ALB protein. ConclusionXiaoyaosan may restore erectile function in diabetic rats by modulating targets such as ALB， Akt1， IL-6， and TNF， and through the RAGE/PI3K/Akt signaling pathway， and its main active component is likely β-sitosterol.

Objective:To observe the effect of early goal directed sedation (EGDS) on cerebral oxygen metabolism in patients with acute brain injury.Methods:A prospective cohort study was conducted. A total of 108 patients with acute brain injury admitted to the intensive care unit (ICU) of the Third Medical Center of the PLA General Hospital from January 2015 to December 2019 were enrolled. According to the patient's condition, dexmedetomidine contraindication and tolerance, and combined with the wishes of patients' families, they were divided into EGDS group and on-demand sedation group. Routine treatments such as surgery, mechanical ventilation, dehydration and reduction of intracranial pressure with mannitol, hemostasis or antiplatelets therapy were given according to the patient's condition. All patients were continuously given sufentanil by intravenous infusion for analgesia. Patients in the EGDS group were sedated by continuously intravenous infusion of dexmedetomidine (0.2-0.7 μg·kg -1·min -1) for 72 consecutive hours. Patients in the on-demand sedation group received intravenous bolus of propofol (0.5-1.0 mg/kg) when treatments were interfered due to agitation. Hemodynamic indexes [heart rate (HR), mean arterial pressure (MAP), cerebral perfusion pressure (CPP), intracranial pressure (ICP)], sedation indexes [bispectral index (BIS)], severity indexes [acute physiology and chronic health evaluation Ⅱ (APACHE Ⅱ) score, Glasgow coma score (GCS)] and cerebral oxygen metabolism indexes [jugular venous blood lactate (Lac), jugular venous oxygen saturation (SjvO 2), cerebral arterial oxygen content (CaO 2), cerebral extraction rate of oxygen (CERO 2), cerebral arteriovenous blood oxygen content difference (a-vDO 2)] were compared between the two groups before sedation and at 24, 48 and 72 hours of sedation. Results:① Among the 108 patients, 3 patients with cerebral hemorrhage received secondary surgery or had worsening of cerebral hernia were excluded. 105 patients were enrolled in the study, including 54 patients in the EGDS group and 51 patients in the on-demand sedation group. There were no statistically significant differences in gender, age, type of craniocerebral injury, GCS score, proportion of mechanical ventilation and operation ratio between the two groups. ② Compared with before sedation, Lac, CERO 2 and a-vDO 2 of both groups gradually reduced over time of sedation while SjvO 2 and CaO 2 were gradually higher. Those changes were more quickly in the EGDS group, Lac, SjO 2, CERO 2 and a-vDO 2 significantly improved at 24 hours of sedation compared with those before sedation. Above indexes at 72 hours of sedation in the EGDS group were obviously better than those in the on-demand sedation group [Lac (mmol/L): 1.81±0.31 vs. 2.19±0.12, SjvO 2: 0.714±0.125 vs. 0.683±0.132, CaO 2 (mL/L): 201.21±15.25 vs. 179.65±14.07, CERO 2: (27.87±3.66)% vs. (33.00±2.58)%, a-vDO 2 (mL/L): 44.32±5.68 vs. 48.57±8.22, all P < 0.05]. ③ Compared with before sedation, HR, MAP and ICP decreased in the two groups over time while CPP, BIS and GCS score showed increasing trend, especially more quickly in the EGDS group, HR at 24 hours of sedation, MAP, CPP, BIS and GCS score at 48 hours significantly improved as compared with those before sedation. Hemodynamics and sedation related parameters and GCS score at 72 hours of sedation in the EGDS group were significantly better than those in the on-demand sedation group [HR (bpm): 70.69±7.80 vs. 79.85±9.77, MAP (mmHg, 1 mmHg = 0.133 kPa): 84.23±8.76 vs. 89.97±9.48, ICP (mmHg): 14.23±8.76 vs. 15.97±9.48, BIS: 60.56±24.58 vs. 56.86±33.44, GCS score: 8.06±3.63 vs. 7.86±2.98, all P < 0.05]. The APACHE Ⅱ scores were significantly reduced at 72 hours of sedation in both groups as compared with those before sedation, while there was no statistical difference between the two groups. Conclusion:Compared with the on-demand sedation, EGDS could reduce cerebral oxygen metabolism, improve the coma degree, and reduce the severity of the disease in patients with acute brain injury.

Humans ; Anodontia/genetics* ; Tooth Abnormalities/genetics* ; Mutation ; Malocclusion/genetics* ; Wnt Proteins/genetics*

Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The recombinant E2 protein was CSFV-specific as proved by Western blotting and indirect ELISA. The rabbits immunized with the recombinant E2 can be protect from the challenge of hog cholera lapinized virus. This is the first report that E2 gene is expressed with high level expression in E. coli. In conclusion, it is an effective measure that mutate the CSFV E2 gene to increase its expression level in E. coli. The recombinant CSFV E2 protein possess fine immunonicity and can be used the antigen for the detection of CSFV antibody.
Antigens, Viral ; genetics ; immunology ; metabolism ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Mutagenesis, Site-Directed ; methods ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Viral Envelope Proteins ; genetics ; immunology ; metabolism

To study the effect of mesenchymal stem cell (MSC) on immune function, MSCs were isolated and cultured from human bone marrow cells. The purity of MSCs were identified with the spindle-fibroblastic morphology characterization by microphotograph and the phenotypes were tested by flow cytometry. MSCs were plated in 96-well plates (2,000/well and 1,000/well), and cocultured for 3 days with T cells isolated from cord blood. Cord blood T cells non-cocultured with MSC acted as control group. After cord blood T cells stimulated by PHA for 60 hours, [(3)H]-thymidine was added to each well and T cell proliferation was assessed by [(3)H] thymidine incorporation. The results showed that cord blood T cell proliferation was suppressed when 2,000 MSCs were plated each well and cord blood T cell proliferation was activated when 1,000 MSCs were plated. Our results suggested that the immunomodulatory function of MSC seemed dependent on cell dose. High concentration of MSC most often resulted in inhibition, while low concentration resulted in stimulation.
Antigens, CD ; analysis ; Bone Marrow Cells ; cytology ; Cell Division ; immunology ; Cells, Cultured ; Coculture Techniques ; Fetal Blood ; cytology ; Flow Cytometry ; Humans ; Lymphocyte Activation ; drug effects ; immunology ; Mesoderm ; cytology ; Phytohemagglutinins ; pharmacology ; Stem Cells ; cytology ; T-Lymphocytes ; cytology ; drug effects ; metabolism ; Thymidine ; metabolism ; Tritium

Catheter Ablation/adverse effects* ; Coronary Stenosis ; Coronary Vessels/diagnostic imaging* ; Humans

OBJECTIVETo develop a system to rescue virus by intracellular expression of T7 RNA Polymerase.

METHODSThe gene of T7 RNA Polymerase was amplified and cloned to VR1012 by molecular biological technology. The expression plasmid VR-1a was then identified. VR-1a and EV71 infectious plasmid were co-transfected in Vero cell. CPE was observed and viral gene viral antigen were detected.

RESULTSThe gene of T7 RNA Polymerase was successfully cloned into vector VR1012. Vero cell developed to CPE after being transfected VR-1a and EV71 infectious plasmid. EV71 gene was amplified by RT-PCR from the culture. EV71 antigen was also detected by ELISA.

CONCLUSIONThe method can be used to rescue virus. It could apply to immunologic research of EV71 DNA vaccine.

Animals ; Cercopithecus aethiops ; DNA-Directed RNA Polymerases ; genetics ; metabolism ; Enterovirus A, Human ; genetics ; physiology ; Gene Expression ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; metabolism ; HeLa Cells ; Humans ; Plasmids ; genetics ; metabolism ; Transfection ; Vero Cells ; Viral Proteins ; genetics ; metabolism ; Virus Replication

OBJECTIVETo develop a vector inserted with complete genome of poliovirus strain Sabin I.

METHODSThe 3 fragments of the complete genome of Sabin I was amplified and cloned to pEASY-T3 by molecular biological technology. These cloned pEASY-T3 were then digested by Restriction enzymes and ligated to pWSK29 step by step and identified.

RESULTSThe complete genome of poliovirus strain Sabin I was successfully cloned into vector pWSK29 with 9 nucleotide mutations.

CONCLUSIONThe complete genome plasmid was constructed and it provided a basis for further research of the function of Sabin I.

Cloning, Molecular ; Genetic Vectors ; genetics ; Genome, Viral ; Mutation ; Poliovirus ; genetics